1NTI RNATIONAI, JOURNAL OI' LEI'ROSY^ Volume 68, Number 2

t'rinted in lhe U.S.A.(ISSN 0148-916X)

CURRENT LITERATURE

This department carries selected abstracts of articles published in current medicaijournals dealing with leprosy and other mvcobacterial diseases.

General and Historical

Andrade, V., Virmond, M., Gil Suarez,R., Moreira, T., Fernando, G. andCustodio, A. New approach to acceler-ate the elimination of leprosy. Hansen.Int. 24 (1999) 49-54.

With the existing effective regimens fortreatment (MDT/WHO), it is unbearablethat, in the turn of the century, leprosy isstill an important and worrying problem ofpublic health in Brazil. Although some esti-mation could lead to the conclusion that theincreasing rate of new cases detected in ourcountry is linked to improved quality ofhealth care, the size of the problem is ex-pressive and calls for innovative actions tocope with.

In a country with the size and culturalvariety of Brazil, it is essential to take ad-vantage of the principies of the health sys-tem in Brazil (SUS) to effectively increasecoverage of MDT. In this regard,CONASEMS, being the political represen-tative of secretaries of health of the munici-palities of Brazil, is the most appropriate lo-cus to center a multi-participative taskforcewith technical competence, financial flexi-bility and political support to introduce acarefully designed group of actions aimingat the acceleration of the elimination of lep-rosy as a public health problem. Partnershipis also essential, mainly from the commu-nity and its organized representatives(MORHAM) as well as national and inter-national nongovernmental organizations(ILEP) and intergovernmental organiza-tions (PAHO/WHO).

CONASEMES is the organization withthe power to guarantee the essence of theproposal—to increase extensively the cov-erage of MDT; taking roto considerationthis extensive expansion of care, simplifica-

tion and dismystification of leprosy is es-sential and is another key point in the pro-posal; the new and positive image of lep-rosy is the framework of these initiatives,since we want leprosy diagnosis and treat-ment reaching every patient in every vil-lage.—Authors' Conclusion

Bhatki, W. S. and Singh, M. G. Modifiedleprosy elimination campaign in Muni-bai (Bombay), índia—a report. Lepr.Rev. 70 (1999) 459-464.With appropriate planning and prepara-

tion, a modified leprosy elimination cam-paign (MLEC) was undertaken in BrihanMumbai (Bombay), which lias a populationof around 11 million. For the campaign,4879 nonleprosy paramedical and nonmed-ical personnel were trained and utilized assearchers. The MLEC revealed 1410 newleprosy cases, with a new case detectionrate of 1.83/10,000. Over 80% of ali casesdetected were either single-lesion or pau-cibacillary (PB), and thus of limited signif-icance with regard to transmission. Furtherefforts are required to detect and treat casesof consequence (those with more than livelesions and those with positive skiu smears)and to identify reservoirs of infection.-Authors' Summary

Dharmshaktu, N. S., Barkakaty, I3. N.,Patnaik, P. K. and Arit ., M. A. Progresstowards elimination of leprosy as a pub-lic health problem in India and role ofmodified leprosy elimination campaign.Lepr. Rev. 70 (1999) 430-439.India (population 943 million) lias seen a

highly significant decrease in the preva-lence of leprosy since the introduction of

193

194^ lnternational ,lonrnol of . Leprosr^ 2000

multidrug thcrapy (MDT) in 1981. Fronm aprevalence rate of 57/10,000 of the popula-tion in March 1981, the figure lias declinaito 5.2/10,000 in March 1999. This was pos-sible duo to the creation of a complctelyvertical (specialized) infrastructure for lep-rosy control in the 218 endemic districts ofthe country and skeleton vertical staff in theremaining districts, coupled with the re-cruitment of additional staff on contract ba-sis to provide MDT through vertical staff inendemic districts and mobile treatmentunits in the moderate and low-endemic dis-tricts. Despite ali efforts, however, new casedetection lias not shown a decline over thelast 14 years due to the presence of hidden(and undiagnosed) cases. Therefore, in or-der to intensify and hasten progress towardelimination (less than 1 case per 10,000 ofthe population) in the whole country, it wasdecided to implement a massive leprosyelimination campaign (LEC) in alI thcStates/Union Territories (UTs). The reportsof 22 States/UTs indicate that 415 out of thetotal of 490 districts in the country werecovered by modificd LEC (MLEC), with85% covcrage of the population. The cam-paign used in Incha was moditied fronm thepanem previously described by the WorldHealth Organization. The detection of hid-den or suspected cases took place within ashort, intensive period of 6-7 clays and re-lied heavily on house-to-house searches byGeneral Health Care staff trained in leprosydetection and confirmation was made byappropriately trained staff. This MLEC re-ceived widespread ^government and publicsupport, resulting in the detection of454,290 hidden cases of leprosy, while pro-viding trainine to a large number of GeneralHealth Care staff and volunteers and creat-ing widespread awareness about leprosyand the availability of treatment free ofcharge for ali cases. This program proved tobe one of the most successful health care in-terventions undertaken in Inclia in recentyears, particularly in the states of Biliar andOrissa. Although a few states in Ilidia areunlikely to reach the current WHO goal ofelimination before end of the year 2000, theresults of the MLEC strongly support thepossibility that elimination leveis will beachieved in the majority of states by the endof the year 2000 and at national levei by theend of the year 2002.—Authors' Summary

Patnaik, P. I:. B. Modificd leprosy elimi-nation campaign (MLEC) in the state ofOrissa. índia. Lepr. Rev. 70 (1999)440-447 .

As pari of a count y-wide, modificd lep-rosy elimination campaign (MLEC) carriedout in 21 sclected states in Incha in 1998,thc state of Orissa launched activitics inearly January of that year, cluring which28.9 million people were examinai, giving85% coveraue of the enumeratcd popula-tion. Usine general hcalth care staff andvolunteers, 416.604 suspected cases wereidcntitied and 62,804 of thcse xvere con-firmed as leprosy by expericnced observers.The perimi of intensive search activitylasted 1 weck only, but this was precededby several months of con uuunity mobiliza-tion and involvement, hcalth education,training of government and voluntary staff,media messages and the involvement of aIlrelevam hcalth departments, 01 -Ciciais andpoliticians. Both this and the intensivesearch period were charactcrizcd by a hiehlevei of interest anel cooperation by aIl con-cerned. The total of new cases detected andput on treatment (multidrug thcrapy; MDT)during the period of only 7 days was ap-proximatcly equal to that which, on routinepopulation survey by the leprosy services,would be recorded over a period of 2 years.The MLEC in Orissa is judgcd to have beennot only an historie step forward in the con-trol of leprosy in a state previously classi-fied as highly endemic for leprosy, but alsoone of the most successful state health in-terventions over mounted. In the 5 monthsafter completion of the campaign, the vol-untary reporting rate increased from 50% to90%. As a clirect result of the campaign, fa-cilities for the diagnosis and treatment ofleprosy are now available daily in an addi-tional 1639 institutions, over and abovethosc in existence before the campaien waslaunched. Thc achievements in tcrms of de-tecting hidden (and thus undiagnosed anduntreated) cases exceeded the outset prccfic-tions, undcrlining the importance of contin-ued vigilance and the necd to maintain in-volvement of general hcalth care staff. It isanticipatcd that the riso in prevalence dite tothe addition of 62,884 cases will be reducedby the implemcntation of MDT by 80% byabout March 1999. Overall the results of

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Current Literature^ 195

the MLEC in Orissa strongly support thelikelihood that an elimination levei of lessthan 1 case per 10,000 of the populationwill be reached in this state by the year2000.—Author's Summary

Smith, W. C. S. Future scope and expecta-tions: why, when, and how LECs shouldcontinue. Lepr. Rev. 70 (1999) 498-505.There is a strong case to continue to use

LEC approaches since they are a compre-hensive and cost effective means of deliver-ing the key elements of leprosy control.LECs should be conducted when there isevidence of large numbers of hidden cases.Probably a minimum of two LECs is re-quired but where largo numbers of newcases continue to be detected they could berun on an annual basis. The methodology ofLECs needs to be improved through experi-ence, evaluation and from LECs conductedelsewhere; feedback from the community isalso important. There is room to improveali aspects of LECs: planning, training, ed-ucation, diagnosis and treatment comple-tion.—Author's Summary

Sofola, O. Leprosy elimination campaigns:the Nigerian experience. Lepr. Rev. 70(1999) 465171.In Nigeria leprosy elimination cam-

paigns (LEC) have been found to be a use-ful intervention to improve case detectionand to facilitate the integration of leprosyservices within the general health services.LEC has indeed strengthened our routineleprosy services and enables us to involvenew partners—general health workers andvolunteers in the fight against leprosy.Therefore, their continuation in other se-lected arcas of the country is most justifiedto complete and sustain leprosy eliminationactivities at the subnational level.—Au-thor's Conclusion

Vi,jaykumaran, P., Prabhakar Rao, T.and Krishnamurthy, P. Pace of leprosyelimination and support teams in BiharState, India. Lepr. Rev. 70 (1999)452-458.Despite the extensive implementation of

multiple drug therapy (MDT) in most lep-

rosy-endemic countries worldwide since1982, bringing about a remarkable reduc-tion in prevalence, there are still regions atthe subnational levei where the implemen-tation of MDT remains difficult. The stateof Bihar (population 86.3 million) in Indiais a good example of such a region. Previ-ously rated as one of the most highly en-demic states, it still contributes about 21%of the total caseload in India and about 12%of the global caseload. For various reasons,case finding and drug treatment have laggedbehind the progress made in most otherstates in the country and in 1996 theDamien Foundation India Trust (DFIT) vol-unteered technical support to increase thepace of elimination. Sixteen out of the 39districts in the state were allocated, with apopulation of 41.8 million. Support teams,including a medicai advisor and a nonmed-ical supervisor, both with over 10 years' ex-perience of leprosy work and control pro-grams, were provided to assist and workalongside government staff in case detec-tion, treatment delivery, case holding anddischarge in their respective arcas of opera-tion. New case detection by intensive sur-vey increased by 394% and total new casedetection by 226% durinQ the year1996-1997, with similar trends in the fol-lowing year. Striking improvements werealso observed in MDT coverage, treatmentregularity, monitoring and discharge of pa-tients and in the training of local staff. Thiscollaboration between a nongovernmentagency (DFIT) and the staff of the NacionalLeprosy Eradication Programme in 16 outof 39 districts in the state of Bihar hasclearly been extremely successful. Similarapproaches in the remaining districts of Bi-har, and in other parts of India, where theinfrastructure is available but inadequate,may contribute signifìcantly to achievingthe elimination goal at nacional and subna-tional leveis.—Authors' Summary

Virmond, M. Role of leprosy related re-search and training institutions in man-agement and prevention of disabilitiesand rehabilitation. Hansen. Int. 24(1999) 38-42.As a conclusion, in the field of preven-

tion of disability and rehabilitation, re-search and training institutions should be

196^ International Journal o/ . Lepros.v^ 2000

regarded as specialized units responsible toimantai') and to further deveio!) knowledge.I-lowever, no nwtter the complexity of ac-tions developed in these institutions, theyshould bear in mind that their most relevamrole is to be ready to provide the control

progr m in the field with adequate answersto their nceds to achieve an effective con-trol of Ieprosy as a public health problem,before and beyond the year 2000.—Au-thor's Conclusion

Chemotherapy

Jacobs, M. R. Activity of quinolonesagainst mycobacteria. Drugs 58 Suppl. 2(1999) 19-22.The fluoroquinolones have been shown

to be active in rum against many mycobac-teria! species, including most strains of Mv-cobacterium tubercnlOSLS . complex and M.fortuitunt, and some strains of M. kan.ca .tiii,M. a►'ir nn-intraccUula re (MAI) complexand M. leprae. Ciprofloxacin, ofloxacin andsparfloxacin are the best studied of theseagents to date, and are among the most ac-tive of this group against M. tuherrnlosisand other mycobacteria. Treatment of pa-tients with multidrug-resistant pufmonarytuberculosis usine olloxacin lias resulted inthe selection of quinolone-resistant mutantsin a few patients. Many strains of MAI,however, are resistant to fluoroquinolones,and structure-activity relationships andDNA gyrase studies have been undertakento identify the moieties associatcd with ac-tivity and the lack thereof. The genetic andmolecular basis of quinolone resistance inmycobacteria lias revealed both the recentprogress marfe in these areal and the limita-dons of the quinolones against this uenus.Considerable progress will need to be mudein resolving these issues in order for thequinolones to become clinically useful an-timycobacterial agents.—Author's Abstract

Katoch, K., Natarajan, M., Katoch, V.M., Singh, H. B. and Bhatia, A. S.Chemotherapy trial in paucibacillary lep-rosy using, clofazimine. Indian J. Lepr.71 (1999) 311-324.In a double blind study, 300 paucibacil-

lary patients (sulcar negative, indetermi-nate, tuberculoid and borderline tubercu-loid) were randomly allotted to two regi-mens: the control subjects (150 patients)

received the standard WI-lO multidrug regi-nmcn of six doses of once a month rifampinwith daily dapsone therapy for 6 months,while the study group (150 patients) re-ceived 50 mg of clofatimine daily for 6months in addition to the WHO regimen.After stoppage of therapy all the patientswere followed up on placebo. The regimenswere wel! tolerated. In 7.5c/e of patients onthe clofatimine-containing regimen, the le-sions showed persisting activity at the timeof stoppage of therapy, compared with 16%on the control regimen. This activity sub-sided spontaneously, more rapidly, in thestudy group (80% compared with 30% inthe control group) in 6 months. Two pa-tients in the control group and one patient inthe study group developed late reactions.There were no relapses in the study group;whereas two patients have relapsed in thecontrol group during a follow up of 2.5 to3.5 years.—Authors' Abstract

Prabhakaran, K., Harris, E. B. andRandhawa, B. Bactericida! action ofampicillin/sulbactam against intracellu-lar mycobacteria. Int. J. Antimicrob.Agents 13 (1999) 133-135.The resistance of mycobacteria to [3-lac-

tam antihiotics is attribntecl to their ablhty tosynthesize (3-lactamasc. In our previous stud-ies, (3-lactam/(3-lactamasc-inhibitor combi-nations suppressed the growth of severa!mycobacteria in axenic cultues and ampi-cillin/sulbactam was bactericida! to Mv-cobacverium tuberc u losi• H37Rv in nitroand to M. h'prae multiplying in mouse foot-pads. Since both these organisms multiplyin phagocytic cens in the host, it is impor-tant to know whether the drug combinationis active against mycobacteria multiplyingin macrophages. We teste(' the action of

68, 2^ Current Literature^ 197

ampicillin/sulbactam against four poten-tially pathogcnic ([o humans or to animais)mycobacteria, Al. Ximiae, M. huencophi/uni,NI. cnium, N1. microti, when phagocytosedby mouse nwcrophages. Bacteria were ex-posed to nionolayers of peritoneal nwcro-phages harvested fronm BALB/c coice. Un-phagocytosed baeilli were removed andthese conceni ations of ampicillin/sulbac-tam were tested. Optinnun activity xvas ob-serve(' at 1O0 mg/1 which killed 58Y-97%of the mycobacteria within n u icrophages, asdetermined by the CFU. (3-Lactam/(3-lacta-mase-inhibitors, especially ampicillin/sul-bactanm, might provicle an effective alterna-tive thcrapy against infcctions causei' bymycobacteria resistam to other drues.—Au-thors' Abstract

Sharma, A., Sharma, V. K., Rajwanshi,A., I)as, A., Kaur, 1. and Kumar, B.Presence of Al. lep rae in tissues in slitskin smcar negativo multibacillary (M13)patients after WHO-MBR. Lepr. Rev. 70(1999) 281-2$6.This study looked for M. leprae in the

lymph node, nerve and skin of multibacil-lary (MB) leprosy patients who becomeslit-skin-smear neeative after the comple-tion of W110-MBR_ Twenty-tive WHO-M13R-treated MB leprosy patients werestudied; borderline Iepromatous (BL) lep-rosy (N = I I) and lepromatous (LL) leprosy(N = 14). Fifteen patients had reaction(erythema nodosum leprosum 11, upgrad-inc reaction 4) either at presentation or dur-ing therapy. AII patients attained slit-skin-smear negativity after WHO-MBR (range24-39 months). Sixteen (64%) patientswith MB leprosy showed fragmented baeilliin skin and verve biopsy or lymph node as-pirares after WHO-MBR. Lymph node as-pirares alone revealecl Al. leprae in 7 pa-tients, followed by nerve in 2 and skin in 1patient. Four cases showed N1. leprae at alisites followed by nerve and skin or lymphnode in one case each. A pretreatment bac-terial index (BI) of 4+ or more was signili-cantly associated with the presence of Al.leprae at the end of trealment. Also, signif-icantly more lymph node aspirates con-tained M. leprae in comparison with nerveor skin biopsies. All 7 cases in whom lreat-ment was extended beyond 24 months

showed N1. )aproe in tissues even after at-taining slit-smcar negativity. In conclusion,M. /aproe persist in tissues after 2 years ofW1-1O-MI3R and patients with an initial BIof 4+ or more need to he closely followed upafter stopping MDT. Authors' Summary

'I'omioka, H., Sato, K., Akaki, T., Kaji-tani, 1-1., Kawahara, S. and Sakatani,1\'1. Comparative ia nitro antimicrobialactivities of the newly synthesizedquinolone HSR-903, sitafloxacin (DU-6859a), gatifloxacin (AM-1155), andlevofloxacin against Mvcobacterium tu-herculosis and Mvcabacterium aviem!complex. Antimicrob. Aeents Chemo-ther. 43 (1999) 3001-3004.We compared the ia Litro antimycobacte-

rial activity of a new iluoroquinolone,HSR-903, with strong activity againsteram-positive cocei with these of levo-floxacin (LVFX), sitalloxacin (STFX), andgatifloxacin (GFLX). The MICs of thequinolones for Mvcohacteriunl tuhe reulosisand M. aviam complex were in the orderSTFX SGLX < LVFX < HSR-903 andSTFX < GFLX < HSR-903 < LVFX, re-spectively. HSR-903 effectively eliminatedint anricrophagial M. tuberculosis, as didLVFX, and exhibited bacteriostatic effectsagainst turhercnlo.si.ti . replicating in typell alvcolar cclls.—Authors' Abstract

van Rensburg, C. E. J., ,loone, G. K.,Siegel, F. A., Nlatlola, N. M. andO'Sullivan„J. F. In nitro investigation ofthe antimicrobial activities of novel tet a-methylpiperidine-substituted phenazinesagainst Mvcobacterium tubo rcuiosis.Chemotherapy 46 (2000) 43-48.The nitra- and extracellular activities of

li ve novel tctramethylpiperdine (TMP)-sub-stituted phenazines against Mvcobacteriumtuberculosis 1-137Rv (ATCC 2 7294) weredetermined and compareci with [pose of clo-fazimine and rifampin. Two of these agents,together with clofazimine, were also testedfor their activities against drue-resistamstrains of Al. tuberculosi.s .. "hhree of theTMP-substituted phenazine compoundswere significantly more active than clofaz-imine against Al. tubc/rulosis, includinginultidrug-resistant clinica) strains of this

198^ International Journa! of Leprosv^ 2000

microbial pathogen, demonstrating a lackof crossresistance between the rimino-phenazines and standard antituberculousdrugs. Using M. luberculosis- infected mono-cyte-derived macrophages, ali of the TMP-substituted phenazines were found to possesint acellular activity which was superior tothat of both clofazimine and rifampin. In thismodel of intracellular bioactivity, the experi-mental compounds inhibited bacterial growth

at concentrations which were approximately10-fold lower than the corresponding mini-mal inhibitory concentration values ob-tained usine conventional in litro sensitivitytesting procedures. These results demon-strate that the novel 'EMP phenazines areactive against nmltidrug-resistant M. tuber-culosis strains, and particularly effective in-tracell ularly.—Authors' Abstract

Clinicai Sciences

Anderson, A. M. and Croft, R. P. Relia-bility of Semmes Weinstein monolila-ment and ballpoint sensory testing, andvoluntary muscle testing in Bangladesh.Lepr. Rev. 70 (1999) 305-313.The reliability of methods of testing

verve function is important, since diagnos-tic decision making is a direct function ofthe quality of the test. Three methods ofnerve function testing were investigated atthe Danish Bangladesh Leprosy Mission(DBLM) in north Bangladesh, and assessedfor inter-observed reliability. The threemethods were 1) ballpoint pen test (BPT)for sensory function; 2) graded SemmesWeinstein monofilament test (SWM) forsensory function and 3) voluntary muscletesting (VMT) for motor function. Theweighted kappa (x,,.) statistic was used toexpress inter-observer reliability. Using thisstatistic, O represents agreement no betterthan random, and 1.0 complete agreement.x«, values of ?0.80 are reckoned to be ade-quate for monitoring and research. Fifty-three patients were tested, a senior phys-iotechnician acting as "gold standard"against whom four other staff physiotechni-cians were assessed. All three testing meth-ods were found to have minimal inter-ob-server variation, with the x,, for inter- ob-server agreement using BPT being 0.86, theSWM 0.92, and VMT 094. It is concludedthat in trained and experienced hands, alithree methods are reliable and repeatable toa levei allowing confident use of results ob-tained in monitoring and research.—Au-thors' Summary

Braga, F. J. H. N., Foss, N. T., Ferriolli,E., Pagnano, C., Miranda, ,J. R. I). anddeMoraes, R. The use of bone scintigra-phy to detect active Hansen's disease inmutilated patients. Eur. J. Nucl. Med. 26(1999) 1497-1999.Mutilation of extremities was very fre-

quent in patients affected by leprosy in thepast; although it is now much less common,it is still seen, mainly in patients with long-term disease. In general, mutilation of thenose and cars is caused by the bacillus andmutilation of the hands and feet a conse-quence of chronic trauma. Leprosy must bechronically treated and any decision to in-terrupt therapy is baseei on laboratory testsand biopsy. Scintigraphy is a noninvasiveprocedure which could be of great value indetermining disease activity. We studied 8patients (5 males and 3 females, aged64-73 years) who presented with mutilationof the nose (2), ear (1), feet (3) or foot andhand (2). Conventional three-phase bonescintigraphy (750 MBq) and X-ray exami-nations of the affected arcas were per-formed in ali patients. Bone scintigraphywas abnormal in 4 patients (the presence ofbacilli was confirmed by biopsy in 2 ofthem), and normal in the other 4. In ali pa-tients except for the one with ear mutila-tion, radiography only showed the absenceof bone. We conclude that bone scintigra-phy is very useful to determine disease ac-tivity in cases of mutilation caused by lep-rosy. It seems to be superior to conventionalradiography and may enable bone biopsiesto be avoided.—Authors' Abstract

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Current Literature^ 199

de Almeida, J. A., Vitti, NI. and Garbino,J. A. lAnatomic study of Martin-Gruheranastomosis. 1 I Iansen Int. 24 (1999)15-20. (in Portuguese)

Forty forearms ol human cadavers weredissected and studied. Five (12.5%) werefound to have Martin-Gruber anastomosis(MG.A). lavo on lhe right lide and three onthe left sitie. From the total of live, twocases of this anastomosis occurred anlongthe branches reserved to the flexor digito-rum profundos nmscle, ove from the branchof the anterior interosseous nove, and twodirected fruiu the mediai] to the uivar nerve.In Chis stucly, MG.A vias found in agree-ment with the percentages of the 1itera-ture. Authors' English Sununary

Lemieux, L., Cherian, 1'. A. and Richard,Il.'lhe stapedial reflex as a topographicalnmarker of proximal involvement of thefacial nerve in Ieprosy: a pilot stucly.Lepr. Rev. 70 (1999) 324-332.

This study aimed to determine theparameters necessary for a stucly of stape-dial reflexes in Ieprosy patients to ascertainif the facial nerve is involved more proxi-mally than the stylomastoid foramen. It in-volved Ieprosy patients with and without fa-cial nerve involvement and nonleprosy con-trols. Clinicai examination of the patients'ears, a tympanogram and audiogram to cx-clude conductive and sensorineural deaf-ness, followed by the measurement of astapedial reflex and the acoustic reflexthreshold, were carried out. The number ofabsent reflexes and the acoustic reflexthresholds did not differ between the threegroups of subjects. A delinitive study wouldbe logistically impossible. Suggestions arenlade as to more exacl patient seleclion Illorder to demonslrate any stapedial reflexchanges due to Ieprosy. The findings of thisstudy do not suggest that racial nervepathology extends proximally to lhe stylo-mastoidforamen. unless such proximal in-volvement is subclinical to the detectionmethods used.—Authors' Sumnnary

Lockwood, 1). N. J. Nerve damage in Iep-rosy: a prohlem for patients, doctors and

scientists. (Kellersberger Memorial Lec-ture 1998). Ethiop. Med. J. 37 (1999)133-140.

The pathology, detection and treatmentof leprosy verve damage are discussed.Present knowledge and future develop-ments are considered. Trop. Dis. Boll. 96(1999) 1156

Nadkarni, N. S. and Rege, V. L. Signifi-cance of histopathological classificationin Ieprosy. Indian J. Lepr. 71 (1999)325-332.A retrospective Ninei stucly was carried

out 011 2640 patients of Ieprosy to correlatethe histopathological and clinicai classifìca-tion of Ieprosy usine the criteria laid downby Ridley and Jopling. There was completeagreeinent betwecn histopathological andclinicai classification in 81.8% of the cases,with one-step deviation in 5.1% of thecases. 1-1 istopathological diagnosis of incle-terminate leprosy in high percentayge(15.9%% ) as against 3.3% of indeterminateIeprosy clinically in our series was an inter-esting feature. Type-wise correlation be-tween histopathological with clinicai classi-fication vias very high, it being the highestin LL (98%) followecf by TT (97%), BT,1313 and 13L (95%, 89% and 87%, respec-tively).—Authors' Abstract

Richard, B. M. and ,lacobs, J. M. Facialnerve pathology in Ieprosy: searching forthe proximal extent of the lesto') in facialnerve hiopsies. Lepr. Rev. 70 (1999)333-344.A light and electron microscope study

vias mace of resin-embedded facial nervesin three cases of Ieprosy involving the facialnerve. The patients had irreversible facialnerve palsies and had requested facial re-consLruction. No consistem pattern of nervefiber damage vias found. In one case thetempororygomatic was affected, but thecervical brandi was normal, suggesting thedamage begins distally. In two cases theloss of nerve fibers in the trunk and alibranches was similar, and is likely to cm-anate from damage at a more proximal site.The presence of increased numbers of un-

200^ International Journal of Leprosy^ 2000

myelinated axons, often in clusters, is evi-dence of regeneration. These axons proba-bly have the potential to develop into func-tional myelinated fibers provided that theycan innervate a viable distai tamet such as amuscle graft. These regenerating axons aredistai to the stylomastoid foramen sugg,est-ing that the most proximal levei of involve-ment of the facial verve could be intracra-nial. The tinding of a more proximal leveiof nerve involvement, implies that the mis-reinnvervation seen in partially recoveredfacial nerve palsies in leprosy could be chieto some regenerating axons being misdi-rected at the levei of the main trunk bifurca-tion.—Authors' Summary

Selvasekar, A., Ebenezer, G. .1. andPartheebarajan, M. Lepromatouslymphadenopathy and concomitant tu-berculous axillary lymphadenitis with si-nus; a case report. Lepr. Rev. 70 (1999)345-350.

A 25-year-old inale patient with floridlepromatous leprosy presented with rightaxillary lymphadenopathy and a discharg-ing sinus. He also had scabies with chronicright otitis media. Histopathological exami-nation of the lymph node revealed leproma-tons lymphadenitis coexisting with tubercu-losis. This unusual combination of two dif-ferem clinicai entities is recorded in thiscase report.—Authors' Summary

Sukpanichnant, S., Hargrove, N. S.,Kachintorn, U., Manatsathit, S.,Chanchairujira, T., Siritanaratkul, N.,Akaraviputh, T. and Thakerngpol, K.Clofazimine-induced crystal-storing his-tiocytosis producing chronic abdominalpain in a leprosy patient. Am. J. Surg.Pathol. 34 (2000) 129-135.

Clofazimine-induced, crystal-storing bis-tiocytosis is a rare but well-recognized con-dition in the literature. Besides the commonreddish discoloration of the skin, clofaz-imine produces gastrointestinal distur-bances—sometimes severe abdominal pain,prompting exploratory laparotomy, becausepathologic and radiologic findings can pro-duce diagnostic diffìculties if the pathologicchanges caused by clofazimine are not rec-ognized. The authors report such a case in a

leprosy patient to emphasize the importanceof history taking, the radiologic abnormali-ties of the small intestine, and the patho-logic findings in small intestine and lymphnode biopsies. Clofazimine crystals are redin the frozen section and exhibit bright-redbirefringence. However, they are clear inroutinely processed histologic sections be-cause they dissolve in alcohol and organicsolvents. They also appear as clear crystalspaces during electron microscopic study,but some osmiophilic bodies can be ob-served. Histiocytosis caused by clofaziminecrystals produces infiltrative lesions inradiologic studies mimicking malignantlymphoma or other infiltrative disorders.Associated plasmacytosis in the histologicsections can simulate lymphoplasmacyticlymphoma or multiple myeloma with crys-tal-storing histiocytosis. With the knowl-edge of this rare condition caused by clo-fazimine, appropriate management to avoidan unnecessary laparotomy is possible.-Authors' Abstract

Sundar Rao, P. S. S., Augustine, V. andJoseph, G. A. Being a female leprosypatient in South India. Indian J. Lepr. 71(1999) 279-284.

The problems of women patients as re-vealed by a case study of a woman patientand a questionnaire study of 100 leprosypatients (47 men and 53 women) are pre-sented. These include, besides general oneslike ignorance of facts about the disease,specific ones like lack of privacy duringclinicai examination, indifference towardwomen's feelings and difficulties in com-municating with inale workers. A greatersensibility toward the sentiments and prob-lems of women patients on the part of thehealth service is required to amend the situ-ation. Recruiting more women workersmight help in this regard.—Authors' Ab-stract

Vaquem, N. L., Ortiz, M. C., Soto, I.,Bruni, M. E., Yonadi, V. andWeiberlen, H. [Late reversa) reactionsin Hansen's disease.] Rev. Argent. Der-matol. 80 (1999) 152-156. (in Spanish)

Late reversal reactions are acure eventsthat arise in leprosy patients after finishing

68, 2^ Current Litc rature^ 201

their treatment, when their CMl improves.The differential diagnosis between reversalreaction and relapses is often difficult and itinvolves a different treatment with a newcourse of nuiltidrug therapy in the latter.Several guidelines may help to make the

right diagnosis. Steroids may be a therapeu-tic proof when severe neural involvementexists as is usually seen in reversal reac-tions. lts early use allows one to avoid dis-abilities.—Authors' English Summary

Immuno-Pathology

Ateais, A., Sanchez, F. O., Thuc, N. V.,Lap, V. D., Oberti, J., Lagrange, P. H.,Schurr, E. and Abel, L. Granulomatousreaction to intradermal injection of lep-romin (Mitsuda reaction) is linked to thehuman NRAMPI gene in Vietnameseleprosy sibships. J. infect. Dis. 181(2000) 302-308.The Mitsuda test, which measures the

specific immune response against intrader-mally injected lepromin, has a high prog-nostic value for susceptibi1ity or resistanceto the Iepromatous forni of leprosy. A sib-pair linkage analysis between the Mitsudaresponse and the NRAMPI gene was doneamong 20 nuclear families with leprosy (to-taling 188 sibs) from Ho Chi Minh City,Vietnam. Ali family subjects were geno-typed for several intragenic and flankingNRAMPI markers, leading to the delinitionof a fully inforniative NRAMPI haplotype.Significant linkage was observed betweenNRAMPI and Mitsuda reaction when con-sidered either as a quantitative (p = 0.002)or as a categorical (p = 0.001) trair. Sepa-rate analyses among healthy and affectedsibs showed evidence for linkage in bothsubsaniples, indicating that linkage be-tween the Mitsuda reaction and NRAMP1is independent of leprosy status. These re-sults support the view that NRAMPI playsa regulatory role for the development of ac-quired antimycobacterial immune re-sponses as determined by iii vivo Mitsudatest reaction.—Authors' Abstract

Andreu, D., Carreno, C., Linde, C., Bo-man, H. G. and Andersson, M. identifi-cation of an anti-mycobacterial domainin NK-lysin and granulysin. Biochem. J.344 (1999) 845-849.

NK-Iysin and granulysin are homolo-gous cationic antibacterial peptides pro-duced by pig and human cytolytic lympho-cytes, respectively. The solution structuresof NK-Iysin comprises tive amphipathic al-pha-helices. To investigate the properties ofa helix-loop-helix region postulated to be amemhrane-docking part of NK-lysin, wesynthesized 22- and 29-residue peptides re-producing this region for both NK-Iysin andgranulysin. CD spectroscopy of the syntheticpeptides in a liposomal solution showedspectra typical of alpha-helical peptides. Thepeptides were active against eram-positiveand grani-negative bacteria, with the tvoNK-Iysin peptides showing higher antibacte-rial activities than the two from granulysin.One NK-Iysin peptide was active againstPseuclonionas aeruginosa and Staphvlococ-cus auretrs, tvo organisms against whichNK-Iysin is inactive. Granulysin peptideswere inactive against these bacteria, in con-trast with granulysin, which is known to beactive against them. Both NK-lysin and allsynthetic analogs killed Mycobacteriuui tu-berculosi• and K562 tumor cells, but didnot display hemolytic activity. These resultsidentify a potent anti-mycobacterial domainin NK-Iysin and granulysin consisting of a22-residue (helix 3) sequente plus a disul-fide-consl ained loop.—Authors' Abstract

Batoni, G., Esin, S., Pardini, M., Bottai,D., Senesi, S., Wigzell, H. and Campa,M. identitication of distinct lymphocytesubsets responding to subcellular frac-tions of Mvcobacterium bovis bacilleCalmette-Guerin (BCG). Clin. Exp. Im-munol. 119 (2000) 270-279.In order to investigate the ability of Mv-

cobacterium bons BCG vaccination to in-

202^ International Journal of Lepros.v^ 2000

duce immune responses toward differentclasses ol mycobacterial antigens and thecell populations involved in such responses,proliferation of distinct human lymphocytesubscts fruiu 13CG-vaccinated donors in re-sponse to different subccllular fractions ofBCG was analyzed and compared with thatof not sensitized subjects. Prol,lerationof different cell subscts was evaluated bynow cytometric deterniination of bromo-deoxyuridine incorporated into DNA of di-viding cells and simultaneous identificationof cell surface markers. Although a certaindegree of variability was observei amongdifferent donors, after 6 clays of in nitrostimulation BCG-vaccinated subjects dis-played, as a mcan, a stronger blastogenicresponse to ali the classes of antigens com-pared with nonsensitized oncs. PPD, cultuefilt ates and membrane antigens induced apredominam proliferation of CD4+ T cens.In contrast, prcparations enrichcd in cytoso-lic antigens elicited proliferation ofgamma delta+ T cens which, as a mean,represented 5510 of the prolifcratiog cens.Although to a lesser extent, proliferation ofgamma delta+ T cens was also elicited bypreparations cnriched in membrane and celawall antigens. In response to the latterpreparation proliferation of CD4+ T eensand CD16+/CD3– (natural killer (NK)]cens was observed, as well. In particular,cell wall antigens were found to induce sig-niticantly higher leveis of' proliferation ofNK cens compared with ali the otherclasses of and grens.—Authors' Abstract

Belman, C., Espinosa, E., Poupot, R.,Peyrat, M. A., Guiraud, M., Poquet,Y., Bonneville, M. and Fournie, J. ,J.3-formy1-1-butyl pyrophosphate; anovel mycobacterial metabolite activat-ing human gamma delta T eens. J. Biol.Chenm. 274 (1999) 32079-32084.

Most human blood gamma delta T eensreact without major histocompatibilitycomplex restriction to small phosphorylatednonpeptide antigens (phosphoantigens) thatare abundantly produced by mycobacteriaand several other microbial pathogens. Al-though isopentenyl pyrophosphate has beenidentilied as a mycobacterial antigen forgamma delta T cens, the structure of severalother stimulatiog compounds with bioactiv-

ities around 1000-fold higher than isopen-tenyl pyrophosphate remitis to be eluci-datcd. This paper deseribes the structuralidcntification of 3-formy1-1-butyl-pyro-phosphate as the core of several non-prenylmycobacterial phosphoantigens bioactive atthe nM range. Recognition of this moleculeby gamma delta T cens is very selective andrefles on its aldehyde and pyrophosphategroups. This novel pyrophosporylated alde-hyde most probably corresponds to a meta-bolic intermediate of the non-mevalonatepathway of prenyl phosphate biosynthesisin eubacteria and algae. The reactivity to3-formyl-1-butyl-pyrophosphate supportsthe view that human gamma delta T censare physiologically devoted to antimicro-bial surveiliance.—Authors' Abstract

Boomershine, C. S., Lafuse, W. P. andZw'illing, B. S. Beta 2-adrenergic recep-tor stimulation inhibits nitric oxide gen-eration by MVcobacterium cn 'iuni in-fected macroph ages. J. Neuroimmunol.101 (1999) 68-75.

Catecholamine regulation of nitric oxide(NO) production by interferon gamma(IFN-y)-primed macrophages infected withMvcobacteriuni ariunn was investigated.Epinephrine treatment of IFN-y-primedmacrophages at the time of. M. ai'iunt infec-tion inhibited the anti-mycobacterial activ-ity of the cens. The anti-mycobacterial ac-tivity of macrophages correlated with NOproduction. Using specific adrenergic re-ceptor agonists, the abrogation of mycobac-terial killine and decreased NO productionby catecholamines was shown to be medi-ated via the beta 2-adrenergic receptor. Ele-vation of intracellular cAMP levels mimic-ked the catecholamine-mediated inhibitionof NO in both M. ai'iuni-infected and LPS-stimulated macrophages. Specific inhibitorsof both adenylate cyclase and protein ki-nase A prevented the beta 2-adrenoceptor-mediated inhibition of nitric oxide produc-tion. P2-adrenoreceptor stimulation at thetime of M. ai'iuni infection of IFN-y-primedmacrophages also inhibited expression of1NOS mRNA. These observations showthat catecholamine hormones can affect theoutcome of macrophage-pathogen intente-dons and suggest that one result of sympa-thetic nervous system activation is the sup-

68, 2^ Current Literature^ 203

pression of the capacity of macrophages toproduce antimicrobial effector mole-cules.—Authors' Abstract

Cuevas-Santos, J•, Contreras, F. and Mc-Nutt, N. S. Multibacillary leprosy: le-sions with macropha ges positive forS 100 protein and dendritic cells positivefor Factor 13a. J. Cutan. Pathol. 25(1998) 530-537.'Re cutaneous lesions of 69 patients with

leprosy (42 lepromatous, 5 mid-borderline,and 22 tuberculoid) were evaluated by im-munohistochemistry for the expression ofS 100 protein, CD I a, CD68, muramidase,FILA-DR, and Factor I 3a. The macro-phages from lesions of polar, subpolar andborderline lepromatous leprosy patients ex-pressed S 100 protein intensely and con-stantly. In contrast, the lesions of polar andsubpolar tuberculoid leprosy had very fewcells that were 1mmunoreactive for S100proteins ("S100+") in the granulomas in thedermis. The macrophages in aIl lesionswere reactive for CD68 and muramidase. Inparaffin sections, macrophages of leproma-tons lesions failed to stain for HLA-DR;whereas in tuberculoid lesions, they werestrongly positive for HLA-DR. Three pa-tients with histoid leprosy (relapse lesions)had lesions that were strongly positive forFactor 1 3a and were negative for S 100 pro-tein ("S 100–''). 11 is concluded that giventhe possible chemotactic and migration in-hibition effects of the calcium-binding pro-teins of the S 100 family, these data suggesta possibly important role for S100 proteinin the acccnnulation of macrophaus in lep-romatous leprosy, and also reveal infectionof Factor I 3a + dermal dendritic cells inhistoid leprosy.—Trop. Dis. Bull. 96 (1999)1266

Fafutis-Morris, M., Guillen-Vargas, C.M., Navarro-Fierros, S., Alfaro-Busta-mante, F., Zaitzeva-Petrovna, G.,Daneri-Navarro, A., Santoscoy-Tovar,L. and Armendariz-Borunda, ,J. Addi-tion of anti-CD28 antibodies restoresPBMC proliferation and IFN-gammaproduction in lepromatous leprosy pa-tients. J. interferon Cytokine Res. 19(1999) 1237-1243.

During antigen recognition, T lympho-cytes are prime(' by a physical interactionwith antigen-presentin;g cells (APC). Atleast two signals are neeclecf to activate Tcells. One is provided by T-cell receptor(TCR)/CD3 in the context of the major his-tocompatibility complex (Ml-IC), and an-other signal is mediated by antigen-inde-pendent molecules, that is T-cell mem-brane-bound CD28 and its specific ligandB7-1 (CD80) present in APC. Both signalstrigger a series of metabolic events initiat-ing riLrht at the cell membrane and endingwith activation and proliferation of T cellsas well as specific cytokine synthesis. Ourmain goal was to determine whether defi-ciency in interferon-gamma (IFN-y) pro-duction shown by peripheral blood mono-nuclear cells (PBMC) from lepromatousleprosy (LL) patient could be overcome byreconstituting in nitro the appropriate sig-nals (by means of acldition of anti-CD28and anti-CD80 monoclonal antibodies). Wealso cletermined the stimulation index (SI)in the same PBMC. Our results demon-strated no significant differences in CD80expression of monocytes and B lympho-cytes from LL patients when compare('with healthy subjects. Nonetheless, CD28expression significantly decreased in lym-phocytes from LL patients (p <0.01). Re-garding IFN-y leveis and SI, LL-PBMCfailure before mitounic stimuli could bereversed by further incubation with anti-CD28 antibody, but stimulation by specificantigen of Mvcobac te riam lehrae was notchanced. Addition of anti-CD80 antibodysigniticantly increased IFN-y Ievels in phy-tohemagglutinin (PHA)-stimulated PBMC,although proliferation deficiency persisted.Cells stimulated with specific antigen cfidnot modify either their proliferation or IFN-y Ievels. Authors' Abstract

Faldt, J., Dahlgren, C., Karlsson, A.,Ahmed, A. M. S., Minnikin, D. E. andRidell, M. Activation of human neu-trophils by mycobacterial phenolic gly-colipids. Clin. Exp. Immunol. 118 (1999)253-260.The interaction between mycobacterial

phenolic glycolipids (PGLs) and phago-cytes was studied. Human neutrophils wereallowed to interact with each of four puri-

204^ lnternational.lournal o/'Lepros_v^ 2000

fìed mycobacterial PGLs and the neutrophi1production of reactive oxygen metaboliteswas followed kinetically by luminol-/isolu-minol-amplified chemiluminescence. ThePGLs from Mvcohacterium tuberculosisand M. kansasii, respectively, were shownto stimulate the procuction of oxygenmetabolites, while PGLs from M. nuu inumand M. borla BCC, respectively, were un-able to induce an oxidative response. Perio-date treatment of the M. tuherculosis' PGLdecreased the procuction of oxygen radi-cais, show1ng the importance of the PGLcarbohydrate moiety for the intcraction.The activation, however, could not be in-hibited by rhamnose or fucose, indicating acomplex intcraction which probably in-volves more than one saccharide unit. Thisis in line with the fact that the activatiogPGLs from M. tuberculosis' and M. kansasiicontais tri- and tetrasaccharides, respec-tively, while the nonactivating P0Ls fromM. inarin um and M. bot^is BCG each con-tam a monosaccharide. The complement re-ceptor 3 (CR3) has earlier been shown to beof importance for the phagocyte binding ofmycobacteria, but did not appear to be in-volved in the activation of neutrophils byPGLs. The subcel1ular localization of thereactive oxygen metabolites formed was re-lated to the way in which the `glycolipidswere presente(' to the cells.—Authors' Ab-stract

Fietta, A., Francioli, C. and Grassi, G. G.Mycobacterial lipoarabinomannan af-fects human polymorphonuclear andna)nonuclear phagocyte functions differ-ently. Haematologica 85 (2000) 1 1-18.Background and Objectives. The role of

mycobacterial lipoarabinomannan (LAM)in regulating the granulomatous responsesand its effects on celis invoived in eariy re-sponses to tuberculosis have not beenclearly dehned. The aim of this study wasto acquire further evidence about the mech-anisms by which LAM takes part in thehost response to mycobacterial infections.

Design and Methods. We compareci thein Nitro ability of mannosylated LAM(ManLAM) and LAM lackimg the terminalmannosyl units (AraLAM) to induce dis-tinct responses in human polymorphonu-clear (PMNs) and mononuclear phagocytes

lboth monocytes and 48-hr nwnocyte-de-rived nrtcrophages (MDMs)1. The re-sponses examinai were chemotaxis, tran-sient changes in free cytosolic calcium,phagocytosis and metabolic activation.

Results. AraLAM and ManLAM affectedmononuclear, but not polymorphonuclear,phagocyte functions. Both fornis of LAMwere chemotactic for monocytes andMDMs. 'lhe LAM-induced chentotactic re-sponse required new protein synthesis, didnot incluce a rise in cytosolic free calciumleveis and was partially inhibited (about50%) by genistein, but not by calphostin Cor PD 98059. Lastly, at physiologic dosesManLAM significantly reduced phagocyto-sis of M. tuhcrrulosis and zymosan parti-cies by MDMs.

interpretation and Conclusions. Differentphagocytic cells can exhibit variable re-sponses to AraLAM and ManLAM. More-over, LAMs affect ccll functions throuL,1hdifferent mechanisms. Protein synthesis andactivation of protein tyrosine kinases areiniportant intermediates in the signa) trans-duction pathway of the chemotactic re-sponse of mononuclear phagocytes toAraLAM and ManLAM: whereas Man-LAM-induced inhibition of niacrophagephagocytic ability could depend on thebinding of macrophage mannose receptoraand/or the insertion of this moiecule istocellular plasma membrane. Together thesedata hinhlight the danger of making gener-aiizations rei*arding the activity of LAMson immune defenses.—Authors' Abstract

Fratazzi, C., Arbeit, R. D., Carini, C.,Balcewicz Sablinska, M. K., Keane, ,I•,Kornfeid, I-1. and Remold, H. G.Macrophage apoptosis in mycobacterialinfections. J. Leuk. Biol. 66 (1999)763-764.Mycobacterial diseases are a major pub-

lic health concern. In the case of tuberculo-sis, the problem lias been acerbated due tothe emergence of drupa-resistant strains ofMvcobacteriuin tubeintlosis, and M. agiamis the major opportunistic pathogen in HIV-1infection in the United States. M. tuhcrculo-sis and M. achou replicate in human niacro-phages and induce apoptosis. Incubation offreshly added uninfected autologous niacro-phages with apoptotic M. at'ium-infected

68, 2^ Current Literature^ 205

macrophages results in 90% inhihition ofbacterial growth. Apoptosis also preventsthe release of intracellular componente andthe spread of mycobacterial infection by se-questering the pathogens within apoptoticboches. Consistem with the model that host-ccll apoptosis is a defense mechanismagainst mycobacteria is the linding that thevirulent M. tuberrulosis strain 1-137Rv in-duces substantially Iess nutcrophage apop-tosis than the attenuated strain H37Ra. Eva-sion of apoptosis by this pathogen isachieved by enhanced release of sTNFR2by H57Rv-infected macrophages and sub-sequent formation of 1nactive TNE-alpha-TNFR2 complexes. These observationscontribute to the hypothcsis that apoptosisof the host macropha2e is an important de-fense mechanism in mycobacterial infec-tions, which prevents the spread of the in-fection.—Authors' Abstract

Gonzales, A. C. de O., Silva, T. C., Bar-bosa, A. de A., ,Ir. and Sadigursky, M.Immunohistologic appraisal of infìlt at-ing cells in skin biopsies from young pa-tients clinically suspected of having var-ious forms of leprosy. An. Bras. Derma-101. 74 (I 999) 365-371.

Skin biopsies obtained from 28 untreatedyoung patients from Brasil, with clinicallysupsected leprosy, during 1988-93, wereused to study the surface phenotypes pro-duced by infiltrati cells and demonstratedby immunohistochemistry usine the mono-clonal antibodies LCA; HAM-56; Pan-B;Pan-T; CD4; CD8; Leu7, and the poly-clonal anti-S-100 ',roteio and anti-BCG. Asemiquantitative analysis of the stainedcells was perforrned. In 24 cases thehistopathological diagnosis of leprosycould be mude: 9 indctcrminate (IND), 5 tu-berculoid (TT), 6 borderline tuberculoid(BT) and 4 lepromatous (LL). All caseswere positive for LCA. In the LL group themore conspicuous cells were the macro-phages, follwed by the T lymphocytes. Thesubpopulation TCDB cells were piore fre-quent than the TCD4. 1n the BT group the Tcells were predominant, with CD4 slightlymore frequent than CD8, followed by thenutcroph ages. In the TT group, the T cellwas also predominam; aunong there, theCD4 cells were more conspicuous, fol-

lowed by the macrophages. The IND groupwas heterogeneous; T cells were the mostfrequent, with CD4 and CD8 cells havingapproximately the same frequency as themacrophages. it is concluded that theT cells were the most frequent cells in theTT/BT groups and there were more suchedis in the TT and BT than in LL cases, in-dicating immunological reactivity consis-tem with 'i'-cell presence and activity. Anti-boclies against T-cell subpopulationsshowed that in the TT/BT cases moreCD4-positive cells and in LL patients moreCD8-positive cells proliferated. Indetermi-nate forms disclosed gradation in the com-position of the inliltrates. It is concludedthat the skin lesions of young patients withearly indeterminate leprosy and with vari-ous debilite forms of leprosy diffcr with theregard to the inlilt ating cells.—Trop. Dis.Bull. 96 (1999) 1267

Leal, I. S., Smedegard, B,, Andersen, P.and Appelberg, R. Interleukin-6 and in-terleukin-12 participate in induction of atype 1 protective T-cell response duringvaccination with a tuberculosis subunitvaccine. Infect. moiam. 67 (1999)5747-5754.

We examined the role of cytokines in thedevelopment of gamma interferon (IFN-y)-secreting protective T cells following im-munization with a culture filtrate subunitvaccine against Mvcohacterium tuberrule-sis containing the adjuvam dimethyldioc-tadecylammonitn bromide (DDA). Deple-tion of either interleukin-6 (iL-6) or iL-12with spccific neutralizing antibodies duringvaccination rcduced the priming of T cellsfor antigen-spccific proliferation and IFN-ysecretion. Such reduction was also oh-served in IL-6 gene-disrupted coice as com-pareci to wild-type animais. 1L-6 was foundto play a role in the iniciai differentiation of.

Th 1 cells but not in their expansion. Thedefect found after 1L-6 depletion or in IL-6-knockout mico was compensated by the in-clusion of recombinant mouse IL-12 in thevaccinc. The induction of protective immu-nity against an inl avenous or an aerosolchallenge with live, virulent M. tnherrnlosiswas markedly reduccd by neutralizing ei-ther IL-6 or iL-12 during immunizationwith the vaccine. Likewise, the effects of

206^ International Journal of Leprosy^ 2000

IL-6 neutralization were partially reversedby including 1L-12 in the vaccine. Our datapoliu to an important role of 1L-6 and IL-12in the generation aí cell-mediated immunityto tuberculosis.—Authors' Abstract

Mitra, 1). K., DeRosa, S. C., Luke, A.,Balamurugan, A., Khaitan, B. K.,7'ung, ,l., Mehra, N. K., Terr, A. i.,O'Garra, A., 1-lerzenherg, L. A. andRoederer, M. Differential representa-tions aí memory "1' cell subsets are char-acteristic of polarized in u iiunity in lep-rosy and atopic diseases. Int. Immunol.11 (1999) 1801-1810.We identifìed functionally polarized sub-

sets of CD4 memory T cens on the basis ofthe expression of CD' ia, CD45RA andCD62L. Within the severa! phenotypicallydistinct subsets of CD4 memory cells aretwo that, upon stimulation, produce primar-ily iL-4 1MT2, CD45RA(–)DC62L(+)CDIIa(dim)1 or primarily 1FN-gamma1 MT 1, CD45RA(–)CD62L(–)CD I 1 a(bright)1.In addition, four other phenotypically dis-tinct subsets of CD4 cens have unique cy-tokine profìles. To determine the clinicairelevance of the representation of these celltypes, we analyzed blood from patientswith the chronic diseases Ieprosy and atopy.These diseases are characterized as im-munologically polarized, since T-cell re-sponses in affected individuais are oftenstrongly biased toward T(h)1 (dominatedby IFN-gamma production) or T(h)2 (IL-4production). We show Itere that this polar-ization reflects homeostatic or differentia-tion mechanisms affecting the representa-tion of the functionally distinct subsets ofmemory CD4 T cens, MT1 and MT2. Sig-nitìcantly, the representation of the MT Iand MT2 subsets differs dramatically be-tween subjects with tuberculoid Ieprosy (aT(h)1 disease), or lepromatous Ieprosy oratopic disease 1T(h) 2 diseasesi. However,there was no difference in the cytokine pra-files of these or any of the other finely re-solved CD4 subsets when compared be-tween individuais across ali disease states.Thus, it is the representation of these sub-sets in peripheral blood that is diagnostic ofthe polarized state of the immune system.-Authors' Abstract

Moraes, M. O., Sarno, E. N., Almeida, A.S., Saraiva, B. C. C., Nery, J. A. C.,Martins, R. C. L. and Sampaio, E. P.Cytokine niRNA expression in Ieprosy: apossible role for interferon-ganna andinterleukin-12 in reactions (RR andENL). Scand. J. Immunol. 50 (1999)541-549.

Leprosy patients during the naturalcourse of the disease may deveio') reac-tional episodes, namely, reversal reaction(RR) and erythenia nodosum Ieprosum(ENL). Immunological events descrihed asoccurring during RR indicate upregulationof the immune response; whereas in ENLthe events are not fully understood. The aimof this study was to analyze the ia viro pat-tern of cytokine gene expression in the re-actional states of Ieprosy. Peripheral bloodmononuclear cells (PBMC, N = 14) and tis-sue samples (N = 17) obtained from pa-tients with ENL and RR were obtained andassayed by RT-PCR. PBMC obtained fromunreactional patients (N = 15) and normalindividuais (N = 5) were also assessed. Ex-pression of interferon (IFN) gannna, granu-locyte-macrophage colony stimulating fac-tor (GM-CSF), interleukin (IL)-2Rp55, per-forin and iL-1 beta mRNA in PBMC weredetected mostly in ENL/RR patients, butnot in unreactional patients. Likewise, cy-tokines such as IL-6, IL-8, tumor necrosisfactor (TNF)-alpha and TNF-beta were alsopresent in reactional and tuberculoid pa-tients as opposed to lepromatous leprosy(BL/LL). interestingly, the majority ofENL/RR patients showed messages forIL-6, IL-10, IL-12 and TNF-alpha in theskin. IFN-gamma was detected in 84.6%(ENL) and 100% (RR) of the patients;whereas iL-4 was detected only in few indi-viduais (38.5% and 25%, respectively). AI-though mRNA expression and protein Iev-els may be different, the data reported inthis study suggest a cytokine mRNA protilethat seems to be indistinguishable for RRand ENL. in addition, it shows upregulationof immunoinflammatory cytokines in theblood and tissue of the same patient exam-Med before and during reaction. Further-more, it is suggested that Chis pattern ofresponse results from an immunological re-activation that might lead to an acate in-

68, 2^ Cerrrent Literatnre^ 207

I anunatory response in hoth reactionalepisodcs.—Authors' Abstract

Mustafa, A. S. HLA-restrictcd immune re-sponse to mycobacterial antigens: rele-vante to vaccine design. Hum. hnmunol.61 Special Issue (2000) 166-171.

Identilication of mycobacterial anti^zensthat are recognized by CD4+ Th 1 cells in anHLA-nonrestricted mamler or In associa-tion with nuiltiple allelic products is re-quired to develop universally effective vac-cines against mycobacterial diseases. Ourstudies in this direction have shown thatsevera! recombinant mycobacterial antigensof cytosolic and eulture tiltrate oriwn arerecognized hy CD4+ Th 1 cells. Mapping ofT-ce11 epitopes with overlapping syntheticpeptides covering the estire sequente ofthese antigens identitied peptide sequentesstinudatory for Th 1 cells. HLA-restrictionanalysis showed that in addition to 1ILA-DRB 1 products (serologically definedHLA-DR 1 to HLA-DR l O, the HLA mole-cules encoded by HLA-DRB3 (HLA-DR52) and HLA-DRB4 (HLA-DR53) areimportant in presentation of mycobacterialantigens and epitopes to T cells. Dependingon the T-cell donor, the presentation of agiven antigen or peptide could be restrictedby HLA-DRB 1, HLA-DRB3, and/or 1-ILA-DRB4 products. In addition stimulation ofTh 1 cells by some antigens and peptides inthe presente of autologous and HLA-DRmismatched allogeneic APC suggestedpromiscuous presentation. These resultstaken together suggest that from HLA-re-striction perspective, severa! mycobacterialantigens qua!ify as candidates for suhunit orrecomhlnant vaccine design against my-cobacterial diseases.—Author's Abstract

Mustafa, A. S., Lundin, K. E. A., Meloen,R. 1I., Shinnick , T. M. and Oftung, F.Identifìcation of promiscuous epitopesfrom the mycobacterial 65-kilodaltorrheat shock protein recognized by humanCD4+ T ce11s of the Mvcobacteriium lep-rue memory repertoire. 1nIect. Immun.67 (1999) 5683-5689.

By using a synthetic peptide approach,we mapped epitopes from the mycohacte-

ria! 65-kDa heat shock protein (HSP65)recognized by human T cclls belonging tothe Mvrobacteriuwn leprue memory reper-toire. A pane! of 1-ISP65 reactive CD4+ T-ccll lises and clones were estahlished fromhealthy donors 8 years after inununizationwith heat-killed M. leprue and then testedfor prol i ferati ve reactivity against overlap-ping peptides comprising hoth the M. lep-rue and M. Iuberculosis HSP65 sequentes.The results showed that the antigen-speciticT-cell lises and clones established re-sponded to 12 mycobacterial HSP65 pep-tides, of which 9 peptides representcd epi-topes crossreactive between Lhe M. tiubercn-losis and M. /eprue HSP65 (amino acids[aal 6! to 75. 141 to 155, I51 to 165, 331 to345, 371 to 355, 411 to 425, 431 to 445,441 to 455, and 501 to 515) and 3 peptides(aa 343 to 355, 417 to 429, and 522 to 534)represented M. leprue HSP65-spccific epi-topes. Major histocompatibi1ity complexrestriction analysis showed that presenta-tion of 9 of thc 12 peptides to T cells wererestrictcd by 1 of the 2 HLA-DR moleculesexpressed from self HLA-DRB 1 genes;whercas 3 pcptides with sequentes com-pletely identical between Lhe M. leprae andM. tuberculosis HSP65 were presentcd toT cells hy multiple HLA-DR moleculcs:peptide (aa 61 to 75) was presentcd hyHLA-DIZ 1, -DR2, and -DR7, peptide (aa141 to 155) was presentcd hy 1-ILA-DR2,-DR7, and -DR53; whercas hoth HLA-DR2and -DR4 (Dw4 and Dw 14) were atile topresent peptide (aa 501 to 515) to T cells. Inaddition, Lhe T-cell tines responding tothese peptides in prol i feration assaysshowed cytotoxic activity against autolo-gous monocytes/macrophages pulscd withthe same HSP65 peptides. In conclusion,we demonstratcd that promiscuous peptideepitopes from the mycobacterial HSP65antigen can serve as targets for cytotoxicCD4+ T cells which belong to the humanmemory T-ceia repertoire against M. leprae.The results suggest that such epitopes mighthe used in the pcptide-haseá design of sub-unit vaccines against mycobacterial dis-eases.—Authors' Abstract

Naik, M., Matsuoka, M., Ohara, N., No-maguchi , H. and Yamada, T. The anti-

208^ International 101n -nal u/ Lepros.y^ 2000

gen 85 complex vaccine against experi-mental Mvcobacterium leprae infectionin mice. Vaccine 18 (1999) 795-798.

The proteins in cultue filtrate dcrivedfrom 13acillus Calmette-Guerin (BCG) wereexamined for protection against infectionby Mycohacterinm leprae. Immunizationwith the major secreted proteins, antigen 85complex (A`g85) A, B and C, induced effec-tive protective II11111Ulllty against multiplica-tion of M. lepra(' in the foot paris of mice.The most effective protection was observaiwhen mice were immunized with Ag85A. Asingle imnlunization with Ag85 could in-duce anti`gen-specific interferon gammasynthesis and more effective protectionthan live E3CG vaccine. This study demon-strates that Ag85 is an important immuno-protective molecule against leprosy infec-tion.—Authors' Abstract

Namer, I. J. and Steibel, ,I• Antibody di-rected against mannan of the Mvcobac-terimn tuherculosis cell envelope pro-vokes blood-brain barrier breakdown. J.Neuroimmunol. 103 (2000) 63-68.

Previous studies denlonstrated thatblood-brain barrier (BBB) breakdown ob-serve- at the cerebral levei during experi-mental allergic encephalomyelitis (EAE)arises from the presence of Mvcobacterimntuberculosis in Freund's adjuvant and notonly from the encephalitogenic antigens.The nlain objective of this study uvas tocheck if the mannan moiety of lipoarabino-mannan present in the mycobacterial cellenvelope is responsible for an immune re-sponse provoking a BBB breakdown. Theresults showed that: firstly, the completeFreund's adjuvant (CFA) contains a high re-lease of polysaccharides; secondly, the ratsimmunized with the CFA present an impor-tant serum concentration of anti-mannanantibody; and finally, a single 2-mg dose ofanti-mannan antibody injected intra-venously in naive rats provokes an immedi-ate and reversible BBB breakdown. Theseresults suggest that mannan arising from thesolubilization of the mycobacterial cell wallin Freund's adjuvant induces a high produc-tion of anti-mannan antibody which, inturn, provokes a BBB breakdown and pos-sibly facilitates the induction of EAE.-Authors' Abstract

Rook, G. A. W. and Barker, R. Cortisolmetabolism, cortisol sensitivity and thepathogenesis of leprosy reactions. Trop.Med. Int. Health 4 (1999) 493-498.

A description is given of how local rcgu-lation of effective cortisol concentrations islargely independent of circulating cortisolconcentrations, and is itself regulated by lo-cal cytokine production. The ways in whichcortisol affects the function of macrophagesand T cells most relevant to mycobacterialdisease is also outlined. It is suggested thatthe hypothesis that this mechanism under-lies events triggcring leprosy reactions isreadily testable, and if correct could lead tonew dia^g nostic tests and treatnlents.—Trop.Dis. Bull. 96 (1999) 1264

Santos, A. R., Degrave, W. M. and Suffys,P. N. Use of polymerase chain reaction(PCR) in leprosy research. Indian J.Lepr. 71 (1999) 101-110.

This conference paper considers the di-agnosis, treatment, case finding and earlydetection of leprosy and describes experi-ence of using nucleic acid-based tools forleprosy control. 11 is concluded that PCRcould be a pronlising complementary diag-nostic tool for leprosy and has a certain po-tencial for the detection of subclinical infec-tion and early stages of the disease. How-ever, it is suggested that some importantconsiderations on the use of the techniquehave to be made.—Trop. Dis. Bull. 96(1999) 1163

Shetty, V. P., Shetty, K. T., Save, M. P.and Anta, N. H. M. leprae-induced aI-teration in the neurofilament phosphory-lation leads to demyelination in leprousnerves: a hypothesis. Indian J. Lepr. 71(1999) 121-135.

A preliminary study was conducted to in-vestigate the mechanism of demyelinationin leprosy. Nerves were obtained from 2normal subjects (amputated limbs) and 64leprosy patients (total of 64 leprous nerves)[in Indial. SMI-31, a specific antibodyknown to bind to the phosphorylated epi-tope of the carboxy terminal region of neu-rofilaments, was used as primary antibodyand stained sections were examined torecord the panem and intensity of staining.

68, 2^ Cru rani Literatura^ 209

The 2 normal nerves showed a linear pat-tern of SMI-31 staining in the longitudinalsections and uniform and intenso stainingwas observed in ali the axons in the trans-verse sections. In both tuberculoid and lep-romatous nerves that showed mild-to-mod-erate pathology there was a severe Ioss ofSMI staining. It is sug*`gested that these ob-servations marfe 011 peripheral nerves inleprosy indicate hypophosphorylation ofneurofilament proteins on carboxy terminalregions.—Trop. Dis. Bull. 96 (1999) 1 163

Stanford, J. L., 'limpa, N., Bati, A. N.,Torres, 1'. and Singh, M. Antibodiesagainst heat shock proteins in long-standing leprosy patients, with its proba-ble association with deteriorating tissueautoimmunity and the results of the ap-plication of immunotherapy with inacti-vated Mvcobacierium vaccae. Rev. Lep-rol. Fontilles 22 (1999) 265-274.In this small study of chronic leprosy

patients randomized to receive an infectionof killed Mvcobacteriunl vaccae in thepast, igG and IgA antibody titers have beenmeasured to the 65 kDa and 70 kDa heatshock proteins (hsp) of BCG. It was foundthat, in common with other inllammatorydiseases with disregulated cellular immu-nity, patients had higher leveis of igG tothese antigens than did healthy staff mem-bers. Immunotherapy was associated witliantibody titers reduced toward normal lev-els. For IgG to hsp 65 kDa this was statisti-cally significam in the Iranian patients, re-ceiving the immunotherapy a year or morebefore sera were taken. In the Spanish pa-tients receiving immunotherapy IO yearsbefore the sera were taken, although notreaching statistical significance, there werestrong trends toward the same finding forhsp 65 kDa, also seen in IgA titers. The re-lationship of these findings to autoimmunedisease and to a switch from Th2 to Th 1helper T-cell activity is discussed.—Au-thors' Summary

Teitelbaum, R., Cammer, M., Maitland,M. L., Freitag, N. E., Condeelis, ,J. andBloom, B. R. Mycobacterial infection ofmacrophages results in membrane-per-meable phagosomes. Proc. Natl. Acad.Sci. U.S.A. 96 (1999) 15190-15195.

Cell-mediated immunity is criticai forhost resistance to tuberculosis. T lympho-cytes recognizing antigens presented by themajor histocompatibility complex (MHC)class 1 and class II molecules have beenfound to be necessary for control of my-cobacterial infection. Mice genetically deli-ciem in the generation of MHC class I andclass 1 a responses are susceptible to my-cobacterial infection. Although soluble pro-tein antigens are ^generally presented bymacrophaces to T cells through MHC class[I molecules, macrophages infected withMvcohacteriunl tuberculosis or bacille Cal-mette-Guerin have been shown to facilitatepresentation of ovalbumin through theMHC class I presentation pathway via aTAP-dependent mechanism. How my-cobacteria, thought to reside within mem-brane-bound vacuoles, facilitate communi-cation with the cytoplasm and enable MHCclass 1 presentation presents a paradox. Byusingr confocal microscopy to study the lo-calization of fluorescent-tag`ged dextrans ofvarying size microinjected intracytoplasmi-cally into macrophages infected withbacille Calmette-Guerin expressing thegreen fluorescent protein, molecules aslari2e as 70 kiiodaltons were shown to ;2ainaccess to the mycobacterial phagosome.Possible biological consequences of thepermeabilization of vacuolar membranes bymycobacteria would be pathogen access tohost-cell nutrients within the cytoplasm,perhaps contributing to bacterial patho^gen-esis, and access of microbial antigens to theMHC class 1 presentation pathway, con-tributing to host protective immune me-sponses.—Authors' Abstract

Tilley, P. A. G. and Menon, J. N. Detec-tion of Mvcobacteriuni-speci fie inter-feron-gamma-producing human T lym-phocytes by flow cytometry. APMIS 108(2000) 57-66.Flow cytometry lias proveu to be a use-

fuI tool for the investigation of cytokinesynthesis by selected cell subpopulations.while most reports have used mitogen stim-ulation or lonu-term cultues with antigen,we describe here a novel method to allowthe detection of rare mycobacterial antigen-specific cytokine synthesizin^g cells withinune day. The most important feature of this

210^ lnternational Journal n/ Lcpros_v^ 2000

method is the use of an FITC-conjugatedisotype-matched control antibody to iden-tify and exclude cclls which Iluoresce non-specilically. With this techniyue, wedemonstrate interferon-ganinla (IFN-y)staining in 785 cens per 1 x 1 (Y '1' eenscoonted in mycobacterial autigen-stimu-lated peripheral blood ntononuclear censfrom a BCG-vaccinated subject. In campar-ison, only 14 IFN-y-staining T cens wereseen in the eultores not stiiatlated by my-cobacterial antigen. Less than 10 eens per 1x 10' T cens are stained by an irrelevantcontrol antibody. Specific responses are de-tectable after 12 hr of in litro culture, andpeak at 24 hr. 1n volunteer health cateworkers, IFN-y staining correlated withIFN-y production using a publishedELISPO'i' assay (r = 0.927). IFN-y stainingwas also higher in P13MC from Mantouxskin test-positive volunteers compared tocens from skin test-negative subjects (p =0.0045). How cytomet y following short-term culture can thus be used for enumera-tion of antigen specific lFN-y synthesizingcells.—Authors' Abstract

Tomimori-Yamashita, j., Cruad, P.,Rotta, O. and Lagrange, P. 1-1. Anti-body-based enzyme-linked 1mmunosor-bent assay for determination of anti-PGL-I specific circulating immune com-plex in leprosy patients. Lepr. Rev. 70(1999) 261-271.

A serological study was performed in122 individuais: 75 leprosy patients and 47healthy controls. The ELISA test was per-formed for IgG and IgM using the glyco-lipid PGL-I antigen from Mvcobacterirun1ehrae. Circulating immune complexes(CIC) were isolated by PEG 6000 precipita-tion method and after dissociation with anacid solution, the IgG and lgM specificagainst PGL-I were tested with the ELISA.The multibacillary patients had high leveisof antibodies compared vvith paucibaci1larypatients and controls. The antibodies iso-lated from the CIC presented a similar spec-trum spectral distribution as the serology. Apositive correlation between the leveis offree and CIC-bound antibodies was ob-served. In contrast with tuberculosis pa-tients, specific antibodies present in CICwere not responsible for false-negative re-sults found in some multibacillary patients'

serology lince no or very low leveis of spe-cific antibodies were founcl in PEG precipi-tated serum of these patients. No relationwas observed with speci fie antibody leveisdetected in CIC during leprosy reactions.-Authors' Summary

7'ominlori-Yamashita, ,I., Nguyen, '1'. NI.,Maeda, S. M., Flageul, 13., Rotta, O.and Cruaud, P. Anti-phenolic glyco-lipid-1 (PGL-I) determination usingblood collection on tilter paper in leprosypatients. Rev. Inst. Med.'l'rop. Sao Paulo41(1999) 239-242.

A total of 70 leprosy patients and 20healthy individuais from Brazil were stud-ied for specific antibodies against the nativephenolic glycolipid-I (PGL-I) from Mv-cobac tcriuni lchrac, comparing the tradi-tional sera collection method with the tin-ger-prick blood and conservation on tilterpaper method. The finger-prick blood driedon tilter paper was eluated in phosphatebuffered salino (PBS) cantaining 0.5%gelatin. The classical method for nativePGL 1 was performed for these eluates andcompared with antibody determination forsera. Results showed a siraight correlationbetween these two methods, although thetiters founcl for the eluates were lower thanthese obtained for serology. It is suggestedthat this blood collection method coold beuseful for investigation of new leprosycases in the field.—Trop. Dis. Bull. 96(1999) 1266-1267

van den Bos, I. C., Khanolkar-Young, S.,Das, P. K. and Loekwood, D. N. J. Im-munohistochemical detection of PGL-I,LAM, 30 kD and 65 kD antigens in lep-rosy infected paraflìn presenved skin andverve sections. Lepr. Rev. 70 (1999)272-280.

A panei of lipid, carbohydrate and pra-teia antibodies were optimized for use indetecting M. leprae antigens in paraffin em-bedded material. Skin and nerve biopsiesfrom 13 patients across the leprosy spec-trum were studied. All antibodies detectedantigen in tissues with a 131 >1. Phenolic-gly-colipid was not detected in bacteriologicallynegative tissue but lipoarabinomannan(LAM) and protein antigens were detected.

68, 2^ Crrrent Literature^ 211

Staining with LAM was strongest and `laveleast background. The transfer of this im-nlunohistochcniical technique to paraffin-embedded material will allow examinationof tissuc with bctter n u>rphology and fromclinics without access to tissue freezing fa-cilities.—Authors' Sumniary

Watson, V. 1?., Ilill, 1.. L., O en Sebaub,L. B., Davis, I). \V., NIcConkey, I). ,1.,Jagannath, C., Hunter, R. L. anel Ac-tor, ,1. K. Apoptosis in Myc'uhucteriunrtuherrulnsis infection in mice exhibitingvaried inununopathoiogy. J. Pathol. 190(2000) 211-220.This study examined mcchanisnus con-

tributing to pulmonary immunopathologyfollowing acute Mvcohcceterium tuberculo-.si.c ( MT13) infection in giro in a murineniodel. A/J and C5713L/6 mice were intra-venously infected with MTB (Erdman).Pathological differences were found be-tween strains, unrelated to pulmonary loadof bacilli. A/J mice developed proeressiveinterstitial pneunumitis, while C5713L/6mice malntained gr ntdoma formation. Thecontt ibution of FAS and FAS ligand-medi-ated apoptosis 'as assessed via biolumines-cent reverse transcription-polymcrase chainreaction (RT-PCR), immunohistochemicalstaining, and TUNEL assessment of 1)NAfragmentation. Cytokine messages forpulmonary tumor necrosis factor-alpha(TN1'-(1) and interferon-eamma (IFN-y), aswell as for the lytic moleculcs perforin andgranzyme 13, were quantified. Immunohis-tochemicai staining for CD3 receptor wasperformed to monitor lymphocytic lung in-filtration. Soon after infection. A/J mice ex-hibited increased pulmonary iFN-y mes-sage, concurrent with the appearance ofCi)3+ lymphocytes distributed throughoutthe lung. C5713L/6 mice exhibited perivas-cular euftìng, with no accompanying in-crease in IFN-y message. A/J mice also hadelevated leveis of FAS and FAS ligand mes-sage and protein early after infection, whilethe C57BL/6 mice had no increased expres-sion of these molecules. I3oth strains exhib-ited qualitatively similar numbers ofTUNEL-positive edis throughout infection,with a marked increase on day 7. Apoptoticcells appeared to co-localize with acid-fastbacilli. It is therefore proposed that apopto-sis durine iniba! granuloma formation fol-

lowing MTB infection may occur through aFAS/FAS ligand-independent pathway.Moreover, a failure of completion of theFAS/FAS 1igand-mediated apoptosis path-way in the A/J mice n rty contribute to inef-ticient elimination of lymphocytes, thusfurther aggravating pulmonary pathol-ogy.—Authors' Abstract

Yang, X., Wang, S., Fan, Y. and Zhu, L.Systemic mycobacterial infection in-hihits antigen-specifie 1mtuunoglobulinE production, bronchial mucus produc-tion and eosinophilic inflammation in-duced by allergen. Immunology 98(1999) 329-337.As the burden of infectious diseases be-

comes reduced in many countries, a re-markable increase in the incidence of aller-eies lias occurred. The hasis for the rise inatopic disorders as a correlate of the declinein 1nfectious diseases lias not been delineei.In the present study, we testei! experimen-tally whether prior systemic infection withMvcohacterium hm'i• bacillus Calmette-Guerin (I3CG) had any effcct on ovalbumin(OVA) AI-(OH), (alum)-induced im-munoelobulin E (IgE) production, airwaymucus production and eosinophilic inflam-nrUion. The data showed that allereen-spe-cifie IgE production and OVA-inducedeosinophilia and goblet ccll developmentwere significantly inhibited by prior infec-tion with BCG. Correspondingly, followingimmunization with OVA aluiu, I3CG-in-fected mice exhibited sienilicantly higherleveis of allergen-driven interferon-gamma(IFN-y) production than the mice withoutinfection. The ratio of IFN-y : interleukin(iL)-d production was higher in OVA-sensi-tized mice with prior I3CG infection than inthose without infection. The abrogation ofOVA-induccd mucus production and pul-monary cosinophilia in I3CG-infected micecorrelated with significantiy decreased IL-5production and increased IFN-y and IL-12production. "I'hese data provide direct evi-dence that intracelluiar bacterial infection(i.e., BCG) can inhibit antigen-specilic IgEand airway reactivity induced by environ-mental allereen. Furthermore, the resultssuggest that changes in cytokine-producingpatterns of T lymphocytes and other cellsnuay be the nuechanism by which infectionsinfluence allergies.—Authors' Abstract

212^ lnternotional Journal of . Leprosy^ 2000

Microbiology

Bancrjee, S. K., Bhatt, K., Nlisra, I'. andCbakraborti, P. K. Involvemcnt of anatural transport system in the process ol'cfilux-mcdiated clrug resistance in Mv-c.a/metcriul!! sme,çnluti.S. MoI. Gen.Genet. 262 (2000) 949-956.

The phosphate-speci fie transporter 1(Pst) in hacteria is a multi-subunit systemwhich belongs to the ABC family of trans-porters. The gene forros pai t of an operonand it is invoivcd in phosphate uptake inprokaryotes. lis import function is known tobe operativo only under conclitions of phos-phate starvation. However, we found over-expression of this transporter in a Mvcobac-terium sinegn!(1I1.s . strain selected forciprofloxacin resistance (CIPr) which wasgrown under conclitions in which the phos-phate-scavenging function of this operonwas inoperative. 111 CIPr cens, active effluxof the drug plays a predominam role in con-ferring high leveis of fluoroquinolone resis-tance. We thereforc investigated the role ofthis transporter in the process of efflux-mc-diated drug resistance by inactivating thepst operou in the CIPr strain. Phenotypiccharacterization of the resulting strain,CIPrd, showed a strriking reduction in theminimal inhibitory concentration (MIC) ofciprofloxacin and in the drug extrusion pro-file as well. Genotype analysis, on the otherhand, revealed partia! disruption of the pstoperon in CIPrd as a consequence of trans-porter gene amplification. Furthermore, dis-ruption of this operou in wild-type cells re-sulted in hypersensitivity to ciprofloxacinand other xenobiotics to which CIPr cellsexhibited cross-resistance. Thus our resultsprovide strong evidence that Pst is a naturalmembrane transport system that has theability to promote drug efflux in addition toits phosphate-scavenging function in theCIPr strain.—Authors' Abstract

Billman-Jacobe, H., Haites, R. E. andCoppel, R. L. Characterization of a Mv-cobacterimn smegmatis mutant lackinggpenicillin binding protein 1. Antimicrob.Agents Chemother. 43 (1999) 3011-3013.

The ponA gene of Mvcobac ieriun! sn!eg-nlcllis encodes a 95-kDa penicillin hindinrprotein, PI3P1, that is similar to P1311 ofM. nlberc!llosis and M. lepras. Transposondisruption of ponA in M. .Snleginati.c re-sultai in a P13P1-deficient imitam that wassensitive to f3-lactam antihiotics, was morepermeable to glycine, and grew slowly inliquid sulfure.—Authors' Abstract

Delogu, G. and Brennan, NI. ,I. Functionaldomains present in the mycobacterialheina ^g^gl utinin, 11131-IA. J. Bacteriol. 181(1999) 7464-7469.

Identilication and characterization ofmycobacterial adhesins and complementaryhost receptors required for colonization anddissemination of mycobacteria in host tis-sues are needed for a more complete under-standing of the pathogenesis of diseasescaused by three bacteria and for the deve!-opment of effective vaccines. Previous in-vestigations have deinonstrated that a 28-kDa heparin-binding mycobacterial surfaceprotein, HBHA, can agglutinate erythro-cytes and promote mycobacterial aggrega-tion in i'itro. In this study, further molecularand biochemical analysis of HBHA dcmon-strates that it has three functional domains:a transmembrane domain of 18 amino acidsresiding near the N terminus, a large do-niain of 81 amino acids consistent with anor-helical coiled-coil region, and a Lys-Pro-Ala-rich C-terminal domais that mediatesbinding to proteoglycans. Using His-taggedrecombinant HBHA proteins and nickelchromatography, we demonstrate thatHBHA polypeptides which contais thecoiled-coil region forro multimers. This ten-dency to oligomerize may be responsiblefor the induction of mycobacterial aggrega-tion since a truncated N-terminal HBHAfragment containing the coiled-coil domaisprotnotes mycobacterial aggregation. Con-versely, a truncated C-terminal HBHA frag-nment which contains Lys-Pro-Ala-rich re-peats binds to the proteoglycan decorin.These results indicate that HBHA containsat least three distinct domains which facili-tate intercalation isto surface membranes,

68, 2^

Current Literatn rc^ 213

pronuote bacterium-bacterium interactions,and mediate the attachment to sulfated gly-coconjugates found in host tissues. Au-thors' Abstract

De Smet, K. A. 1.., Weston A., Brown, I.N., Young, D. B. anel Robertson, B. I)."I'hree pathways for trehalose biosynthe-sis in mycobacteria. Microbiology 1-16(2000) 199-208.Trehalose is present as a free disaccha-

ride in the cytoplasm of mycobacteria andas a component of ccll-wall glycolipids im-plicated in tissue dama`ae associated withmycobacterial infection. To obtain anoverview of trehalose metabolism, we ana-lyzed data from the Mvrobacierium tuber-culosis genome project and identitied ORFswith homolo`ay to genes encoding en/ymesfrom three trehalose biosynthesis pathwayspreviously characterited in other bacteria.Functional assays usine mycobacterial ex-tracts and recombinant enzymes derivedfrom these ORFs den uonst ated that my-cobacteria can produce trehalose from glu-cose 6-phosphate and UDP-glucose (theOtsA-OtsB pathway) from glycogen-likealpha (1—>4)-linked glucose polymers (theTreY-TreZ pathway) and from maltose (theTreS pathway). Each of the pathways wasfound to be active in both rapid-growing M.sme,tumatis and slow-growing M. bot'isBCG. The presence of a disrupted treZ genein M. lcprae suggests that this pathway isnot functional in this organism. The pres-ence of multiple biosynthetic pathways in-dicates that trehalose plays an importantrole in mycobacterial physiology.—Au-thors' Abstract

Harboe, M., Christensen, A., Haile, Y.,Ulvund, G., Ahmad, S., Mustafa, A. S.and Wiker, H. G. Demonst ation of ex-pression of six proteins of the mam-manai] cell entry (mccl) operon of Mv-cobacterium tuberculosi.c by anti-pepticleantibodies, enzyme-linked immunosor-bent assay and reverse transcription-polymerase chain reaction. Scand. J. Im-munol. 50 (1999) 519-527.Polyclonal rabbit antibodies were gener-

ated to synthetic peptides corresponding topredicted B-cell epitopes of six proteins of

the mce 1 operou of Mvenhacterium tuher-culosis. Antipeptide antibodies reacted withMce 1 A and Mcc 1 E fusion proteins in soni-cates of recombinant Ese/jerichia coli aswell as with distinct bands in sonicates, butnot in culture fluids of M. tuberculosis andM. horis bacill us Calmette-Guerin (BCG).Polyvalent rabbit antibodies generated byimmunitation with sonicates of BCG bacillireacted with synthetic peptides from the sixMce 1 proteins on the solid phase in enzyme-Iinked immunosorbent assay (ELISA), albeitwith different frequencies. The Mce l A pep-tide (pl24-140) reacted most frequently,with 7 of the 9 antibodies tested, while theMcelF peptide (p329-343) reacted with 2.Used as a control, 20 polyclonal rabbit anti-boclies to 12 isolated proteins of M. tuber-culosi.s. and M. bons BCG cfid not reactwith any of the six synthetic peptides, ex-cept in une case. mRNA expression of thesix Ince I A–mce2F genes of Al. tuhc'rci,Io.s•i.cwas demunstrated by reverse t anscription-polymerase chain reaction (RT-PCR).These data indicate that all Mce 1 A–Mce l Eproteins of the mce 1 operou are expressedby in I'it ro-grown M. tuberculosis and M.bons BCG.—Authors' Abstract

Katoch, V. 1b1. Molecular techniques forleprosy: present applications and futureperspectives. Indian J. Lepr. 71 (1999)45-49.This conference paper considers molec-

ular techniques for detection of nucleicacids and their therapeutic application,molecular techniques for epidemiology, ex-pression of M. leprae genes by recombinantDNA techniques and sequencing of thegenome of M. lehrae.—Trop. Dis. Bull. 96(1999) 1 162

Leoni, E., Legnani, P., Mucci, M. T. andPirani, R. Prevalence of mycobacteria ina swin uning pool environment. J. Appl.Microbiol. 87 (1999) 683-688.A study was performed to evaluate the

prevalence of nontubercular mycobacteriain swimming pool environments. The bac-teria in question were found in 88.2% ofpool water samples. The most frequent spe-cies were Mvcobacte rium gordonac (73.5%of samples: range 1-840 cfu 100 ml-';

214^ International Journal o/ . Leprosv^ 2000

M. cluelonei (382% 2-360 cfu 100 ml '; andM. fortuitum (35.3% 2-250 cfu 100 ml '. Thesame species were also recovered from thewater at the different phases of the treat-ment cycle, with relative percentages simi-lar to those of the 1)001 water. Shower floorsand pool edges also presented high concen-trations of the mycobacteria (100% of sam-pies) and M. mcnrinum was isolated from thesurfaces of pool edges on two occasions(4.5% of samples). The swimming 1)001 en-vironment provides a suitable habitat forthe survival and reproduction of mycobac-teria. Althoui/h mycobacteria are commonin swimming pools, human mycobacterialdisease associated with their use is rare.Apart from superficial infections with M.marinum, the risk of more serious diseasesin subjects with weakened in unune systemsshould not be underestimated, given thewidespread presence of mycobacteria thatare possible opportunistic pathogens andthe direct contact bathers have with the wa-ter and aerosol.—Authors' Abstract

Matsumoto, S., Furugen, M., Yukitakc,II. and Yamada, T. The gene encodingmycobacterial DNA-binding protein 1(MDPI) transformed rapidly growingbacteria to slowly growing bacteria.FEMS Microbiol. Lett. 182 (2000)297-301.Pathogenic species of Mycobacterimn

are slowly growing intracellular bacteria.Slow growth is important for the parasitismof these organisms and chronicity of thedisease, but its precise mechanism has notbeen elucidated. Recently, we found that anovel DNA-binding protein (MDPI) wasexpressed (7%-10% in total protein) in my-cobacteria, such as M. boris bacillus Cal-mette-Guerin M. tuberculosis, and M. lep-rae. In this study, we observed that MDPIinterfered with replication, transcription,and translation in the analysis in in vitro Es-cherichia coli cell-free macromolecularbiosynthesizing systems. Furthermore,MDPI inhibited the rapid growth of both E.coli and M. smegniatis, and NH2-terminalsecond amino acid, asparagine, was ob-served to be important in terras of this func-tion. These data suggest an important roleof MDPI for suppression of growth rates ofmycobacteria.—Authors' Abstract

Matsunu►to, S., Yukitakc, 1-1., Furugen,NI., Nlatsuo, 7'., Mineta, 'I'. and Ya-nuula, 7'. identification of a novel DNA-binding protein from Mvcobacverium Io-ris bacillus Calmette-Guerin. Microbiol.Inununol. 43 (1999) 1027-1036.A novel DNA-binding protein express-

ing (8%-10% in total protein) in Mvrobac-terium bovis bacillus Calmette-Guerin wasobserved. This protein was designated my-cobacteria) DNA-binding protein 1(MDP1), MDP1 recognized bases, sugarmoieties, phosphate-backbone on DNA andpreferentially bound to DNA guanine andcytosine. In the gel retardation assay,MDPi preferentially bound to closed circu-lar plasmid DNA than to open circular andlinear form plasmid DNA and also bound toRNA. MDP1 forme(' a highly polymerizedstructure and localized not only in the nu-cleoid but also at the 50S ribosomal sub-units and cell surface. MDP1 was con-served in Mvcobacterium thus far examinedand the expression was entrance(' in station-ary growth phases. These results will pra-vide a reasonable basis for further study ofthe function of MDPi in living mycobacte-ria.—Authors' Abstract

Mistry, N. F. and Antia, N. H. IncompletekiIIing of intracellular mycobacteria-speculation on the creation of persisters.Indian J. Lepr. 71 (1999) 69-74.A brief overview is given from interdis-

ciplinary viewpoints of the phenomenon ofincomplete intracellular of distinctspecies of mycobacteria. M_vcobacte riumleprae and M. tuberculosis infection areused as examples of disease persistenceduring chemotherapy.—Trop. Dis. Bull. 96(1999) 1151

Miyamoto, M., Yamaguchi, Y. andSasatsu, M. Disinfectant effects of hotwater, ultraviolet light, silver ions andchlorine on strains of Legionella andnontuberculous mycobacteria. Microbios101 (2000) 7-13.The disinfectant effects on Legionella

and nontuberculous mycobacteria of hotwater, ultraviolet light, silver ions and chlo-rine were evaluated. The bacterial strains

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Current Lite rature^ 215

Legionella pneu/nophi/a ATCC33152 andMvcobacterium aviam ATCC25291 andstrains of L. pnewnopinla and M. aviamwhich had been isolated from a 24 hr bathwere examined for their resistance to treat-ments. All strains were killcd within 3 minon exposure to hot water at 70°C and expo-sure to ultraviolet light at 90 mW.s/cm .The strains of L. pnewnophila tested werekillcd within 6 hr on exposure to a solutionof silver íons at 50 pg/I. The number of vi-able cclls of strains of M. avium fell from10' CFU/nll to 10` CFU/ml after exposureto an aqueous solution of silver ions at 100µcg/I for 24 hr. Chlorine effectively killedstrains of Legionella which were exposed toan aqueous solution of chlorine at 2 mg/1within 3 mm, but strains of Mvcobacteriumsurvived exposure to chlorine at 4 mia/1 formore than 60 min.—Authors' Abstract

Mollenkopf, H. J., Jungblut, P. R., Rau-pach, B., Mattow, J., Lamer, S., ZimnyArndt, U., Schaible, U. E. and Kauf-mann, S. H. E. A dynamic two-dimen-sional polyacrylamide gel electrophore-sis database: the mycobacterial proteomevia internet. Electrophoresis 20 (1999)2172-2180.

Proteonle analysis by two-dimensionalpolyacrylamide gel electrophoresis (2-DPAGE) and mass spectrometry, in combina-tion with protein chemical methods, is apowerful approach for the analysis of theprotein composition of complex biologicalsamples. Data oruanization is imperativefor efficient handling of the vast amount ofinformation generated. Thus, we have con-structed a 2-D PAGE database to store andcompare protein patterns of cell-associatedand culture-supernatant proteins of differentnlycobacterial strains. in accordance withthe guidelines for federated 2-DE data-bases, we developed a program that gener-ates a dynamic 2-D PAGE database for theWorld-Wide-Web to organize and publish,via the Internet, our results from proteomeanalysis of different Mvcobacterilan tuber-culosis as well as M. bovis BCG strains.The uniform resource locator for the data-base is http://www.mpiib-berlin.mpg. de/2D-PAGE and can be read with a Java com-patible browser. The interactive hypertextmarkup language docunlents displayed are

generated dynamically in each individualsession from a rational data file, a 2-1) gelimage file and a map file describing, the pro-tein spots as polygons. The program con-sists of conlmon gateway interface scriptswritten in PERL, nunimizing the adminis-trative workload of the database. Further-more, the database facilitares not only inter-active use, but also worldwide active partic-ipation of other scientific groups with theirown data, requiring only minimal conlputerhardware and knowledge of informationtechnology.—Authors' Abstract

Nakata, N. The absence of catalase func-tion in Mvcobacterium leprae. Indian J.Lepr. 71 (1999) 87-92.

A study was conducted to prove the ab-sence or presence of a catalase peroxidasegene (kai(;) in N/. leprae. Using a PCR am-plified kaiG fragnlent, DNA fragmentswere cloned covering the whole kaiG re-gion and the complete nucleotide sequencewas determined. Although the Al. lepraekatG sequence showed approxinlately 70%honlology to the M. tuberculosis katG gene,severa! lesions were found in it. It is sug,-gested that Chis linding indicates that thekatG gene iras been inactivated in Al. lep-rae. The 2 deletions in the M. laprac katGsequence were found in all 7 different M.Ieprae isolares examined. To confiem thatthe Al. Ieprae katG region was not func-tional, it was examined whether the kaiGreuion of M. Ieprae could produce a func-tional catalase-peroxidase protein by usinean in vitro protein synthesis system. In thein nitro translation experiment, no specificprotein was produced from the M. IepraekatG fragnlents while a 75 kDa protein wasobtained from the correspondine M. tuber-culosis katG frawnents.—Trop. Dis. Bull.96 (1999) 1162-1163

Narayanan, R. 13. Recombinant proteinsfrom Mvcobacterium Ieprae: current sai-ais. Indian J. Lepr. 71 (1999) 93-99.

In this overview it is conclucfed that re-combinant DNA technolocy is a potentialtool to generate proteins from M. lepraeand other related organisnls. Proteins of di-agnostic, therapeutic and functional impor-tance can be produced in largo quantities

216^ Internationa/ loura/ o/ Lehros.v^ 2000

and easily purilied for research use.—Trop.Dis. Bull. 96 (1999) 1 163

Nopponpunth, V., Sirawaraporn, '1'.,(:reene, I'..I. and Santi, 1). A'. Clon iand expression of Mvcobacterimn niber-culosisv and Mcobacterium leprae dihy-dropteroate synthase in Escherichia co/i.J. Bacteriol. 181 ( 1999) 6814-6821.The genes for dihydropteroate synthase

of Meohac ierium ncberculosis and M. lep-rae were isolated by hyhridization with',robes amplified from the genomic DNA Ii-braries. DNA sequencing revealed an openreading frame of 840 bp encoding a proteinof 280 amino acids for M. tuberculosis di-hydropteroate synthase and an open readingtrame of 852 bp encoding a protein of 284amino acids for M. lepra« dihydropteroatesynthase. The dihydropteroate synthaseswere expressed under control of the T5 pro-meter in a dihydropteroate synthase-deli-cient strain of E_.schcrichia cali. Using threechromatography steps, we purilied both M.tubeirulosis and M. leira« dihydropteroatesynthases to >98%/o homogeneity. Sodiumdodecyl sulfate-polyacrylamide gel elcc-trophoresis revealed molecular masses of29 kDa for M. tuherculosis dihydropteroatesynthase and 30 kDa for M. lepra« dihy-dropteroate synthase. Gel tiII ation of bothenzymes showed a molecular mass of ca.60 kDa, indicating that the nativo enzymesexist as dimers of two identical subunits.Steady-state kinetic parameters for dihy-dropteroate synthases from both M. ncber-culosis and M. leprae were determined.Representative sulfonamides and dapsonewere potent inhibitors of the mycobacterialdihydropteroate synthases, but the antimy-cobacterial agem p-aminosalicylate, a puta-tive dihydropteroate synthase inhibitor, wasa poor inhibitor of the enzymes.—Authors'Abstract

Oftung, F., Mustafa, A. S. and «'iker, H.G. Extensive sequence homology be-tween the Mvcobarterium Icprac LSR (12kDa) antigen and its Mvcobac icrium tu-berculosis counterpart. FEMS Immunol.Med. Microbiol. 27 (2000) 87-89.The Mvcobacte rium leprae LSR (12

kDa) protein antigen has heen reported to

mimic whole-ceII M. leprae in T-cell re-sponses across the leprosy spectrum. In ad-dition, I3-cell responses to specific se-quences within the LSR antigen have heenshown to be associated with inun u nopatho-logical responses in leprosy patients witherythema nodosum leprosum. We have inthe present stucfy applied the Al. lepra« LSR1)NA sequence as query to search for thepresence of homologous genes within therccently completed Al. tuheicvlosis genomedatabase (Sanger Centre, U.K.) By usingthe BLASTN search 1001, a homologousAl. tuberculosis open reading frame (336bp), encoding a protein antigen of 12.1kDa, was identilied within the cosmidIVITCY071-1713.25. The gene is designatedRv3597c within the M. Hebe rrulosi.ti H37Rvgenome. Sequence alignment revealed 93r/cidentity between the Al. lepra« and M. tu-berrulosi.^ anticens at the amino acid se-quence levei. The finding that some B- and'1'-cell epitopes were localized to regionswith amino acid substitutions may accountfor the putative differential responsivenessto this antigen in tuberculosis and leprosy.—Authors' Abstract

Ronning, D. R., Klabunde, T., Besra, G. S.,Vissa, V. I)., fletiste, J. T. and Sacchet-tini, J. C. Crystal structure of the secretedforni of antigen 85C reveals potencial tar-gets for mycobaclerial drugs and vac-cines. Nat. Strue. Biol. 7 (2000) 141-146.The antigen 85 (Ag85) complex, com-

posed of three proteins (Ag8.5A, B and C),is a major protein component of the Mv-cobacieriunr tuberculosis cell wall. Eachprotein possessas a mycolyltransferase ac-tivity required for the biogenesis of tre-halose dimycolate (corá factor), a dominamstructure necessary for maintaining cellwall integrity. The crystal structure of re-combinant Ag85C from Al. tuberculosis, re-finei' to a resolution of 1.5 Angstrom, re-veals an alpha/beta-hydrolase polypeptidefold, and a catalytic triad formed by Ser124. Glu 228 and His 260. Ag85C com-plexed with a covalent inhibitor implicatesresidues Leu 40 and Met 125 as compo-nente of the oxyanion bole. A hydrophobicpocket and tunnel extending 21 Angstromroto the core of the protein indicares the lo-cation of a probable trehalose monomyco-

68,2^

Current Literature^ 217

late binding site. Also, a lavre region ofconserved surface residues among Ag85A,13 and C is a probable site for the interactionof Ag85 proteins with human libronectin.-Authors' Abstract

Schaefl'er, M. L., Khoo, K. H., Besra, G. S.,Chatterjee, ll., Brennan, P. J., Deliste, J.'E and Inamine, J. M. The piml3 gene ofMycobacveriunl tuhcrcnlosis encodes amannosyltransferase involved in lipoara-binomannan biosynthesis. J. Biol. Chem.274 (1999) 31625– 31631.The biosynthesis of 1ipoarabinomannan

(LAM), a key mycobacterial Iipoulycanthat has been implicated in numerous im-munoregulatory functions, was examinedutilizing D-mannosamine (ManN) as a toolto identify mannosyltransferase genes in-volved in LAM synthesis. CeIl-free reac-tions utilizing cellular membranes of my-cobacteria as the enzyme source indicatedthat ManN inhibited the synthesis of phos-phatidylinositol mannosides, early pre-cursors to LAM. A selection strategy wasdevised to screen a Mycobactcrium tuber-culosis genomic library in M. siuegmatis forclones conferring conditional resistance toManN, with the rationale that overexpres-sion of the gene(s) encoding a target ofManN would impart a ManN-resistant phe-notype under these conditions. This strategyled to the identification of pimB, whosededuced amino acid sequence shows simi-larity to mannosyltransferases and otherglycosyltransferascs. Partially purified re-combinant PimB protein from Escherichiacoli or membranes from M. smegmatis over-expressing the pimB gene were used in cell-free assays to show that PimB catalyzes theformation of triacylphosphatidylinositol di-mannoside from GDP-mannose and tria-cylphosphatidylinositol monomannoside.-Authors' Abstract

Sengupta, U. Recombinant antigens: cur-rent situation and future. Indian J. Lepr.71 (1999) 111-120.This conference paper describes how

gene cloning techniques have enabled sev-eral Mvcobacterium lehrae genes to befused to those of other organisms, particu-larly Escherichia coli, and has led to the iso-

lation, purilication and characterization ofseveral cloned proteins. The cloned pro-teins, mostly heat shock proteins, are de-scribed and their role in T-cell stimulation isdiscussed.—Trop. Dis. Bull. 96 (1999) 1163

\1'ei, J., 1.)ahl, ,J. L., Moulder, J. W.,Roberts, E. A., O'Gaora, P., Young, I).B. and Friedman, R. L. ldenti(ìcation ofa Mvcohacterium tuherculosis gene thatenhances mycobacterial survival inmacrophages. J. Bacteriol. 182 (2000)377-384.Intracellular survival plays a central role

in the pathogenesis of Mvcobacteriun► tu-berculo.sis. To identify M. tuberculosisgenes required for intracellular survivalwithin macrophages, an M. tube rculosisH37Rv plasniid library was constructed byusing the simule vector pOLYG. This plas-mid library was electroporated into M.siuegnultis I-2c, and the transformants wereused to infect the human macrophage-likecell line U-937. Because M. sanegn►atisdoes not readily survive within macro-phages, any increascd intracellular survivalis likely due to cloned M. tuberculosisH37Rv DNA. After six sequential passaresof M. slneglnalis transformants throughU-937 cens, one clone (p69) was enrichedmore than 70% as determined by both re-striction enzyme and PCR analyses. P69demonstrated significantly enhanced sur-vival compared to that of the vector control,ranging from 2.4- to 5.3-fold at both 24 and48 hr after infection. DNA sequence analy-sis revealcd three open reading frames(ORFs) in the insert of p69. ORF2 (1.2 kb)was the only une which contained a puta-tive promoter region and a ribosome-bind-ing sito. Delction analysis of the p69 inseriDNA showed that disruption of ORF2 re-sulted in complete loss of the enhanced in-tracellular survival phenotype. This genewas named the enhanced intracellular sur-vival (eis) gene. By using an internai regionof eis as a probe for Southern analysis, eiswas found in the genomic DNA of variousM. tiherculosis strains and of M. bcn'isBCG but not in that of M. svnegn►atis or 10other nonpathogenic mycobacterial species.Sodium dodecyl sulfate-polyacrylamide gelelectrophoretic analysis showed that ali M.s!uegmatis eis-containing, construas ex-

218^ International Journal of Leprosy^ 2000

pressed a unique protein of 42 kDa, the pre-dicted size of eis. The expression of this 42-kDa protein directly correiated to the en-hanced survival of M. snu'gmati• p69 in U-937 cells. These results suggest a possiblerole for eis and its protein product in the in-tracellular survival of M. tuberculosis.-Authors' Abstract

Weinstein, I). E., Freedman, V. II. andKaplan, G. Molecular mechanism ofverve infcction in leprosy. Trends Micro-biol. 7 (1999) 185-186.

This paper discusses research roto themolecular mechanisms that facilitate theentry of Mrcobacterium leprae isto the pe-ripheral serves and subsequent infcctionof Schwann cells. 11 considers the bindingof M. leprae to Schwann cells, M. leprae-infected blood monocytes as the likely vec-tor of mycobacterium entry isto the verveand nerve degeneration.—Trop. Dis. 13u11.96 (1999) 1 160

Experimental Infections

Williams, D. L. and CiIIis, T. P. Detectionof drug-resistant M vcobacterium lepra('using molecular methods. Indian J. Lepr.71 (1999) 137-153.

This overview considers the develop-ment of antileprosy drugs and drug resis-tance in M. leprae and discusses techniquesused to detect drug resistance.—Trop. Dis.Bull. 96 (1999) 1163-1164

Franco, R., Fliess, E., Cecere, L., Llopis,L. and Bandim), R. [Experimental lep-rosy in Dasvpus hvbridus in Argentina.!Rev. Hosp. Nac. Baldomero Sommer 2(1999) 135-143. (in Spanish)

Leprosy bacilli could be obtained onlyfrom biopsy specimens of untreated pa-tients in Argentina. In order to assue thecontinuous production of lepromin we de-cide(' to focus the research on the ar-madillo. In the light of the internationalexperience, the multiplication of Mvcobac-teriunr leprae in armadillo is the least ex-pensive and complicated in viro cultuemethod. The susceptibility to M. leprae hasalready been studied in Dasvpus hrbridus.Seven animais were infected with an inocu-lum prepared with a human leproma ob-tained from an untreated patient. One ml ofthis inoculum adjusted to 10 8 bacilli/ml wasinoculated to each animal. The inoculumwas injected under anesthesia (sodium pen-tobarbital, intraperitoneal) in the externai

femoral or subclavian veias. Intracardiacinoculation was performed only if it wasnot possible to inject any of these veias.One armadillo showed disseminated lep-rosy 26 month after inl avenous inocula-tion. Abundam solid bacilli appeared inskin ulcer (leproma), liver, spleen andlymph rodes. It was possible to purifybacilli from the infected tissues. The inocu-lation of bacilli into mouse foot pad andpyridine extraction were positive. The cul-tues in different medium were negative.Puritication of M. leprae was performed asdescribed by Draper. Only highly purifiedbacilli were employed to prepare 500 mllepromin.—Authors' English Abstract

Nogueira, M. E. S., Coelho, K. I. R.,Fleury, R. N. and de Arruda, M. S. P.[ Inocu1ation of Mvcobacteriunr lepraeisto the cheek pouch of hamsters.1!Jansen. Int. 24 (1999) 5-14. (in Por-tuguese)

The use of the hamster cheek pouch in ex-perimental leprosy was evaluated throughthe inoculation of 6.0 x 10 8 M. leprae/ml inits subepithelial tissue in 60 animais. A con-trol group of 12 hamsters was inoculated inthe foot pad. The animais were sacrificed20 and 48 hours, 7, 14, 21 and 28 days afterinoculation. Histological sections stainedwith hematoxylin-eosin and Faraco-Fitewere used to evaluate the evolution of the

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Current Literature^ 219

lesions. The evaluation of the bacilli viabil-ity in the cheek pouch was done by a bacillirecovery test using mice inoculated in thefoot pad and sacrificed 6 months after inoc-ulatiou. The results led us to the followingconclusions: a) after the exudative phase,the lesions evolvcd i s to nulcrophagic eran-uloni formation similar to lepromatousleprosy in humans: h) there was a remark-able increase of the bacterioloL, ic index, as

opposed to the biological characteristics ofM. leprae, a fact that was interpreted as alocal concentration after reabsorption ofedema and congestion; c) the lesion in thefoot pad evoluted finto epithelioid granulo-mas similar to borderline leprosy; d) the M.leprae recovery test from the foot pad ofmice suggests that there was no multiplica-tion of the bacilli—Authors' English Sum-mary

Epidemiology and Prevention

Ohara, N., Matsuoka, M., Nomaguchi,Ii., Naito, M. and Mamada, T. Inhibi-tion of multiplication of Mvcohacteriunlleprae in mouse foot pads by recombi-nant Bacillus Calmette-Guerin (BCG).Vaccine 18 (2000) 1294-1297.Immunization of mice with recombinant

Mycobacteriunl boi'is Bacillus Calmette-Guerin (rBCG) which overproduces a puta-tive protective antigen candidate, the Acomponent of antigen 85 complex (Ag85A),reduced the multiplication of Al. leprae inthe foot pads of coice. The inhibition by thisrBCG (rBCG/85A) was more evident thanthat with parental BCG. Repeated rBCG/85A immunization significantly could re-doce Al. leprae multiplication in coice. Thisis the first report of rBCG to control my-cobacterial infection in an animal model.Therefore, the rBCG technique may be use-ful for the development of a more effectivemycobacteria vaccine.—Authors' Abstract

LeBlanc, S. B., Naik, E. G., Jacobson, L.and Kaslow, R. A. Association ofDRB 1=1501 with disseminated Mvco-baCterll/nl al'll nll complex infection inNorth American AIDS patients. TissueAntigens 55 (2000) 17-23.The HLA class II anele, DR2 (DRB 1'

1501), has been repeatedly found to be as-sociated with development of tuberculosisand leprosy. We searched for associationsof there and other class II alicies with dis-seminated Mvcobacte riam ai'ium complex

(DMAC) infection in North American Cau-casian homosexual AIDS patients. Molec-ular typing for HLA-DRB 1 and -DQB 1 al-leles in 176 cases of DMAC and 176matched controls showed an association ofaccelerated onset of disease with DRB 1 *1501 (and the closely linked DQB 1 =0602)that was stronger upon adjustment for thedegree and duration of CD4+ cell deli-ciency (p = 0.04) and in multivariate analy-sis (p = 0.02) than in unadjusted analysis. Asimilar trend was seen with DRB1 =0701,and no other alteie showed a relationship ofsimilar magnitude. Al. cn 'ium complex or-ganisms may more effectively evade hostdefenses in individuais carrying an 1-ILApolymorphism identical to that associatedwith M. tuberculosis and Al. leprae.—Au-thors' Abstract

Lechat, M. F. [The program for the elimi-nation of leprosy: ambitious challenge,innovative approaches, questions inabeyance.1 Bull. Mem. Acad. R. Med.Belg. 154 (1999) 201-207. (in French)The WHO-sponsored program on the

"elimination of leprosy as a public healthproblem by the year 2000" has been highlysuccessful. Over 9 million patients weretreated by multiple drug therapy. The num-ber of patients worldwide has been reducedby more than 90% over the last 10 years,being at present less than 800,000. Trans-mission, however, the ultimate goal of theprogram, has not been interrupted. The

220^ International .lourncil o/ . Lepre.ti_v^ 2000

number of new cases detected per year isstill high. 'Tis observation raises seriousquestions regarding the future of the pro-gram.—Author's English Sunu uary

Naik, S. S., Thakar, U. H., Phrande, A.M. and Canapati, R. Survey of leprosyin unapproachablc and uncovered arcas.hulian J. Lepr. 71 (1999) 333-335.Leprosy surveys in tribal population,

fishcrmen and laborers engaged in con-struction work revealed prevalence rates of32/10,000, 109/10,000 and 20/10,000 re-spectively, suggesting that systematic sur-veys have to be carried out in such popula-tion groups to reach the goal of a "worldwithout leprosy."—Authors' Abstract

Ramos, J.-M. H. [The Araguaia project inBrazil: some significam findings.1 Rev.Leprol. Fontilles 22 (1999) 257-264. (inPortuguese)The Araguaya Project for the eradication

of leprosy, started in 1997 in the northernstate of Mato Grosso (Brazil) ali extensiveand isolated part of the country, is described.The total arca covered by the project is ap-proximately 75.000 km' and a populationestimated in 34,000. The work was carriedout mainly in 3 villages: Sao Felix doAraguaia, Santa Terezinha and Porto Alegredo Norte. The number of multibacillarycases (59.8%) is greater than the paucibacil-lary (40.2%) with 109 and 73 patients,

respectively, and 76 new cases detectedthis year and a detection rate of 19.8 cases/10,000 population. The prevalence rate is53.8/10,000 population, an indication of avery endemic arca. 'lhe incidente of leprosyin children Iess then 14 years is very high,with 11 cases/I0,000 population. The proj-ect will continue under the management andsupervisiou of the work by the Fontillesmedicai staff.—Author's English Summary

Sclvasekar, A., Geetha, ,J., Nisha, K., Na-trajan, M., Jesudasan, K., SondarRao, P. S. S. Childhood leprosy in an cn-demic arca. Lepr. Rev. 70 (1999) 21-27.A study was conclucted 01'794 new cases

of leprosy among children (aged 0-14years) detected and trcated with multidrugthcrapy during 1990-1995 in GudiyathamTaluk, Tamil Nadu, Ilidia. Incidence rates ofleprosy and proportion of multibacillarycases incrcased with age, with the maxi-mum incidente amonu children aged 10-14years. I3orderlinc tuberculoid was the mostcominou type of leprosy encountered in thechildren; 83.1(70 had a single patch andmost children were detectes through sur-veys, 29.8%/c had history of household con-tacts with leprosy, mostly parents or grand-parents. Reactions and relapses were notuncommon. The (inclines emphasize theneed fòr more careful surveys for case de-tection and better follow up in case man-agement.—Trop. Dis. Bull. 96 (1999) 1049

Rehabilitation

Zodpey, S. P., Bansod, B. S., Shrikhande,S. N., Maldhure, B. R. and Kulkarni,S. W. Protective efiect of Bacillus Cal-mett-Guerin (BCG) against leprosy: apopulation-based case-control study inNagpur, India. Lepr. Rev. 70 (1999)287-294.

A population-based, pair-matched, case-control study was carried out in ali urbancommunity, Nagpur, índia, to estimate theeffectiveness of BCG vaccination in the

prevention of leprosy. The study included212 cases of leprosy (diaunosed by WHOcriteria), below the age of 35 years, de-tected during a leprosy survey conclucted bythe governnient of Maharashtra over apopulation of 2,003,325. Each case waspair-matched with one neighborhood con-trol for age, sex and socioeconomic status.A significam protective association betweenBCG and leprosy was observed (OR = 0.40,95% CI = 0.23-0.68). The overall vaccineeffectiveness (VE) was estimated to be 60%

68, 2^ Current Literature^ 221

(95% Cl = 32-77). The BCG effectivenessagainst multibaci1lary and paucibaci1laryleprosy was 72% (95% Cl = 3.5-88) and45% (95% Cl = 3-73), respectively. Vac-cine was more effective during the firstdecade of life, among females and in lowersocioeconomic st ata. The overal1 pre-vented fraction was 39% (95% CI = 16-58). In conclusion, this lirst-ever popula-tiou-based case-control study performed incentral Ilidia identified a beneficial role of .

BCG vaccination in prevention of leprosyin a study population.—Authors' Summary

Andreotti, C. G. and Fantoni, R. D. 1Pe-ripheral neuropathy in Hansen's dis-ease.1 Rev. Hosp. Nac. Baldomero Som-mer 2 (1999) 153-157. (in Spanish)The subject of the present paper is to de-

scribe the peripheral neuropathy inHansen's disease across the clinicai spec-trum. Multiple mononeuritis is the usualpresentation. A population of 400 leprosypatients was studied by clinic examinationand clectromyography. The patients wereasked about: age, gender, time of disease,clinicai aspects, reactions, trealments, asso-ciated diseases (diabetes, chronic renal in-sufficiency, alcoholism) nerve micro-surgery. Electromyography was performedin ali patients. No difference in peripheralnerve injury was found between tuberculoidand lepromatous leprosy patients. Neitherwas found a significam increase of the lep-rosy polyneuropathy in patients with an-other associated pathology (diabetes,chronic renal insufficiency, and alco-holism).—Authors' English Summary

Reine, A. O. Modification of the "wraparound" tendon anastomosis of faseialata graft with a slim double tendon ofpalmaris longos. Indian J. Lepr. 71 (1999)337-340.A modification of Brand's "wrap

around'' technique of anastomosis is de-scribed, which allows joining a double ten-don or split tendon of palmaris longos tofaseia lata graft, when une of the slim ten-dons would not allow performance of theBrand tendon anastomosis. Four such caseshave been done successfully.—Author'sAbstract

Dong, L., Li, F., Jiang, J. and Zhang, G.Techniques for covering soft tissue de-fects resulting from plantar ulcers in lep-rosy: Part II—First toe web and dorsalfoot flaps. Indian J. Lepr. 71 (1999)297-309.

The first toe web flap consists of the skinand subcutaneous tissues of . the contiguoussidos between the great and second toes. Itis based on the first dorsal metatarsal arteryor the common plantar digital artery. Thisflap was used as an artery pedicled islandgraft to reconslruct losses of skin and softtissue cushion in the ball of the foot in thefirst and second metatarsal head region in16 cases. Follow-up examination revealedthat ulceration had recorre(' in one case dueto dehiscence of the flap margin 12 monthspost-operatively. The other 15 patients havedone well without recurrence at 48 to 124months follow-up examination.

The dorsal flap of the foot based on thedorsalis pedis artery, the correspondingveins and the deep peroneal nerve was de-signe(' in 1974 to resurface skin and soft tis-sue defects in the sole of the foot. This flapwas used in 30 cases of. leprosy with excel-lent results. During follow up 36 to 120months after surgery the plantar ulcer hadrecorre(' in only one case. All the othershave done well. The long-terra curative ef-fect has thus proved satisfactory.—Authors'Abstract

Dong, L., Li, F., Wang, Z., Jiang, J•,Zhang, G., Peng, J., Zhang, J. and Ye,Y. Techniques for covering soft tissuedefects resulting from plantar ulcers inleprosy: Part I—General considerationsand sununary of results. Indian J. Lepr.71 (1999) 285-295.

Recurrent plantar ulceration is a com-mon and serious complication occurringconsequent to impairment of the tibialnerve in leprosy patients. In spite of manytherapies and a long therapeutic course, it isextremely difficult to abolish this complica-tion in many cases because of extensiveskin and soft tissue cushion loss due to re-peated infection. Since the early 1970s wehave been usino microscopic surgical tech-niques to reconstruct the ulcerated arca us-ing eight types of the flaps. In this series of

222^ /nternational .1ournal o/ Leprosy^ 2000

papers we review our experience (76 pa-tients). Post-operatively, the flaps survivedin ali cases, the lona-term results haveproved satisfactory, and recurrent ulcerationoccurred in only three patients.—Authors'Abstract

Duerksen, F., Oprornolla, D. V. A., Vir-mond, NI. and Garbino, ,I. Teachingand training for surgical rehabilitation inhanseniasis: results of 20 years of activi-ties of the Instituto Lauro de Souza Limain South America. Hansen. Int. 24 (1999)55-60.In summary, we can say, based on 20

years of experience, that with education andinformation—waking up a motivation,training in arcas of each team member,keeping continuity by regular supervisionvisits and allowing and furthering growththrough courses and congresses, stable andeffective rehabilitation teams for hansenia-sis can be established and maintained at avery low cost. More than half of the pro-grams listed have worked with the sameteam mcmbers for over 10 years.—Fromthe Article

Ogheiwi, O. and Nash, J. What wouldmake your life better'? A needs analysisof leprosy settlements in the middle beltregion of Nigeria. Lepr. Rev. 70 (1999)295-304.A needs analysis using rural appraisal

and matrix ranking techniques was done insix leprosy communities in the middle beltregion of Nigeria. Asked "What wouldmake their life better?" whole villa`gegroups were made to list, prioritize andrank their expressed needs by voting in amatrix table drawn on the ground. Out of atotal of 504 votes, 31% was for health careor drugs for their general ailments, 23.6%for money and less than 10% for otherneeds that ranked from water, trade andhousing to love and, least, mobility aids.Health care was prioritized in ali communi-ties but got the highest votes in three com-munities; money got the highest in the onlytwo communities where it was prioritizedand water in one. The need ranked the high-est in each settlement seemed to be a reflec-tion of its peculiar socioeconomic situation.

Apart from the similar priorities of healthcare and money, men's differing prioritieswere water, housing, clothes and assistancewith farming, and women's, school fees forchildren, family, trado and food. These re-tlect their difierent tradicional roles. Con-sidering the variety of needs, we think thatthere is no one solution to rehabilitation inthe Nigerian context, but the situation andcontext of individual settlements should beconsiderai, looking at general health care,income generation or loan schemes, school-ing and water supply.—Authors' Summary

Paschoal, V. I). and Soler, Z. A. S. G. IAcolor system for the biopsychosocialcharacterization of leprosy patients in re-action. I Hansen. Int. 24 (1999) 21-31.(in Portuguese)This study's objective is to analyze the

issues related to nursing assistance Hansen'sdisease, especially when there is a reac-tional phenomenon. It was aimcd at charac-terizing a sample of patients with reactionalHansen's disease, emphasizing aspects ofthe disease and the reaction and investigate,by means of a color system, the majorbiopsychosocial changes. It is a descriptive-investigatory study carried out at a univer-sity hospital developing a Program forHansen's Disease Control, with 28 adultmate and female patients with reactionalHansen's disease participating in the pro-gram. Two data collection instruments wereused: ai] interview using a form as a guide,with questions to be orally answered by thepatients and a list of the changes experi-enced by the patients resulting from the re-actional crisis, where he/she made a sum-mary, based on the color systems. The datacollection period was from October 1977 toMarch 1998. We concluded that the reac-tional crisis occurs both in female and inalepatients with Hansen's disease, with no re-lation to their age and clinicai manifestationof the disease, and were seen throughout upto 5 years of follow up after chemotherapydischarge. It was observed that the reac-tional crises significantly change the pa-tients' life quality, signaled by the record-ings in green, yellow and red, showing thediffculties found to deal with the issues inquestion, such as living with the pain, fu-ture expectations, feeding, and sleep, among

68, 2^ Current Literature^ 223

others, in their daily lives. The color systemused facilitated the anamnesis and theanalysis of the iteras, providing, a basis formore effective nursing and aimed at the in-dividual needs of the patients.—Authors'English Abstract

van Brakel, W. H., Anderson, A. M.,Worpel, F. C., Saiju, R., Bk, H. B.,Sherpa, S., Sunwar, S. K., Gurung, J.,de Boer, M. and Scholten, E. A scale toassess activities of daily living in personsaffected by leprosy. Lepr. Rev. 70 (1999)314-323.The aim of this study was to develop

scale for identifyint!, disability amongpeople in the rural arcas of developingcounlries. The studies were carried out inthe Green Pastures Hospital and the leprosyfield program of the Western Region ofNepal. With the help of staff experienced inworking with people with disability, a 68-question questionnaire was nade based onthe International Classitication of Impair-ments, Activities and Participation (ICIDH-2). A survey was carried out of 269 peopleaffected by leprosy who had impairments,as well as a sample of those who wereunimpaired. The survey results were used to

develop the questionnaire into a scale, us-ing standard scale development methods.This included checking of criterion validity,discrimination and reliability and stabilityusing weighted kappa statistics. Of the 68questions, 38 were included in the seconddraft of the instrument. Eight questionswere added to identify difficulty in relation-ships, about the use of aids and about occu-pation and employment. The sum score ofthe scale against the expert score gave aSpearman correlation coeffìcient of 0.72.Infra- and inter-interviewer reliability coef-ticients were 0.77 (95% CI 0.73-0.81) and0.61 (95% Cl 0.56-0.67), respectively. Thestability test gave an overall kappa of 0.76(95% CI 0.70-0.82). Four questions withparticularly poor results were omitted fromthe final draft of the instrument. An inter-view-based instrument was developed foridentifying limitations in activities of dailyliving (disability) in people living in a ruralsetting in a developing country—the GreenPastures Activity Scale (GPAS). The scaleperformed well during validity and reliabil-ity testing. 11 consists of 34 activity ques-tions, 5 rclationship questions, and 3 ques-tions on the use of aids, occupation and em-ployment.—Authors' Summary

Other Mycobacterial Diseases and Related Entities

Ahmad, S., Akbar, P. K., Wiker, H. G.,Harboe, M. and Mustafa, A. S.Cloning, expression and immunologicalreactivity of two mammalian cell entryproteins encoded by the meei operon ofMycobacterium tuberculosis. Scand. J.Immunol. 50 (1999) 510-518.

The DNA segments corresponding totwo members of the mammalian ccll entryoperon 1 (mce 1) encoding Mce 1 A andMcelE proteins were amplitiied from Mv-cobacterium tuberculosis genomic DNA bypolymerase chain reaction, cloned and sub-cloned into pGEM-T and pGEX-4T-3 vec-tors, respectively, and expressed in Es-cherichia coli as fusion proteins with glu-

tathione-S-transferase (GST) of Schisto-sonm japonicum as the fusion partner. Therecombinant proteins appeared as majorcellular proteins in SDS-PAGE gels at theexpected molecular mass of 68 kDa and 64kDa for GST-Mce 1 A and GST-Mce 1 E, re-spectively. The identify of each fusion pro-tein was conlirmed by reactivity vvith anti-GST antibodies in Western immunoblots.The fusion proteins were purified to nearhomogeneity by affinity chromatography,and puri fied Mce IA and Mce l E, free of thefusion partner, were recovered followingspecific proteolytic cleavage of the GSTportion by thrombin protease. PuritiedMce 1 E appeared as a single band of 38kDa; whereas purified Mce IA tended to ex-

224^ International Jourual of . Leprosy ^ 2000

ist in degradai as wcll as aggregated fornisof different sizes. The fusion proteins, freeGST and monomeric Mce 1 A and Mcc 1 Ercacted in Western immunoblots with anti-boclies in pools of human sera from 6 to 11tuberculosis paticnts. Similar analysisshowed the presence of antibodies to GSTand Mce 1 A in pools of human sera from M.hovis BCG-vaccinated healthy subjccts.Whcn pure Mce I E was blottcd against indi-vidual sera, antibodies in 4 of 10 sera fromtuberculosis paticnts rcacted; whereas noreaction was seen with 10 individual serafrom M. bovis BCG-vaccinated healthysubjccts. i-lowever, whcn the same serawere tested for rcactivity to the purifiedpreparation of Mce 1 A, 8 of 10 sera fromboth tuberculosis paticnts and M. bot'isBCG-vaccinated healthy subjccts showedpositive reactivity. These lindings demon-strate that both Mce l A and Mce 1 E are ex-pressed and immunogenic during naturalinfection with M. tuberculosis.—Authors'Abstract

Alderson, M. R., Bement, '1'., Day, C. H.,Zhu, L. Q., Molesh, D., Skeiky, Y. A.W., Coler, R., Lewinsohn, D. M., Reed,S. G. and Dillon, D. C. Expressioncloning of an immunodominant familyof MVcobacterium tuberculosis antigensusing human CD4+ T Mis. J. Exp. Med.191 (2000) 551-559.

Development of a subunit vaccine forMycobacteriwn tuberculosis (Mtb) is likelyto be dependent on the identification ofT-cell antigens that inducc strong prolifera-tion and interferon gamma (IFN-y) produc-tion from healthy purified protein derivativa(PPD+) donors. We have developed a sensi-tive and rapid technique for screening anMtb genomic library expressed in Es-cherichia coli using Mtb-specific CD4+ Tcells. Using this technique, we identified afamily of highly relatei! Mtb antigens. Thegene of one family member encodes a 9.9-kD antigen, termed Mtb9.9A. RecombinantMtb9.9A protein, expressed and purifiedfrom E. coli, elicited strong T-cell prolifera-tion and IFN-y production by peripheralblood mononuclear cells from PPD+ butnot PPD– individuais. Southern blot analy-sis and examination of the Mtb genome se-qucnce revealed a family of highly related

genes. A T-cell line fruiu a PPD+ donor thatfailed to react with recombinant Mtb9.9Arecognized une of the other family mem-bcrs, Mtb9.9C. Synthetic peptides wereused to map the T-cell epitopc rccognizedby this line, and rcvealed a single aminoacid substitution in this region when com-pareci with Mtb9.9A. The direct idcntifìca-tion of antigens using T cells from immunedonors will undoubtcdly be criticai for thedevelopment of vaccines to several intracel-lular pathogens.—Authors' Abstract

Andersen, P. and Snwdegaard, B. CD4+T-cell subsets that mediate immunologi-cal memory to Mvcobacteriunn tubercu-losis infection in mice. Infect. Immun.68 (2000) 621-629.

We have studied CD4+ T cells that medi-ate immunological memory to an intra-venous infection with Mvcobacteriunr tu-berculosis. The studies were conductedwith a mouse model of memory immunityin which mico are rendered immune by aprima y infection followed by antibiotictreatment and rest. Shortly after reinfcction,tuberculosis-specific memory cells were re-cruited from the recirculating pool, leadingto rapidly increasing precursor frequenciesin the liver and a simultaneous dccrease inthe blood. A smal1 subset of the infiltratingT cells was rapidly activated (<20 hr) andexpressed high leveis of intracellulargamma interferon and the T-cell activationmarkers CD69 and CD25. These memoryeffector T cells expressed intermediate lev-els of CD45RB and were heterogeneouswith regard to the L-selectin and CD44markers. By adoptive transfer into nudemice, the highest levei of resistance to achallenge with M. tuberculosis was medi-ated by CD45RB(high), L-selectin(high),CD44(low) cells. Taken together, these twolines of evidence support an important rolefor memory cells which have reverted to anaive phenotype in the long-term protectionagainst M. tuberculosis.—Authors' Abstract

Appelberg, R., Leal, I. S., Pais, T. F., Pe-drosa, J. and Florido, M. Diffcrences inresistance of C57BL/6 and C57BL/10mice to infection by Mycobacterium

68, 2^ Current Literatura^ 225

al'irrnr are independent of gamma inter-feron. Infect. Immun. 68 (2000) 19-23.After infection with a low-virulence

strain of Mvcobacterium avim, C57BL/6and C57BL/10 coice had clear differencesin the-control of the infection in their liversand spleens. This difference in susceptibil-ity was not associated with differences inthe H-2 complex. It was dependent on theactivity of CD4+ T cells but unrelated to theability of those cells to secrete gamma in-terferon or to the development of delayed-type hypersensitivity responses at 3 weeksof infection. It was associated with lowertotal numbers of CD4+ cells present in in-fected spleens and was related to an earlierinduction of protective T cells, as measuredby adoptive-transfer assays. These data fur-ther strengthen the notion of gamma-inter-feron-independent mechanisms of protec-tion against mycobacteria.—Authors' Ah-stract

AvGay, Y., Jamil, S. and Drews, S. J. Ex-pression and characterization of the Mr-cobacterirun tuberculosis serine/threo-nine protein kinase PKnB. Infect. Im-mun. 67 (1999) 5676-5682.PknB is a member of the newly discov-

ered eukaryotic-like protein serine/threo-nine kinase (PSTK) family of proteins. ThepknB gene was cloned and expressed in Es-cherichia coli. The active recombinant pro-tein was purified and shown to be reactivewith antiphosphoserine antibodies, as wellas with antibodies to the phosphorylated eu-karyotic Ser/Thr kinases, mitogen-activatedprotein kinase, kinase 3 and 6, P38, andCreb. In litro kinase assays demonstratedthat PknB is a functional kinase that is au-tophosphorylated on serine/threonine resi-dues and is also able to phosphorylate thepeptide substrate myelin basic protein.Analysis of pknB expression in Mvcobac-terium tuberculosis indicates the presenceof pknB mRNA in (a) organisms grown ir►vitro in bacteriological media, (b) a murinemacrophage ia nitro infection model, and(c) ia vivo alveolar macrophages from a pa-tient with tuberculosis.—Authors' Abstract

Banales, J. L., Rivera Martinez, E., PerezGonzalez, L., Selman, M., Raymond,

Y. and Nava, A. Evaluation of adenosinedeaminase activity in the Mvcobacteriunttuberculosis culture supernatants. Arch.Med. Res. 30 (1999) 358-359.Background. Adenosine deaminase

(ADA) catalyzes hydrolytic and irreversibledeamination of deoxyadenosine into de-oxyinosine and of adenosine into inosine,and is related to lymphocytie proliferationand differentiation. The measurement ofADA activity in body fluids is a useful toolin the evaluation of mycobacterial infec-tions. Elevated ADA activity has beenfound in pleural effusions of patients withpleural tuberculosis relative to those frompatients with nontuberculous pleural dis-eases, and is mainly associated with cellularhost factors such as monocyte-macrophagesor lymphocytes. In contrast, there is littleinformation about ADA activity measure-ment in mycobacteria culture supernatants.

Methods. We evaluated ADA activity asdescribed by Giusti in the culture super-natants of eight i%1 cobacterir un tubcrrulo-sis isolates.

Results. Mycobacteria culture super-natants did not display any ADA activity.

Conclusions. This result supports the no-tion that M. tuberculosis is not the source ofADA activity. However, increased ADA ac-tivity in biological fluids from tuberculosispatients might be due to the interaction ofthe mycobacterium with host factors.—Au-thors' Abstract

Bodman, 1'., Miltner, E. and Bermudez,L. E. Mvcobacterium aviam resists ex-posure to the acidic conditions of thestomach. FEMS Microbiol. Lett. 182(2000) 45-49.Or<eanisms of the Mvcobacterium a►'ium

complex are common pathogens immuno-suppressed patients such as individuais withAIDS. There is evidence that in AIDS pa-tients, the main route for M. aviam infectionis the gastrointestinal tract. The stomach isa formidable barrier to pathogens and theability to resist exposure to pl1 lower than 3has been shown to be a virulence determi-nant of enteric pathogens. Incubation ofthree clinicai isolates of M. al'iunl underacidic pH revealed resistance of M. aviamgrown both to the exponential and station-

226^ International.101njnal o/'Lepsos.v^ 2000

ary phase at p1-1 2.2 for 2 hr. Inhibition ofprotein synthesis had no effect ort lhe acidtolerance. When the duration of the incuba-tion at pH 2.2 was ex[ended to 24 hr, bacte-ria grown to the stationary phase hacl a sig-nificantly greater tolerance to acid than ex-ponential phase bacteria. /19. chilrarincubated with acid ia the presence of waterwas significantly more resistam to p1-1 2.2than M. avim)! ia the presence of huffer.Pre-adaptatloa ia \valer prior to exposure toacidic conditions was also associated withincreased resistance to pH 2.2. Isoosmolar-ity of Hanks balanced salt solution appearsto be responsible for the inlpaired resistanceto acid between 2 and 24 lir of incuba[ion.These findings indicate that M. ai'iuln isnaturally toleram [o pl-1 <3 and that pre-adapta[ion under conditions similar to theconditions where M. arinnl is found ira theenvironment results ia increased acid resis-tance.—Authors' Abstract

Brandt, L., Elhay, M., Rosenkrands, 1.,Lindblad, E. B. and Andersen, P.ESAT-6 subunit vaccination against Mv-cobacteriunl tuberculosis. Infect. 1m-mun. 68 (2000) 791-795.The ESAT-6 anligen from Mvcobac-

terium tnberculosis is a dominar[ target forcell-mediated immunity ia the early phaseof tuberculosis (TB) ia TB pa[ients as Oveslas ia various animal models. The purposeof our study was to evaluate the potencial ofESAT-6 ia an experimental TB vaccine. Westarted out using dimethyl dioctadecylam-monium bromide (DDA), an adjuvantwhich lias bem] demonstra[ed to be efficientfor the induction of cellular immune re-sponses and lias been used successfully be-fore as a delivery system for TB vaccines.Here we demonstrate that, whereas immuneresponses to both short-term-culture tiltrateand Ag85B are efficiently induced withDDA, Chis adjuvant was inefficient for theinduction of immune responses to ESAT-6.Therefore, we investigated the modulatoryeffect of monophosphoryl lipid A (MPL),an immunomodulator which ia differentcombinations has demonsl ated strong ad-juvant activity for both cellular and hu-moral immune responses. We show in thepresent study that vaccination with ESAT-6delivered in a combination of MPL and

DDA elicited a strong ESAT-6-specificT-cell response and protective immunitycomparable to that achieved with M. boi'isBCG. Authors' Abstract

Camacho, L. R., Ensergueix, D., Perez,E., Gicyucl, B. and Guilhot, C. [dentifi-cation of a virulence gene cluster of Mv-cohuc•teriunl tuberculosis by signalure-tagged transposon mutagenesis. MoI.Microbiol. 34 (1999) 257-267.Tuberculosis remains the greatest cause

of death Ovorldwide chie to a singlepathogen. In order [o identify the genes re-quired for lhe paihogenicity of Mvcohac-tel'illlll uIhcrculo.vis', a functional genonlicapproach was developed. A library of signa-ture-[ag`ged ll'alltiposon mntants of this bac-terium was constructed and screened forthose affected ira their multiplication withinthe lungs of mice. From 1927 mutantstested, 16 were attenuated for their viru-lence. The insertions harbored by the se-Iected nurlants were mapped ora lhe Al. tu-berculosi.v genome and most of the mutatedsoei appeared to be involved ia lipid metab-olism or transpor[ across the membrane.Four independent mutations identified acluster of virulence genes located ora a 50kb chromosomal region. These genes mightbe involved ia the production of phthioceroland phenolphthiocerol derivali 'es, a groupof molecules restricted to eight mycobacte-rial species, seven of them being ei[herstrict or opportunistic pathogens. The inter-action of tive nlutant strains with mousebone marrow nlacrophages was investi-gateei. These tive nlutan[s were still able tomultiply in this cell [ype. However, ia theeecases, there was a grow[h defect in compar-ison with the wild-type strain. The othertwo strains exhibited no clear differencefrom the virulent strain, MT103, in thismodel. This study, which is the first globalresearch of virulence factors of M. tubercu-lo.vi.v, operas the way to a better understand-ing of the molecules that are key players irathe interac[ion of chis pa[hogen with itshost.—Authors' Sununary

Chetchotisakd, P., Mootsikapun, P.,Anunnatsiri, S., Jirarattanapochai, K.,Choonhakarn, C., Chaiprasert, A.,

68, 2^ Current Literature^ 227

Ubol, P. N., Wheat, L. J. and Davis, T.E. Dissemination infection due torapidly growing mycobacteria in im-munocompctent hosts preventiog withchronic lymphadenopathy: a prcviouslyunrecognized clinicai entity. Clin. Infect.Dis. 30 (2000) 29-34.Disseminated infection due to rapidly

growing mycobacteria is uncommon andoccurs mosily in immunocompromised pa-tients. We report 16 cases of such infectionwith an unusual presentation seen at Srina-garind Hospital, a university hospital innortheastern Thailand. The clinicai featureswere different from those in previous re-ports. All of the patients presented withchronic bilateral cervical lymphadenopathy.Twelve had mycobacteria) involvement ofother organs (sinuses, 6 patients; lungs, 4;liver, 4; spleen, 3; skin, 3; bone and joint, 2;and tonsils, 2). An interesting occurrence in11 patients was 14 episodes of reactive skinmanifestations (Sweet's syndrome, 9; gen-eralized pustulosis and erytheina nodosum,2 each; and pustular psoriasis, 1). No iden-titiable predisposinsy, factors, including hu-man inununodeficiency disease, were foundin these patients. 1-lowever, 8 patients had11 episodes of prior infection or coinfectionwith other opportunistic pathogens (salmo-nellosis, 4; penicilliosis, 3; pulmonary tu-berculosis, 2; and melioidosis and crypto-coccosis, 1 each). These findings suggestthat cell-mediated immunity is defective inthese patients.—Authors' Abstract

Colangeli, R., Spencer, J. S., Bifani, P.,Williams, A., Lyashchenko, K., Keen,M. A., Hill, P. J., Belisle, J. and Gen-naro, M. L. MTSA- 10, the product ofthe Rv3874 gene of Mvcobacteriunn tu-berculosis, elicits tuberculosis-specific,delayed-type hypersensitivity in guineapigs. Infect. Immun. 68 (2000) 990-993.in a search for new skin-test reagents

specific for tuberculosis, we found that theantigen encoded by gene Rv3874 of My-cobacteriu,n tuberculosis elicited delayed-type hypersensitivity in M. tuberculosis-in-fected guinea pigs but not in control ani-mais immunized with M. bovis bacillusCalmette-Guerin (BCG) or M. aviam. Theantigen, which was named MTSA-10 (for

M. tuberculosis-specific antigen 10), is aprime candidate for a component of a newtuberculin that will allow discrimination bya skin test of latent M. tuberculosis infec-tion from vaccination with BCG or fromsensitization with environmental, nontuber-culous mycobacteria.—Authors' Abstract

Datta, M., Vallishayee, S. R. S., and Di-wakara, S. A. M. Fifteen year follow upof trial of BCG vaccines in South Indiafor tuberculosis prevention. Indian J.Med. Res. 110 (1999) 56-69.A large-scale, community-based, double-

blind, randomized controlled trial was car-ried out in the Chingleput district of SouthIndia to evaluate the protective effect ofBCG against bacillary fornis of pulmonarytuberculosis. Fron1 among 366,625 individ-uais registered, 281,161 persons were vac-cinated with BCG or placebo by random al-location. Two strains of BCG were used,the French and Danish, with a high dose(0.1 mg/0.1 ml) and a low dose (0.01 mg/0.1ml) in each strain. The entire populationwas followed up for 15 years by means ofresurveys every 30 months, and selectivefollow up every 10 months and continuouspassive case finding. There were 560 cases(189, 191 and 180 from the high dose, lowdose and placebo groups, respectively) aris-ing over 15 years anions2 109,873 personswho were tuberculin negative and had anormal chest X-ray at intake. This repre-senta a small fraction of the total incidenceof 2.6 per 1000 person-years, most of whichcarne from those who were initially tuber-culin positive. The incidente rates in thethree "vaccination" groups were similar,confirming the complete lack of protectiveefficacy seen at the end of 7'/ years. BCGoffered no overall protection in aduits and alow levei of overall protection (27%; 95%C.I. –8% to 50%) in children. This lack ofprotection could not be explained bymethodological flaws, or the influente ofprior sensitization by nonspecific sensitiv-ity, or because most of the cases arose as aresult of exogenous re-infection. The find-ings at 15 years show that in this populationwith high infection rates and high nonspe-cific sensitivity, BCG did not offer any pro-tection against adult forms of bacillary pul-monary tuberculosis.—Authors' Abstract

228^ International Journal o/ I_epros.r^ 2000

Demissie, A., Ravn, P., Olobo, .1., I)o-herty, 'I'. NI., Eguale, T., Geletu, NI.,Ilailu, W., Andersen, P. and Britton, S.T-cell recognition of Mvcohactcrium tu-bercu losis culture filtrate fractions in tu-berculosis patients and their householdcontacts. Infect. Immun. 67 (1999)5967-5971.We examined the immune responses of

patients with active pulmonary tuberculosis(TB) and their healthy household contactsto short-terra culture filtrate (ST-CF) of Mv-cohacterium tuberculosis or molecularmass fractions derived from it. Our goalwas to identify fractions strongly recog-nized by donors and differences among thedonor groups of possible relevance for vac-cine development. The study populationconsisted of 65 human immunodeficiencyvirus-negative donors from the HossanaRegional Hospital, Hossana, Ethiopia. Pe-ripheral blood leukocytes from the donorswere stimulated with different antigens andimmune responses were determined.Household contacts produced si`gniticantlyhigher leveis of gamma interferon (IFN-y)than the TB patients in response to antigenspresent in ST-CF and the 10 narrow-moiec-ular-mass fractions. A similar difference inleukocyte proliferative responses to theantigens between the two groups was alsofound. In general, while ali fractions stimu-lated immune responses, the highest activ-ity was seen with the low-molecular-massfractions, which include well-defined TBantigens such as ESAT-6. Leukocytes fromcontacts of TB patients with severe diseaseproduced higher leveis of antigen-specificIFN-y than those from contacts of patientswith minimal disease. Both groups of con-tacts exhibited higher cell-mediated re-sponses than the patients themselves. Theenhanced immune response of healthy con-tacts, especially those of patients with se-vere disease, to secreted mycobacteria) anti-gens is suggestive of an eariy stage of in-fection by M. tuberculosis, which could intime result in overt disease or containmentof the infection. This possibility is currentlybeing investigated by follow-up studies ofthe household contacts.—Authors' Abstract

De Smet, K. A. L., Kempsell, K. E., Gal-lagher, A., Duncan, K. and Young, D.

B. AIleration of a single amino acidresidue reverses fosfomycin resistance ofrecombinant MurA from Msrobacteriunrtuberculosis. Microbiology 145 (1999)3177-3184.Mvcobacterium tuberculosis lias in na te

resistance to a range of broad-spectrum an-timicrobial agents. This may in part reflectthe relative impermeability of the mycobac-terial ccll wall, but additional specificmechanisms may also be important. In thecase of fosfomycin, it lias been suggestedthat a key difference in the active cite of theM. tuberculosis MurA enzyme might conterresistance. In Escherichia coli, fosfomycincovalently binds to a cysteine normally in-volved in the enzymic activity, while pro-tela alignments predict an aspartate at thisposition in the M. tuberculosis MurA. In thepresent study, it is demonstrated that thewild-type M. tuberculosis MurA is indeedresistam to fosfomycin, and that it becomessensitive following replacement of the as-partate residue in position 117 by a cys-teine. In addition, the study illustrates theuse of an inducible expression system inmycobacteria to allow functional character-ization of an M. tuberculosis enzyme that isunstable during constitutive expression.-Authors' Abstract

De Voss, .1..1., Rutter, K., Schroeder, B.G., Su, H., Zhu, Y. Q. and Barry, C. E.The salicylate-derived mycobactin si-derophores of Mvcobacteriunr tuberculo-sis are essential for growth in macro-phages. Proc. Natl. Acad. Sci. U.S.A. 97(2000) 1252-1257.Mvcobacteriunl tuberculosis is an impor-

tant pathogen of mammals that relies on2-hydroxyphenyloxazoline-containing si-derophore molecules callcd mycobactinsfor the acquisition of iron in the restrictiveenvironment of the mammalian macro-phage. These compounds have been pro-posed to be biosynthesized through the ac-tion of a cluster of genes that include bothnonribosomal peptide synthase and polyke-tide synthase components. One of thesegenes encodes a protein, MbtB, that puta-tively couples activated salicylic acid withserine or threonine and then cyclizes thisprecursor to the phenyloxazoline ring sys-

68, 2^ Curre nt Lite ran u re^ 229

tem. We have used gene replacementthrough homologous recombination todelete the mbtB gene and replace this witha hygromycin-resistance cassette in the vir-ulent strain of M. tubcrrulosis H37Rv. Theresulting mutant is restricted for growth iniron-limited media but grows normally iniron-repleto media. Analysis of siderophoreproduction by this organism revealed thatthe biosynthesis of ali salicylate-derivedsiderophores was interrupted. The mutantwas found to be impaired for growth inniacrophage-like THP-1 cells, suggestingthat siderophore production is required forvirulence of M. tuberculosis. These resultsprovide conclusive evidence linking thisgenetic locus to siderophore production.-Authors' Abstract

Ehlers, S., Kutsch, S., Benini, J., Cooper,A., Hahn, C., Derdes, ,J., Orme, 1.,Martiu, C. and Rietschel, E. 'I'. NOS2-derived nitris oxide regulates the size,quantity and quality of granuloma for-mation in Mvcobacteriuwn uricuu-in-fected mice without affecting bacterialloads. Immunology 98 (1999) 313-323.

Granuloma formation in response to my-cobacterial infections is associated with in-creased expression of inducible nitric oxidesynthase (NOS2) within granuloma macro-phages and 1ncreased leveis of . nitrate/nitritein the sera of infected mice. Continuoustreatment with 5 mM or 10nmM L-N-6-(1-imino-ethyl)-lysine (L-NIL), a selectiveNOS2-inhibitor, in acidified drinking waterfor up to 7 weeks consistently reduced in-fection-induced nitrate/n1trite to back-ground leveis in mycobacteria-infectedBALB/c mice. Oral treatment with 5 mML-NIL initiated at the time of infection si<g-nifìcantly exacerbated growth of Mvcobac-terium tuberculo.tiis, but had no effect on M.avim colony-forming unit development inthe liver, spleen and lungs of intravenouslyinfected mice. In order to examine the roleof nitric oxide in mycobacteria-inducedgranulomatous 1nllammation in the absenceof any effect on the bacterial load, M.ai'ium-infected mice were treated with 5mM L-NIL from day 1 through 38 and thedevelopment of granulomatous lesions inthe liver was assessed by histology, im-nulnohistology and reverse-transcription-

polymerase chain reaction (RT-PCR). Com-puter- and video-assisted molphometly per-formed at 4 and 7 weeks post-infectionshowed that treatment with L-NIL led tomarkedly 1ncreased number, cellularity andsize of granulomatous lesions in infectedcoice regardless of the virulence of the M.afiam isolate used for infection. Imnulno-histology of the liver revealed that in coicetreated with L-NIL, the numbers of CD3+ Tcens, CD21/35+ B eel ls, CD 1 1 b+ macro-phages and RB6-8C5+ granulocytes assoei-ateei with granulomatous lesions were in-creased. RT-PCR of the liver showed that inL-NIL-treated mice infected with M. anil ou,mRNA leveis of tumor necrosis factor, in-terleukin-12p40, interferon-gamma, inter-leukin-10 and interferon-gamma-inducibleprotein-10 (IP-IO) were upregulated, whilemRNA leveis of interleukin-I I, monocytechemotactic protein-1 (MCP-1) and MCP-5were similar to those in untreated controlinfected mice. When M. anium-infectedmice were treated with 5 mM L-NIL be-tween the 5111 and 121h weeks of infection,similar changes in granuloma number andsize were found in the absence of any effect00 the bacterial load. These findingsdemonstrate that nitric oxide regulates thenumber, size and cellular composition of M.anium-induced granulomas independentlyof antibacterial effects by modulating thecytokine prolile within infected tissues.-Authors' Abstract

Ehrenpreis, E. D., Kane, S. V., Cohen, L.B., Cohen, R. D. and Hanauer, S. B.Thalidomide therapy for patients with re-fractory Crohn's disease: an open-labeltrial. Gastroenterology 117 (1999) 1271-1277.

Background & Aims: Inhibition of tumornecrosis factor is a proposed mechanism forthe anti-ioilammatory properties of [balido-mide. We performed an open-label mal ofthalidomide in refractory Crohn's disease.

Methods: Twenty-two patients with re-fractory Crohn's disease (Crohn's DiseaseActivity Index [CDAI[ >200 and/or drain-ing perianal disease) initiated therapy withthalidomide, 200 mg at bedtime (18 pa-tients), or 300 mg at bedtime (4 patients).CDAI and goal interval scores (GIS) wereassessed at weeks 0, 4, and 12. Clinicai re-

230^ International Journal of Leprosy^ 2000

sponse for patients with luminal diseasewas defined as reduction in CDAI score of>150 points and for fistula patients was twoscores of greater than or equal to 1+ in theeeparameters of the GIS. Clinica) remissionwas define(' as a total CDAI <150 (luminalpatients) or ?2+ for ali parameters of theGIS (fistula patients).

Results: Nine patients with luminal dis-case and 13 with fistulas (16 mate, 6 fe-male) were enrollcd. The mediar CDAIscore at entry was 371 (95-468). Sixteenpatients compieted 4 weeks of treatment(12 clinica) responses, 4 clinica) remis-sions). Ali 14 patients completing 12 weeksmet criteria for clinicai response. Nineachieved clinica) remission (3 luminal, 6fistula patients). The mediar CDAI scorewas 175 (30-468; p <0.001 vs. baseline).

Conclusions: Thalidomide is effìcaciousin sorne patients with reiractory Crohn'sdisease.—Authors' Abstract

Eisen, 1'., Boshoff, C., Mak, I., Sapunar,F., Vaughan, M. M., Pyle, L., John-ston, S. R. D., Ahern, R., Smith, 1. E.and Gore, M. E. Continuous low dosethalidomide: a phase II study in ad-vanced melanoma, renal ccll, ovarianand breast cancer. Br. J. Cancer 82(2000) 812-817.To grow and metastasize, solid tumors

must develop their own blood supply byneo-angiogenesis. Thalidomide inhibits theprocessing of mRNA encoding peptidemolecules including tumor necrosis factor-alpha (TNF-y) and the angiogenic factorvascular endothelial growth factor (VEGF).This study investigated the use of continu-ous low-dose thalidomide in patients with avaricty of advanced malignancies. Sixty-sixpatients (37 women and 29 men; medianage, 48 years; range 33-62 years) with ad-vanced measurable cancer (19 ovarian, 18renal, 17 melanoma, 12 breast cancer) re-ceived thalidomide 100 mg orally everynight until disease progression or unaccept-able toxicity was encountered. Three of 18patients with renal cancer showed partia)responses and a further 3 patients experi-enced stabilization of their disease for up to6 months. Although no objective responseswere seen in the other tumor types, therewere significant improvements in patients'

sleeping (p <0.05) and uulintained appetite(p <0.05). Serum and urine concentrationsof basic libroblast growth factor (bFGF),TNF-y and VEGF were mcasured duringtreatment and higher leveis were associatedwith progressive disease. Thalidomide waswell tolerated: 2 patients developed WI-IOgrade 2 peripheral neuropathy and 8 pa-tients developed WI-lO grade 2 lethargy.No patients developed WI-1O grade 3 or 4toxicity. Further studies evaluating the useof thalidomide at higher doses as a singleagem for advanced renal cancer and incombination with biochemotherapy regi-mens are warranted.—Authors' Abstract

Fernandez Roblas, R., Esteban, J.,Cabila, F., Lopez, ,1. C., ,limenez, M.S. and Soriano, F. in nitro susceptibili-ties of rapidly growing mycobacteria totelithromycin (HMR 3647) and sevenother antimicrobials. Antimicrob. AgentsChemother. 44 (2000) 181-182.The antimicrobial activities of telithro-

mycin (HMR 3647) and seven other antimi-crobials against 94 strains of rapidly growingmycobacteria were determined. Telithro-mycin at a concentration of 1 µg/ml inhib-ited Mvcobacte riam peregrimnn (10O%),M. chelonae (80%), M. abscessos-M. muco-genicun (44.4%), and M.,fnrtuimn (2.1%).AII or most strains of M. pere,ç'rin un t, M.fortuitum, and M. nncogenicun were inhib-ited by 2 of quinolones per ml.—Au-thors' Abstract

George, K. M., Pascopella, L., Welty, D.M. and Sma11, P. L. C. A Mycobac-terium ulccrans toxin, mycolactone,causes apoptosis in guinea pig ulcers andtissue culture cells. Infect. Immun. 68(2000) 877-883.Mycobacterium ulcerans is the causative

agent of Buruli ulcer, a tropical ulcerativeskin disease. One of the most intriguing as-pects of this disease is the presence of ex-tensive tissue damage in the absence of anacute inflammatoly response. We recentlypurified and characterized a macrolidetoxin, mycolactone, from M. ulccrans. In-jection of this molecule into euinea pig skinreproduced cell death and lack of acute in-flammatory response similar to that seen

68,2^

Curro nt Literature^ 231

following the injection of viable bacteria.We also showed that mycolactone causes acytopathic effect on nlouse libroblast L929cells that is characterized by cytoskeletalrearrangements and growth arrest within 48hr. I-iowever, these results could not ac-count for the extensive cell death which oc-curs in Buruli ulcer. The results presentedhere demonstrate that L929 and J774mouse macrophage cells die via apoptosisafter 3 to 5 days of exposure to mycolac-tone. Treatment of cells with a pan-caspaseinhibitor can inhibit mycolactone-inducedapoptosis. We demonstrate that injection ofmycolactone into guinea paz skin results incell death via apoptosis and that the extentof apoptosis increases as the lesion pro-gresses. These results may help to explainwhy tissue damage in Buruli ulcer is not ac-companied by an acate inflammatory re-sponse.—Authors' Abstract

Glatman hreedman, A., Mednick, A. J.,Lendvai, N. and Casadevall, A. Clear-ance and organ distribution of Mvcohac-teriunl tuberculosis lipoarabinomannan(LAM) in the presence and absence ofLAM-binding immunoglobulin M. In-fect. Immun. 68 (2000) 335-341.Lipoarabinomannan (LAM) is a compo-

nent of the mycobacterial surface which hasbeen associated with a variety of deleteri-ous effects on immune system function.Despite the importance of LAM to thepathogenesis of mycobacterial infection,there is no infornmation available on its falein vivo. In Chis study, we determined thepharmacokinetics and tissue distribution ofexogenously administered LAM in mice.For measurements of serum and tissueLAM concentrations, we developed an en-zyme-linked immunosorbent assay whichused monoclonal antibodies of differentisotypes to capture and detect LAM at con-centrations of >_0.4 tg/ml. Int avenous ad-ministration of LAM to mice resulted intransient serum leveis with organ deposi-tion in the spleen and in the liver. Immuno-histochemical studies localized LAM to thespleen marginal zune macrophages and, toa lesser degree, to tiver macrophages. WhenLAM was administered to mice previouslygiven a LAM-binding in ununoglobulin M(IgM), LAM was very rapidly clearcd from

circulation. In those mice, deposition ofLAM in the spleen was signifìcantly re-duced while LAM deposition in the liver in-creased. Administration of LAM-bindingigM resulted in significam leveis of IgM toLAM in bile consistent with an increasedhepatobiliary excretion of LAM in the pres-ence of specific antibody. Bile, tiver ex-tracts, and bile salts were found to rapidlyinactivate the immunoreactivity of LAM.The results indicate that serum clearanceand organ deposition of LAM in mice areaffected by the presence of LAM-bindingantibody and suggest a mechanism bywhich antibody could modify the course ofmycobacterial infection.—Authors' Ab-stract

Harth, G., Zameenik, P. C., Tang, J. Y.,'I'abatadze, U. and Horwitz, M. A.Treatment of Mvcobacteriurn tuberculo-sis with antisense oligonucieotides toglutamine synthetase mRNA inhibitsglutanmine synthetase activity, formationof the poly-L-glutamate/glutamine cellwall structure, and bacterial replication.Proc. Nat. Acad. Sci. U.S.A. 97 (2000)418-423.New antibiotics to combat the emerging

pandemic of drug-resistant si gins of Mv-cobacterium tuberculosis are urgentlyneeded. We have investigated the effects onM. tuberculosis of phosphorothioate-modi-fied antisense oiigodeoxyribonucleotides(PS-ODNs) against the mRNA of gluta-mine synthetase, an enzyme whose exportis associated with pathogenicity and withthe formation of a poiy-L-glutamate/gluta-mine cell watt structure. Treatment of viru-lent M. tuberculosis svith 10 µm antisensePS-ODNs reduced glutanmine synthetase ac-tivity and expression by 25'7-50% depend-ing on whether une, two, or three differentPS-ODNs were used and the PS-ODNs'specific target sites on the mRNA. Treat-ment with PS-ODNs of a recombinantstrain of M. snlegnnatis expressiog M. tuber-culosis glutamine synthetase selectively in-hibited the recombinant enzyme but not theendogenous enzyme for which the mRNAtranscript was mismatched by 2-4 nt. Treat-ment of M. tuberculosis with the antisensePS-ODNs also reduced the amount of poly-L-glutamate/glutamine in the cell watt by

232^ International.lonr7 u11 of. Lepros_v^ 2000

24%. Finally, treatment \vith antisense PS-ODNs reduccd M. tuberculosis erowth by0.7 Iogs (1 PS-ODN) to 125 logs (3 PS-ODNs) but had no effect on the growth ofM. s^uc^^matis, which does not export gluta-mine synthetase nor possess the poly-L glu-tamate/glutamine (P-L lx) cell \vali struc-ture. The experimenta indicate that the a n ti-sense PS-ODNs enter the cytoplasm of M.tuberculosis and bind to their cognate tar-gets. Although more potent ODN technol-ogy is needed, this study demonstrates thefeasibility of usine antisense ODNs in theantibiotic armamcntarium against M. tuber-culosis.—Authors' Abstract

Hess, J. and Kaufmann, S. H. E. Devel-opment of novel tuberculosis vaccines.C. R. Acad. Sci. III 322 (1999) 953-958.Efficacious control of tuberculosis (TB),

one of the world's major health threats, isbest achieved by a combination of chemo-therapy and vaccination. The current vac-cine, BCG, fails to preveni pulmonary TBin adults, which is the most prevalent formof this disease. Consequently, the design ofnovel vaccines against TB is urgently re-quired. Because the acquired immune re-sponse is mediated by different T-cell sets,an optimal combination of these popula-tions must be stimulated. Since one third ofthe world's population is already infectedwith Mycobacterium tuberculosis, twotypes of vaccine may be required: one foreradication of already established infectionand the other for prompt combat of invad-ing microbes. A rational judement on theefficacy of the different types of vaccinecurrently under development needs to awaitfurther evaluation.—Authors' Abstract

Hou, W., Wu, Q.-X., Yu, H., Yin, Y.-P.,Zhang, Z.-P. and Cai, X.-L. [Evalua-tion of Tb-NT-P-BSA antigen in theserodiagnosis of tuberculosis.] J. Zoo-noses 15 (1999) 43-44. (in Chinese)Sera from 40 tuberculous patients and 52

healthy controls fin China] were assayed byELISA using Tb-NT-P-BSA (containing theterminal triglycosyl unit of the Al. tubercu-losis phenolic glycolipid) as antigen. Thesensitivity and specificity were 94.2%, 20%for IeG and 79.6%, 45% for IgM antibody

activity, respectively. The results show thatthe antigen is not sensitive, which will limitits use in the serodiagnosis of tuberculo-sis.—Trop. Dis. Bull. 96 (1999) 1 154

Huang, K. L., Chang, D. M. and Lu, J. J.Tuberculosis of skeletal muscle in a caseof polymyositis. Scand. J. Rheumatol. 28(1999) 380-382.We describe a patient with polymyositis

receiving corticosteroid therapy, who pre-sente(' with persistem fever and mass lesionat the left thigh. Surgical exploration andmycobacterial cultue proved to be My-cobucteriu/n tuberculosis infection involy-ing the semitendinous muscle of the leftthigh. Suitable sureical debridement, anti-TB medications, and sufficient cortico-steroid administration resulte(' in a goodcontrol of both polymyositis and the tuber-culous infection.—Authors' Abstract

Keane, ,i., Remold, H. G. and Kornfeld,H. Virulent Mvcobacterii un tuberculosisstrains evade apoptosis of infected alve-olar macrophages. J. Immunol. 164(2000) 2016-2020.Human alveolar macrophages (AmO) un-

dergo apoptosis following infection withMvcobacteriuni tuberculosis in vitro. Apop-tosis of cells infected with intracellularpathogens may benefit the host by eliminat-ing a supportive environment for bacterialgrowth. The present study compared Anapoptosis following infection by M. tuber-culosis complex strains of differing viru-lence and by Al. kansasii. Avirulent or at-tenuated bacilli (Al. tuberculosis H37Ra,Al. bovis bacillus Calmette-Guerin, andM. kansassii) induced significantly moreAMO apoptosis than virulent strains (M. tu-berculosis H37Rv, Erdman, Al. tuberculosisclinicai isolate BMC 96.1, and Al. boviswild type). Increased apoptosis was not dueto greater intracellular bacterial replicationbecause virulent strains grew more rapidlyin AMO than attenuated strains despitecausing less apoptosis. These findings sug-eests the existence of mycobacterial virulencedeterminants that modulate the apoptotic re-sponse of AMO to intracellular infectionand support the hypothesis that macrophage

68, 2^ Current Literature^ 233

apoptosis contributos to innate host defensein tuberculosis.—Authors' Abstract

Kim, T. S., Chung, S. W., Kang, B. Y.,Choe, Y. K. and Hwang, S. Y. Inductionof in vivo persistem anti-mycobacterialactivity by interferon-gamma-secreting fi-broblasts. Vaccine 18 (2000) 1067-1073.To determine whether the paracrine se-

cretion of interferon-gamma (1FN-y) can ef-ficiently stimulate the resistance to Myco-bacteriunt aram! complex (MAC) infec-tion, 3T3 fibroblasts were stably transducedto secrete IFN-y (500 units/10 6 cells/48 hr)and their effects on MAC infection were in-vestigated in genetically susceptible BALB/e coice, compared with that of free recombi-nant IFN-y (rIFN-y). lnununization withIFN-y-secreting fibroblasts (3T3-IFN-y)during intranasal infection with MAC re-sulted in a significam decrease in bacterialload of lung during the estire 8-week obser-vation period, while rIFN-y reduced thebacterial load at initial 1 week but not by 8weeks postinfection. Furthermore, immu-nization with the 3T3-IFN-y cells inducedand maintained signifìcantly higher leveisof cytotoxic activity and nitric oxide pro-duction by lung cells than those of rIFN-yimmunizatien. This work suggests thatIFN-y-secreting fibroblasts may serve as avehicle for paracrine secretion of IFN-y inimmunotherapy of MAC infection. Au-thors' Abstract

Kirschner, R. A., Parker, B. C. and Falk-inham, J. O. Humic and fulvic acidsstinu►late the growth of Mycobacteriumavium. FEMS Microbiol. Ecol. 30(1999) 327-332.Mvcobacteriu,n aviam, an environmen-

tal, opportunistic pathogenic mycobac-terium, has been isolated frequently and inhigh numbers from waters in Finland andfrom acid, brown water swamps of thesoutheastern coastal U.S.A. M. avium hasalso been recovered in high numbers fromFinnish drinking water and frequently iso-lated from Finnish AIDS patients. Borealforests and brown water swamps are similarin that they are rich in humic and fulvicacids and of low pH and dissolved oxygen.Growth of representative isolates of M.

at'iom in natural water was stimulatedmarkedly by the addition of humic and ful-vic acids. Further, the M. ariuni isolatesgrew at pH leveis as low as 4.0 and at oxy-gen leveis equal to 4% of atmospheric lev-els. The high numbers of M. aviou, in bo-real waters and brown water swamps arelikely due to their ability to proliferate inthose humic- and fulvic-rich, acidic, micro-aerobic environments.—Authors' Abstract

Kusner, D. J. and Adams, J. ATP-inducedkil ling of virulent Mvcobacterium tuber-culosis within human macrophages re-quires phospholipase D. J. Immunol. 164(2000) 379-388.The global dissemination of antibiotic-

resistant M cobacterium tuberculosis hasunderscored the urgent need to understandthe molecular mechanisms of immunity tothis pathogen. Use of biological im-munomodulatory compounds to enhanceantituberculous therapy has been hamperedby the limited effìcacy of these agents to-ward infected human macrophages and lackof infornmation regarding their mechanismsof activity. We tested the hypotheses thatextracellular ATP (ATP') prometes killingof virulent M. tuberculosis within humanmacrophages, and that activation of a spe-cific macrophage enzyme, phospholipase D(PLD), functions in this response. ATP`treat nent of infected monocyte-derivedmacrophages resulted in 3.5-log reductionin the viability of three different virulentstrains of M. tuberculosis. Stinullation ofmacrophage P2X7 purinergic receptors wasnecessary, but not sufficient, for maximalkilling by primary macrophages or humanTHP-1 promonocytes differentiated to amacrophage phenotype. Induction of tuber-culocidal activity by ATP` was accompa-nied by marked stimulation ef PLD activity,and two mechanistically distinct inhibitorsof PLD produced dose-dependent reduc-tions in ATP`-induced killing of intracellu-lar bacilli. Purified PLD restored centreileveis of mycobacterial killing to inhibitor-treated cells, and potentiated ATP`--depen-dent tuberculocidal activity in controlmacrophages. These results demonstratethat ATP' promotes killing of virulent M.tuberculosis within infected human macro-phages and strongly suggest that activation

234^ International Journal aFLeprosv^ 2000

of PLD plays a key role in this process.-Authors' Abstract

Leuenberger, R. and Bodmer, T. 1Clinicalpresentation and treatment of Mvcobac-teriunr marinum infection as seen in 12cases.' Dtsch. Med. Wochcnschr. 125(2000) 7-10. (in German)Background and Objective: Mvcobac-

terium marinum (M.m.) is the causativepathog^en of skin infections that have beencalled "swimming pool granulomas." Anincreasing number of reports that deepstructures are involved in these infectionswas the reason for studying the clinicai pre-sentation and response of the infection todifferent therapeutic regimens.

Patients and Methods: All patients (8men, 4 women, age range 18-73 years) wereincluded in whom, between January 1991and February 1995, M.m. infection hadbeen prover by culture. The clinicai data ofthese patients were retrospectively obtainedby standardized questionnaire.

Results: The infection was limited to theskin in 4 of the 12 patients, deep structuresonly were involved in 3, and 5 had both. In-fections Iimited to the skin were success-fully treated with sulfamethoxazole, andtrimethoprim or with tetracylcines, while ri-fampin, alone or in combination withethambutol, was efticacious when deepstructures were involved. No sureical inter-vention was—or should be—performed.

Conclusions: Infections with M.m. ofteninvolve deep structures, even in the absenceof the skin being involved. The term"swimminLY pool granuloma" is, therefore,misleading when the infection is limited tothe skin. A history of a chronic and indolentcourse, frequent changes of doctor andstriking polypharmacy in its treatment arepointeis to this infection.—Authors' En-glish Abstract

Lim, J. H., Park, J. K., ,Jo, E. K., Song,C. H., Min, D. L., Song, Y. J. and Kim,H. J. Purification and immunoreactivityof three components from the 30/32 kilo-dalton antigen 85 complex in Mvcobac-teriam tuberculosis. In fect. Immun. 67(1999) 6187-6190.The three proteins of the antigen 85 com-

plex (85A, 85B, and 85C), which are major

secretory products of Mvcohacterium tu-berculosis, were purified to honxogeneity inlarge amounts by a combination of chro-matography on hydroxylapatite, DEAE-Sepharose, and DEAE-Sephacel and gel 1iI-tration from M. tuberculosis culture fìltrate.Then we examined the immunological reac-tivity of the three proteins in tuberculosispatients and healthy controls. Antibody re-sponses to the 8513 and 85A proteins in pa-tients were significantiy greater than re-sponses to the 85C protein. 1n contrast, alithree antigens induced significant lympho-proliferation and gamma-interferon produc-tion in peripheral blood mononuclear censfrom healthy tuberculin reactors.—Au-thors' Abstract

Manabe, Y. C., Saviola, B. J., Sun, L.,Nlurphy, J. R. and Bishai, W. R. Atten-uation of virulence in Mvcohacterium tu-berculosis expressing a constitutively ac-tive irou repressor. Proc. Natl. Acad. Sci.U.S.A. 96 (1999) 12844-12848.Iron is an essential nutrient for the sur-

vival of most organisms and has played acentral role in the virulence of many infec-tions disease pathogens. MycobacterialIdeR is an iron-dependent repressor thatshows 80% identity in the functional do-mains with its corynebacterial homolog,DtxR (diphtheria toxin repressor). We havetransformed Mycobacterium tuberculosiswith a vector expressing an iron-indepen-dent positive dominant, corynebacterialdtxR hyperrepressor, DtxR (E175K). West-ern blots of whole-cell lysates of M. tuber-culosis expressing the dtxR (E175K) generevealed the stable expression of the mutantprotein in mycobacteria. BALB/c micewere infected by tail vein injection with 2 x1W organisms of wild type or M. tuberculo-sis transformed with the dtxR mutant. At 16weeks, there was a 1.2 log reduction in bac-terial survivors in both spleen (p = 0.0002)and lungs (p = 0.006) with M. tuberculosisDtxR (E175K). A phenotypic difference incolonial morphology between the twostrains also was noted. A computerizedsearch of the M. tuberculosis genome forthe palindromic consensus sequence towhich DtxR and IdeR bind revealed six pu-tative "iron boxes" within 200 bp of anORF. Using a gel-shift assay we showed

68, 2^ Current Literature^ 235

that purified DtxR binds to the operator re-gion of tive of those boxes. Attenuation ofM. tuberculosis can be achieved by the in-sertion of a plasmid containing a constitu-tively active, iron-insensitive repressor,DtxR (E175K), which is a homolog ofIdeR. Our results strongly suggest that IdeRcontrols genes essential for virulence in M.tuberculosis.—Authors' Abstract

Martinez, A., Torello, S. and Kolter, R.Sliding motility in mycobacteria. J. Bac-teriol. 181 (1999) 7331-7338.Mycobacteria are nontlagellated grani-

positive microorganisms. Previously thoughtto be nonmotile, we saw here that Mvco-bacterium smegmatis can spread on the sur-face of growth medium by a sliding mecha-nism. M. smegmatis spreads as a monolayerof cens which are arranged in pseudofila-ments by dose cell-to-cell contacts, pre-dominantly along their longitudinal axis.The monolayer noves away from the inoc-ulation point as a unir with only minor re-arrangenents. No extracellular structuressuch as p1li or tìmbriae appear to be in-volved in this process. The ability totranslocate over the surface correlates withthe presence of glycopeptidolipids, a my-cobacterium-specific class of amphiphilicmolecules located in the outermost layer ofthe cell envelope. We present evidence thatsurface motility is not restricted to M.smegmatis but is also a property of theslow-growing opportunistic pathogen M.ai'iiuu. This form of motility could play animportant role in surface colonization bymycobacteria in the environment as well asin the host.—Authors' Abstract

Miller, B. H. and Shinnick, M. Evalua-tion of Mvcobacterium tuberculosisgenes involved in resistance to killing byhuman macrophages. Infect. Immun. 68(2000) 387-390.A coinfection assay was developed to ex-

amine Mvcobacterimn tuberculosis genessuspected to be involved in resistance tokilling by human macrophages. THP-1macrophages were infected with a mixtureof equal numbers of recombinant M. smeg-matis LR222 bacteria expressing an M. tu-berculosis gene and wild-type M. smegma-

tis LR222 bacteria expressing the xylEgene. At various times after infection, theinfected macrophages were lysed and thebacteria were plated. The resulting colonieswere sprayed with catechol to determine thenumber of recombinant colonies and thenumber of xylE-expressing colonies. M.smegmatis bacteria expressing the M. tu-berculosis glutamine synthetase A (g1nA)gene or open reading frame Rv2962c orRv2958c demonstrated signifìcantly in-creased survival rates in THP-1 macro-phages relative to those of xylE-expressingbacteria. M. smegmatis bacteria expressingM. tuberculosis genes for phospholipase C(ptcA and plcB) or for high temperature re-quirement A (htrA) did not.—Authors' Ab-stract

Monastirli, A., Georgiou, S., Bolse'', K.,Pasmatzi, E., Papapanagiotou, A.,Goerz, G., Kalofoutis, A., Merk, H. F.and Tsambaos, D. Treatment of por-phyria cutanea tarda with oral thalido-mide. Skin Pharmacol. Appl. Skin Phys-iol. 12 (1999) 305-311.Eight mate patients with overt clinicai

and biochemical features of porphyria eu-tanea tarda (PCT) were orally treated with300 mg/day thalidomide for 1 week andwith 200 mie/day for 3 more weeks. Alreadyafter the first week of treatment no newvesicles and/or bullae could be observed.Spontaneous btisters completety disap-peared, increased skin fragility subsidedand skin hyperpigmentation receded about2 months after completion of therapy;whereas hypertrichosis persisted. There wasa rapid decrease in the urinary total por-phyrin excretion which reached normal lev-els in ali patients by the end of the fourthweek of therapy; whereas the post-treat-ment chromatographic pattern of urinaryporphyrins revealed a slight reduction ofhigher carboxylated porphyrin metabolitesand an increase in the amount of the ex-creted coproporphyrin, as compared to thepretreatment period. Somnolence, intermit-tent constipation and dry mouth occurred inali patients; two patients additionally experi-enced dizziness. No evidence of peripheralneuropathy could be detected and laboratoryinvestigations revealed no abnormalities, ascompared to the pretreatment period. Dur-

236^ International Journal of . Lepros.v^ 2000

ing the 16- to 28-month follow up of the pa-tients, no clinicai or biochemical relapsewas observed. In view of the encouragingresidis of the present investigation, furtherstudies are now warranted in orcler to deli-nitely answer the question whether oralthalidomide may be regarded as an effec-tive alternative approach to lhe trealment of .

PCT.—Authors' Abstract

Mulder, N. ,I., Powles, R. E., Zappe, H.and Steyn, L. M. The Mvcobacteriumtuberculosis mysB gene product is afunctional equivalem of the Eschericlriacoli sigma factor, KatF. Gene 240 (1999)361-370.Mycobacterium tuberculosis, the causa-

tive agent of tuberculosis, may remain dor-mant within its host for many years. Thenature of chis dornlant or latem state is notknown, but it may be a specialized forro ofthe stationary growth phase. In Escherichiacoli, KatF (or RpoS) is the major stationaryphase sigma factor regulating an array ofgenes expressed in this phase of growth. Apotencial M. tuberculosis katF homolog wascloned using a fragment of the E. coli katFgene as a probe. DNA sequence analysis ofa resultam clone showed 100% identity to afragment of DNA encoding the M. tubercu-losis mysA and mysB genes. Overexpres-sion of mysB in M. bor is BCG resulted inan increase in katG mRNA and catalase andperoxidase activity and an increase irr sensi-tivity of the cells to isoniazid. An increasein katG promoter activity from a reportervector was demonstrated when mysB wasoverexpressed from the same plasmid, indi-cating a direct relationship between MysBand katG expression.—Authors' Abstract

Murugasu Oci, B., Tay, A. and Dick, T.Upregulation of stress response genesand ABC transporters in anaerobic sta-tionary-phase Mvcobacterium smegnur-tis. Mol. Gen. Genet. 262 (1999)677-682.Oxygen starvation triggers an adaptive

stationary-phase response in Mvcobac-terimn smegmatis. During this anaerobicstationary phase, RNA synthesis continuesat a low but signiticant levei. Employing amodified expressed-sequence-tag (EST) ap-

proach in combination with the M. tubercu-losis genome data and comparati va Norlh-ern analysis, we have identilied the firstgenes that show an increase in I anscriptionin M. cells that have entered theanaerobic stationary phase. One gene en-codes the counterpart of the M. tuberculosisNif:S-like protein RN/1464. Two genes arehomologs of M. tuberculosis RN/1460 andRv3368c, of unknown function. Strikingly,several genes induced by oxygen starvationencode putative stress protection proteins(counterparts of M. tuberculosis DnaK,Rv0350; betaine-aldehyde dehydrogenase,Rv768; thioredoxin recluctase, Rv3913) andABC transporters (counterparts of M. tu-berculosis Rv1463, Rv1473, Rv3197). Weconclude that development of general stressresistance and cerlain active transportprocesses might play a role in the survivalof oxygen-starved M. siiRmatis.—Au-thors' Abstract

Nlustal'a, '1'., Phyu, S., Nilsen, R., Bjune,G. and Jonsson, R. Increased expres-sion of Fas ligand on Mvcobacterium tu-berculosis-infected macrophages a po-tencial novel mechanism of immune eva-sion by Mvcobacte riam tuberculosis?Inllammation 23 (1999) 501-521.We have studied the location and mecha-

nism of apoptosis within the granulomas inthe lungs at various stages of slowly pro-gressiva primary murine Mvcobacteriumtuberculosis infection. Parallel sectionswere analyzed for detection of mycobacte-rial antigens, Fas, and Fas ligand (FasL) byimmunohistochemist y, and for apoptoticcells by terminal deoxynucleotidyl-trans-ferase-mediated dUTP-digoxigenin nickend labeling (TUNEL) method. The fre-quency of apoptosis was high in the macro-phage aggregates as compareci to the lym-phocyte ageregates and at the interface be-tween them. Five to seven percent of thevacuolated macrophaues in the granulomasexpressed FasL intensely. These cells con-tained large amounts of mycobacterial anti-gens. These findings suggest that M. tuber-culosis infection can induce increased ex-pression of FasL in a population of infectedmacrophages. As a consequence the in-fected macrophages will be protected fromthe attack of cytotoxic T cells and activa-

68, 2^ Current Literature^ 237

tion of bactericidal mechanisms by Th 1type lymphocytes. This constitutes a novelevasion mechanism for M. tuberculosis,possibly explaining the chronic course ofinfection. Authors' Abstract

Mve Obiang, A., Remacle, ,1., I'alomino,.1. C., Houbion, A. and Portaels, F.Growth and cytotoxic activity by Mv-cobacteriuni ulceraras ira protein-free me-dia. FEMS Microbiol. Lett. 181 (1999)153-157.The pathogenic slow-growing Mvcobac-

terium ulceraras lias, until now, been cul-tural ira liquid media containing albumin.Here, we report the first description of useof Sauton medium and modified Reidmediam, two protein-free media ira whichM. ulceraras was able to grow and produceits toxin, a viajor virulence factor of this en-vironmental orranism which causes a skindisease commonly called Buruli ulcer.These results suggest that Sauton and mod-ified Reid media may be useful for certainfields of Al. ulceraras research requiringprotein-free growth conditions.—Authors'Abstract

Nasca, M. R., O'Toole, E. A., Palicharla,P., West, I). P. and Woodley, D. T.Thalidomide increases human kera-tinocyte migration and proliferation. J.Invest. Demudo'. 113 (1999) 720-724.Thalidomide is reported to have thera-

peutic utility in the treatment of pyodermagangrenosum, Behcet's disease, aphthousulcers. and skin wounds. We investigatedthe effect of thalidomide on human kera-tinocyte proliferation and migration, twoearly and criticai events in the re-epithelial-ization of skin wounds. Thalidomide atconcentrations less than 1 gm did not affectkeratinocyte viability. Using a thymidineincorporation assay, we found that thalido-mide, at therapeutic concentrations, in-duced more than a 2.5-fold increase ira theproliferative potential of the cens. Kera-tinocyte migration was assessed by two in-dependent motility assays: a colloidal goldassay and an ia nitro scratch assay. At opti-mal concentrations, thalidomide increasedkeratinocyte migration on a collagen matrixmore than 2-fold in the colloidal gold assay

and more than 3-fold ira the scratch assayover controls. Although pro-migratory,thalidomide did not alter the levei of metal-loproteinase-9 secreted finto culture me-dium. Thalidomide did, however, induce a2- to 4-fold increase ira keratinocyte-derivedinterleukin-8, a pro-migratooy cellular au-tocrine factor. Human keratinocyte migra-tion and proliferation are essential for re-ep-ithelialization of skin wounds. Interleukin-8increases human keratinocyte migration andproliferation and is chemotactic for kera-tinocytes. Thcrefore, thalidomide may modu-late keratinocyte proliferation and motilityby a chemokine-dependent pathway.—Au-thors' Abstract

Oliver, S. J., Freeman, S. L., Corral, L. G.,()campo, C. J. and Kaplan, G. Thalido-mide analogue CC 1069 inhibits develop-mem of rat adjuvant arthritis. Clin. Exp.Im mmunol. 118 (1999) 315-321.The cytokine tumor necrosis factor-alpha

(TNF-a) has been implicated ira the etiol-ogy of rheumatoid arthritis ira humans aswell as of experimental arthritis ira rodents.Thalidomide, and to a ereater extent thenew thalidomide analog CC 1069, inhibitsmonocyte TNF-a production both ira eive)and ia rimo. The aim of the present study isto establish whether these drugs block pro-duction of TNF-a as well as IL-2 by ratleukocytes and whether this inhibition af-fects the development of rat adjuvant arthri-tis (AA). Cultured splenocytes were stimu-lated with either lipopolysaccharide (LPS)or concanavalin A (ConA) ira the presenceof thalidomide, CC 1069, or solvem. and theproduction of TNF-a and I L-2 were com-pared. Next, adjuvant was injected isto thebase of the tail of rats without or with dailyintraperitoneal injections with 100-200mg/kg per day thalidomide or 50-200me/kg per day CCI069. Disease activity,including ankle swelling, hind limb radio-graphic and histoloeical changes, weightgain, and ankle joint cytokine mRNA lev-els, were monitored. CC 1069, but not theparem dru`g thalidomide, inhibited ia litroproduction of TNF-a and IL-2 by stimu-lated splenocytes ira a dose-dependent man-ner. /n viro, a dose-dependent suppressionof AA disease activity occurred ira theCC 1069-treated animais. In contrast,

238^ International Journal o/ Lcpros.v^ 2000

thalidomide-treated rats experienced com-parable arthritis severity to placebo-treatedanimais. There was also a reduction inTNF-a and IL-2 mRNA leveis in the anklejoints of CC 1069-treated nus comparedwith thalidomide- and placebo-treatedarthritic rats. Early initiation of CC 1069treatment suppressed AA inflammationmore efficiently than delayed treatment. Weconclude that thalidomide, which did notsuppress TNF-a or IL-2 production in i'itroby Lewis rat cells, did not suppress devei-opment of rat AA. However, the develop-ment of rat AA can be blocked by thethalidomide analog CC1069, which is aneffìcient inhibitor of TNF-a production andIL-2 üt nitro.—Authors' Abstract

Pais, T. F. and Appelberg, R. Macrophagecontrol of mycobacterialgrowth inducedby picolinic acid is dependent on hostcell apoptosis. J. Immunol. 164 (2000)389-397.The effects of picolinic acid (PA) on the

intramacrophagic growth of Mvcobac-terium avimun were studied. PA reduced M.aviam growth inside mouse macrophagesand led to a complete control of mycobacte-rial growth when added together with IFN-gamma. The mechanism involved did notrequire TNF-alpha, NO, or the respiratoryburst, and was not dependent on eithèr ironor zinc withholding. The mycobacterio-static activity of the macrophages was asso-ciated with the induction of morphologicalchanges that culminated in apoptosis at day4 of treatment. PA alone induced apoptosisin macrophages, and this effect was in-creased by IFN-gamma treatment. Apopto-sis at day 4 of infection was reduced by in-hibitins.; macrophage activation with theprostaelandin 15 deoxyprostaglandin J(2)or by treating the cens with the antioxidantN-acetylcysteine. Mycobacterial growthwas partially restored in macrophagestreated with PA and IFN-gamma whenIS deoxy-prostaglandin J(2) was added,concomitant with a delay in apoptosis.N-Acetylcysteine or glutathione could alsocompletely revert the mycobacteriostaticeffects of PA or PA plus IFN-gamma.—Au-thors' Abstract

Pedrosa, .I., Saunders, B. M., Appelbcrg,R., Orme, 1. M., Silva, M. T. andCooper, A. M. Neutrophils play a protec-tive nonphagocytic role in systemic Mv-cobacterium tuberculosis infection ofmice. lnfect. Immun. 68 (2000) 577-583.Evidence showimg that neutrophils play a

protective role in the host response to infec-tion by different intracellular parasites liasbeen published in the past few years. We as-sessed Chis issue with regar(' to the infectionof mice with Mvcobacterium tuberculosis.We found a chronic recruitment of neu-trophils to the infection roei, namely, to theperitoneal cavity after intraperitoneal infec-tion and to the spleen and liver after intra-venous inoculation of the mycobacteria.However, bacilli were never found associ-ated with the recruited neutrophils butrather were found inside macrophages. Theintravenous administation of the antineu-trophil monoclonal antibody RB6-8C5 dur-ing the first week of infection led to selec-tive and severe neutropenia associated withan enhancement of bacillary growth in thetarget organs of the mice infecte(' by the in-travenous route. The neutropenia-associ-ated exacerbation of infection was most im-portant in the liver, where a bacterial load10-fold higher than that in nonneutropenicmice was found; the exacerbation in theliver occurred both during and after theneutropenic period. Early in infection by M.tuberculosis, neutropenic mice expressedlower leveis of mRNAs for gamma inter-feron and inducibie nitric oxide synthase inthe Tiver compared to nondepleted mice.These results point to a protective role ofneutrophils in the host defense mechanismsagainst M. tuberculosis, which occurs earlyin the infection and is not associated withthe phagocytic activity of neutrophils butmay be of an inununoniodu1atory nature.-Authors' Abstract

Piatek, A. S., Telenti, A., Murray, M. R.,El Hajj, H., Jacobs, W. R., Kramer, r.R. and Alland, D. Genotypic analysis ofMvcobacterium tuberculosis in two dis-tinct populations using molecular bea-cons: implications for rapid susceptibilitytesting. Antimicrob. Agents Chemother.44 (2000) 103-110.

68, 2^ Current Literature^ 239

Past genotypic studies of Mycobacteriunztuberculosis may have incorrectly estimatedthe importance of specific drug resistancemutations due to a number of sampling bi-ases including an overrepresentation ofmultidrug-resistant (MDR) isolates. An ac-curate assessment of resistance mutations iscrucial for understanding basic resistancemechanisms and designing genotypic drug,resistance assays. We developed a rapid,closed-tube PCR assay using fluorogenicreporter molecules called molecular bea-cons to detect reportedly common M. tuber-culosis mutations associated with resistanceto isoniazid and rifampin. The assay wasused in a comparative genotypic investiga-tion of two different study populations todetermine whether these known mutationsaccount for most cases of clinicai drug re-sistance. We analyzed samples from a refer-ence laboratory in Madrid, Spain, which re-ceives an overrepresentation of MDR iso-lates similar to prior studies and from acommunity medicai center in New Yorkwhere almost ali of the resistam isolates andan equal number of susceptible controlswere available. The ability of the molecularbeacon assay to predict resistance to isoni-azid and rifampin was also assessed. Theoverall sensitivity and specificity of the as-say for isoniazid resistance were 85% and100%, respectively, and those for rifampinresistance were 98% and 100%, respec-tively. Rifampin resistance mutations weredetected equally well in isolates from bothstudy populations; however, isoniazid resis-tance mutations were detected in 94C/c ofthe isolates from Madrid but in only 76% ofthe isolates from New York (p = 0.02). InNew York, isoniazid resistance mutationswere significantly more common in theMDR isolates (94%) than in single-drug-re-sistant isolates (44%; p <0.001). No assoei-ation between previously described muta-tions in the kasA gene and isoniazid resis-tance was found. The first mutations thatcause isoniazid resistance may often occurin sequences that have not been commonlyassociated with isoniazid resistance, possi-bly in other as yet uncharacterized genes.The molecular beacon assay was simple,rapid, and highly sensitive for the detectionof rifampin-resistant M. tuberculosis iso-lates and for the detection of isoniazid re-

sistance in MDR isolates.—Authors' Ab-stract

Saunders, B. M., Frank, A. A. and Orme,I. M. Granuloma formation is required tocontais bacillus growth and delay mor-tality in mice chronically infected withMrcobacteriunr tuberculosis. Immunol-ogy 98 (1999) 324-328.Previous studies in this laboratory have

shown that mice with a gene disruption tothe int acellular adhesion molecule-1(ICAM-K/O) express normal cell-mediatedimmunity but cannot mount delayed-typehypersensitivity reactions following Mv-cobacterium tuberculosis infection. How-ever, even in the absence of any appreciablegranuloma formation, these mice controlbacterial growth for at least 90 days. Whilenot required to control the infection ini-tially, we hypothesized that granuloma for-mation was required to control chronic in-fection, acting by surrounding infected censto prevent bacterial dissemination. To testthis, ICAM-1 KO mice were infected with alow dose aerosol of M. tuberculosis Erdmanand were found to succumb to infection 136± 30 days later, displaying highly elevatedbacterial loads compared to wild-type mice.Lung tissue from ICAM-K/O mice dis-played extensive cellular infiltration andwidespread tissue necrosis, but no orga-nized granulomatous lesions were evident;whereas the control mice displayed orga-nized compact granulomas. These datademonstrate that while a <granulomatous re-sponse is not required initially to control M.tuberculosis infection, absence of granulo-mas during chronic infection leads to in-creased bacterial growth and host death.Thus, these data support the hypothesis thatgranuloma formation is required to controlchronic infection, acting by surroundingand walling off cites of infection to preventbacterial dissemination and maintain a stateof chronic infection.—Authors' Abstract

Schon, T., Gebre, N., Sundqvist, T., Ader-aye, G. and Britton, S. Effects of HIVco-infection and chemotherapy on theurinary leveis of nitric oxide metabolitesin patients with pulmonary tuberculosis.Scand. J. Infect. Dis. 31 (1999) 123-126.

240^ lnternational ./ournol of Lepros_v^ 2000

The metabolites of nitric oxide NO (ni-trite (NO,-) and nitrate (NO S-)) in urinefrom Ethi-opian patients suffering from tu-berculosis were measured. The urinarylevei of NO , /NO; in a group of healthyEthiopians was 1020 ± 471 tM (N = 22).Untreated HIV-negative patients with activepulmonary tuberculosis (1574 ± 588 µM, p<0.01, N = 12) and household contacts totuberculosis patients (1949 ± 812 ltM, p =0.006, N = 7) had signiticantly higher leveisof urinary NO, -/N0,- than the controlgroup. Untreated HIV-positive patients withpulmonary tuberculosis did not have in-creased leveis of urinary NO, -/NOS (1101± 614 µM, N = 6). Some of the HIV-nega-tive untreated patients with pulmonary tu-berculosis (1710 ± 519 µM, N = 6) werefollowed up after treatment and showed areduction in the leveis of urinary NO, -/NO,- 1 week after treatment (945 ± 599µM, p <0.05). It is concluded that HIV-neg-ative patients with active pulmonary tuber-culosis have increased urinary leveis of ni-tric oxide metabolites with a reductionfollowing specific antituberculous chemo-therapy.—Trop. Dis. Bull. 96 (1999) 1152

Shannon, E. J., Aseffa, A., Pankey, G.,Sandoval, F. and Lutz, B. Thalido-mide's ability to augment the synthesisof IL-2 in litro in HIV-infected patientsis associated with the percentage ofCD4+ cens in their blood. Immunophar-macology 46 (2000) 175-179.

Thalidomide is used for treating erythemanodosum leprosum. It is also used to treataphthous ulcers in HIV-infected patients.The mcchanism of action of this drug is notyet fully understood, but modulation ofinflammatory cytokines like IL-2 and TNF-alpha may play a role. We investigated theeffect of thalidomide on the production ofIL-2 and TNF-alpha by staphylococcal en-terotoxin A (SEA) stimulated peripheralblood mononuclear cens (PBMC) fromHIV-infected patients. The PBMC from 20patients was incubated in the presence of4.0 gg/ml of thalidomide and 50 ng/ml ofSEA. After 18 hr, the culture supernatantwas assayed for IL-2 and TNF-alpha. ThePBMC incubated with thalidomide andSEA produced significantly more IL-2 thanthose incubated with SEA alone. The TNF-

alpha secreted by the same cells incubatedwith thalidomide and SEA was not signifi-cantly different from that secreted by thecens incubated with SEA alone. Theamount of IL-2 produced in the thalidomideand SEA treated cultures was directly corre-lated with the percentage of CD4+ cens inblood and inversely correlated with the per-centage of CD8+ cens in blood. No statisti-cally significam correlations were foundwhen comparing the amount of TNF-alphaproduced in the thalidomide and SEAtreated cultures with the percentage ofCD4+ or CD8+ cens in the blood. Thalido-mide can act, in nitro, as an additional stim-ulant to augment the synthesis of IL-2 inH1V-infected patients. Increased productionof IL-2 by activated T cens may be a mecha-nism through which it exerts its immuno-modulatory effects.—Authors' Abstract

Singhal, S., Mehta, ,1., Desikan, R., Ayers,D., Roberson, P., Eddlemon, P., Mun-shi, N., Anaissie, E., Wilson, C., Dho-dapkar, M., Zeldis, J., Barlogie, B.,Siegel, D. and Crowley, J. Antitumoractivity of thalidomide in refractory mul-tiple myeloma. N. Engl. J. Med. 341(1999) 1565-1571.

Background: Patients with myelomawho relapse after high-dose chemotherapyhave few therapeutic options. Since in-creased bone marrow vascularity imparts apoor prognosis in myeloma, we evaluatedthe efficacy of thalidomide, which has an-tiangiogenic properties, in patients with re-fractory disease.

Methods: Eighty-four previously treatedpatients with refractory myeloma (76 with arelapse after high-dose chemotherapy) re-ceived oral thalidomide as a single agent fora median of 80 days (range 2 to 465). Thestarting dose was 200 mg daily, and thedose was increased by 200 mg every 2weeks until it reached 800 mg per day. Re-sponse was assessed on the basis of a re-duction of the myeloma protein in serum orBence Jones protein in urine that lasted forat least 6 weeks.

Results: The serum or urine leveis ofparaprotein were reduced by at least 90% in8 patients (2 had a complete remission), atleast 75% in 6 patients, at least 50% in 7 pa-tients, and at least 25% in 6 patients, for a

68, 2^ Current Literatura^ 241

total rate of response of 32%. Reductions inthe paraprotein leveis were apparent within2 months in 78% of the patients with a re-sponse and were associated with decreasednumbers of plasma cells in bone marrowand increased hemoglobin leveis. The mi-crovascular density of bone marrow did notchange significantly in patients with a re-sponse. At least one third of the patients hadmild or moderate constipation, weakness orfatigue, or somnolence. More severo ad-verse effects were infrequent (occurring inless than 10% of patients), and hematologiceffects were rare. As of the most recent fol-low up, 36 patients had died (30 with no re-sponse and 6 with a response). After 12months of follow up, Kaplan-Meier esti-mates of the mean (±S.E.) rates of event-free survival and overali survival for alipatients were 22 ± 55 and 58 ± 5%, respec-tively.

Conclusions: Thalidomide is activeagainst advanced myeloma. It can inducemarked and durable responses in some pa-tients with multiple myeloma, includingthose who relapse after high-dose chemo-therapy.—Authors' Abstract

South Africa, Durban ImmunotherapyTrial Group. Immunotherapy with Mv-cobacterium vaccae in patients withnewly diagnosed pulmonary tuberculo-sis: a randomised controlled trial. Lancet(Br.) 354 (1999) 116-119.The hypothesis that the addition of M.

vaccae to standard short-course antituber-culosis chemotherapy would decrease thetime to achieve a negative sputum culturewas tested in a South African study. Pa-tients with newly diagnosed tuberculosiswere enrolled between 1994 and 1996 andrandomly assigned an injection of saline(placebo) or M. vaccae (10'') on treatmentday 8. All patients received antituberculosischemotherapy with rifampin, isoniazid,pyrazinamide, and ethambutol. Sputumsamples were checked by microscopy andculture every week for the first 8 weeks andmonthly until the end of chemotherapy at 6months. The primary outcome was the timeto a negative sputum culture in the first 8weeks. Intention-to-treat analysis was usedand time to sputum clearance was assessedby log-rank test and Cox's proportional-

hazards regression; 172 patients receivedM. vaccae and 175 patients receivedplacebo. At 8 weeks, 70 patients in the M.vaccae group and 65 patients in the placebogroup had a negative culture; there was nodifference between groups in the time to anegative culture (p = 0.83). There was nointeraction between HIV status and treat-ment. It is concluded that M. vaccae im-munotherapy has no benetit when added tostandard antituberculosis chemotherapy.-Trop. Dis. Bull. 96 (1999) 1262

Sun, Z. H. and Zhang, Y. Spent culture su-pernatant of Mvcobacterium tuberculosisH37Ra improves viability of aged cul-tures of this strain and allows small inoc-ula to initiate growth. J. Bacteriol. 181(1999) 7626-7628.Spent culture supernatant from early sta-

tionary-phase Mycobacteriu,n tubcrc u losisH37Ra cultures increased the viability ofbacilli from aged cultures of this strain andallowed small inocula to initiate growth inliquid culture. The resuscitation factor wasacid labile and heat stable, with a mass ofless than 1,375 Da.—Authors' Abstract

Triccas, J. A., Berthet, F.-X., Pelicic, V.and Gicquel, B. Use of fluorescence in-duction and sucrose counterselection toidentify Mvcobacterium tuberculosisgenes expressed within host cells. Mi-crobiology 145 (1999) 2923-2930.The identification of Mvcobacterium tu-

berculosis genes expressed within host cellswould contribute greatly to the develop-ment of new strategies to combat tuberculo-sis. By combining the natural fluorescenceof the Aequoria viciaria green tluorescentprotein (GFP) with the counterselectableproperty of . the Bacillus subtilis SacB pro-tein, M. tuberculosis promoters displayingenhanced in viro activity have been iso-lated. Macrophages were infected with re-combinant Al. bovis bacille Calmette-Guérin containing a library of M. tuberculo-sis promoters controlling gfp and sacBexpression, and fluorescent bacteria recov-ered by fluorescence-activated cell sorting.The expression of sacf was used to elimi-nate clones with strong promoter activityoutside the macrophage, resulting in the

242^ International Journal of Leprosy^ 2000

isolation of seven clones containing M. tu-berculosis promoters with greater activityintracellularly. The gene products identitieddisplayed similarity to proteins from otherorganisms whose functions include nutrientutilization, protection from oxidative stressand defense against xenobiotics. These pro-posed functions are consistent with condi-tions encountered within the host cell and,lhos, suggest that the augmented activity ofthe isolated promoters/genes may representstratecies employed by M. tuberculosas toenhance intracel1ular survival and promoteinfection.—Authors' Abstract

Underhill, D. M., Ozinsky, A., Smith, K.D. and Aderem, A. Toll-like receptor-2mediates mycobacteria-induced proin-flammatory sienalintz in macrophages.Proc. Natl. Acad. Sci. U.S.A. 96 (1999)14459-14463.

The recognition of mycobacterial cellwall components causes macrophages to se-create tumor necrosis factor-alpha (TNF-a)and other cytokines that are essential for thedevelopment of a protective inflammatoryresponse. We show that toll-like receptorsare required for the induction of' TNF-a inmacrophages by Mycobacteriuin tuberculo-sis. Expression of a dominant negative forniof MyD88 (9 signaling components requiredfor toll-like receptor signaling) in a ìnousemacrophage cell line blocks TNF-a produc-tion induced by M. tuberculosis. We iden-tify toll-like receptor-2 (TLR2) as the spe-cific toll-like receptor required for this in-duction by showing that expression of aninhibitory TLR2 (TLR2-P681 H) blocksTNF-a production induced by whole M. tu-be rculosis. Further, we show that TLR2-de-pendem signaling mediates responses tomycobacterial cell wall fractions enrichedfor 1ipoarabinomannan, mycolylarabino-galactan-peptidoglycan complex, or M. tu-berculosis total lipids. Thus, although manymycobacterial cell wall fractions are identi-fied to be inflammatory, all require TLR2for induction of TNF-a in macrophages.These data suggest that TLR2 is essentialfor the induction of a protective immune re-sponse to mycobacteria. Authors' Abstract

Vasiliauskas, E. A., Kam, L. Y., AbreuMartin, M. T., Hassard, P. V., Pa-

padakis, K. A., Yang, H. Y., Zeldis,Il. and Targan, S. R. An open-label pi-lot study of low-dose thalidomide inchronically active, steroid-dependentCrohn's disease. Gastroenterology 117(1999) 1278-1287.

Background and Aims: Thalidomide de-creases production of tumor necrosis factor-alpha, a proinflammatory cytokine assoei-ated with Crohn's disease (CD). In thisstudy the safety, tolerance, and efficacy oflow-dose thalidomide were evaluated fortreatment of moderate-to-severe, steroid-dependem CD.

Methods: Twelve adult male patientswith Crohn's Disease Activity Index(CDAI) scores of ?250 and __500 despite>_20 m`g prednisone/day were enrolled. Thefirst 6 patients received 50 mg thalidomideevery night, the next 6 received 100 mgevery night. Steroid doses were stable dur-ing the first 4 weeks of treatment, then ta-pered during weeks 5-12. CDAI was usedto assess response.

Results: (1) Disease activity improvedconsistently in all patients during weeks1-4: 58% response, 17% remission. (2)Clinicai improvement was generally main-tained despite steroid taper during weeks5-12. All patients were able to reducesteroids by 50%. Forty-four percent discon-tinued steroids entirely. In weeks 5-12,70% of patients responded and 20%achieved remission. (3) Side effects weremild and mostly transient, with the mostcommon being drowsiness, peripheral neu-ropathy, edema, and dermatitis.

Conclusions: Low-dose thalidomide ap-pears to be well tolerated and effective overa 12-week period. Results of this pilot studysupport the need for controlled multicentertrials of thalidomide for treatment of CD.-Authors' Abstract

Wallis, R. S., Patil, S., Cheon, S.-H., Ed-monds, K., Phillips, M., Perkins, M.D., Joloba, M., Namale, A., Johnson, J.L., Teixeira, L., Dietze, R., Siddigi, S.,Mugerwa, R. D., Eisenach, K. and Ell-ner, ,J. ,J. Drug tolerance in Mycobac-terium tuberculosis. Antimicrob. AgentsChemother. 43 (1999) 2600-2606.

Although Mvcobacterium tuberculosis iseradicated rapidly during therapy in some

68, 2^ Current Literature^ 243

patients with pulmonary tuberculosis, it canpersist for many months in others. Thisstudy examined the relationship betweenmycobacterial drug tolerance (delayedkilling ia nitro), persistence, and relapse. Itwas performed with 39 fully drug-suscepti-ble isolates from a prospective trial of stan-dard short-course antituberculous therapywith sputum smear-positive, human im-nuinodeficiency virus-uninfected subjectswith pulmonary tuberculosis in Brazil andUganda. The rate of killing ia nitro was de-termined by monitoring the growth index(GI) in BACTEC I 2B medium after addi-tion of drug to established cultures and wasmeasured as the number of days requiredfor 99°lí sterilization. Druws differed signifi-cantly in bactericidal activity, in the follow-ing order from greatest to least, rifampin > iso-niazid-cthambutol > ethambutol (p <0.001).Isolates from subjects svho had relapses(N = 2) or in whom persistence was pro-Ionged (N = 1) were significantly more tol-erant of isoniazid-ethambutol and rifampinthan isolates from other subjects (p <0.01).More generally, the duration of persistenceduring therapy was predicted by strain tol-erance to isoniazid and ri fampin (p = 0.012and 0.026, respectively). Tolerance to isoni-azid-ethambutol and tolerance to rifampinwere highly correlated (p <0.001). Tolerantisolates did not differ from others with re-spect to the MIC of isoniazid; the rate ofkilling of a toleram isolate by isoniazid-ethambutol was not increased at higherdrug concentrations. These observationssuggest that tolerance may not be due todrug-specific mechanisms. Tolerance wasof the phenotypic type, although increasedtolerance appeared to emerge after pro-longed drug exposure ia viro. This studysuggests that drug tolerance may be ara im-portant determinant of the outcome of ther-apy for tuberculosis.—Authors' Abstract

Walsh, D. S., Meyers, W. M., Krieg, R. E.and Walsh, G. P. Transmission of My-cobacterinm ulceraras to the nine-bandedarmadillo. Am. J. Trop. Med. Hyg. 61(1999) 694-697.

Animal enodeis for Mvcobacterium ul-ceraras infections (Buruli ulcer) includeguinea pigs, rats, and coice, but each haslimitations in replicating the spectrum of

human disease. Here, 19 adult nine-bandedarmadillos were inocuiated intradermallywith M. ulceraras. Injection sites were ex-aminei and skin samples obtained for histo-logic and microbiology studies. Necropsieswere conducted to assess systemic involve-ment. In group I (N = 4), 2 animais devel-oped progressive skin ulcers with under-mined borders at the injection sites within6-10 weeks. Biopsies showed features sim-ilar to human disease including extensivenecrosis in the deep dermis and subcuta-neous fat, mixed cellular infiltrates, andacid-fast bacilli (AFB). In group 2 (N = 15),5 animais developed progressive skin ul-cers; 3 had evanescent papulo-nodules, 3died shortly after inoculation of unknowncauses, and 4 showed no silms of infection.Lesion samples from 3 animais with pro-gressive ulcers were cultue positive forAFB. Our findings indicate that nine-banded armadillos are susceptible to M. ul-ceraras and may develop cutaneous lesionsthat closely mimic Buruli ulcer.—Authors'Abstract

Wichelhaus, T. A., Schafer, V., Brade, V.and Boddinghaus, B. Molecular charac-terization of ipoB mutations conferrin;cross-resistance to rifamycins ora methi-cillin-resistant Staphylococcus aureus.Antimicrob. Agents Chemother. 43(1999) 2813-2816.

Mutations of the rpoB gene conferringresistance to rifampin were analyzed ira 40methicillin-resistant Staphvlococcus aureusisolates obtained from six countries. Inter-estingly, the majority of clinicai isolatesshowed multiple mutations within rpoB.The amino acid substitution 48I His-*Asnwas the most prevalent one, capable of con-ferring low-levei resistance ou its own.Crossresistance to rifampin, rifabutin, andrifapentine was demonstrated for ali mu-tants identified. The levei of resistance torifamycins correlated with both the muta-tion position and type of amino acid substi-tution.—Authors' Abstract

Wilson, W., DeRisi, J., Kristensen, H. H.,Imhoden, P., Rane, S., Brown, P. O.and Schoolnik, G. K. Exploring drug-induced alterations in gene expression iraMvcobacterium tuberculosis by microar-

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ray hyhridiration. Proc. Natl. Acad. Sci.U.S.A. 96 ( 1999) 12833-12838.

Tuberculosis is a chronic infectious dis-case that is t ansmitted by cough-propellcddroplets that carry the etiologic bacterium,Mvc obac•teriorü tuhcrculo.sis. Although cur-rently available drugs kill most isolates ofAl. nrherculo.sis, strains resistam to each ofthese have emerged, and multiply resistamstrains are increasingly widespread. Thegrowing problem of drug resistance com-hined with a global incidence of seven mil-lion new cases per year underscore the ur-gent need for new antituberculosis thera-pies. The recent publication of the completesequence of. the M. tuberculosas g,enome liasmarfe possible, for the tirst time, a compre-hensive genomic approach to the biology ofthis organism and to the drug discoveryprocess. We used a DNA microarray con-

taining 97%, of the ORFs predicted fromthis sequence to monitor changes in M. tur-hCrru losis gene expression in response tothe antituberculous drug isoniazid. 1lere weshow that isoniarid induced several genesthat encode proteins physiologically rele-vam to the drug's mode of action, includingan operonic cluster of live genes encodingtype II fatty acid synthase enzymes andfbpC, which encodes trehalose diniycolyltransferase. Other genes, not apparentlywithin directly affected biosynthetic path-ways, also were induced. These genes,EfpA, fadE23, fadE24, and ahpC, likelymediate processes that are linked to thetoxic consequences of the drug. Insightsgained from this approach may define newdrug targets and suggest new methods foridentifying compounds that inhibit thosetargets.—Authors' Abstract