INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. (ISSN 0148-916X) CURRENT LITERATURE

This department carries selected abstracts of articles published in current medical journals dealing with leprosy and other mycobacterial diseases.

General & Historical 383 Chemotherapy 384 Clinical Sciences 388 Immuno-Pathology 391 Leprosy 395 Tuberculosis 396 Microbiology 403 Leprosy 405 Tuberculosis 406 Experimental Infections 410 Epidemiology and Prevention 413 Rehabilitation and Social Concerns 414 Other Mycobacterial Diseases 415 Molecular & Genetic Studies 420

General and Historical

Araujo, M. G. [Leprosy in Brazil]. Rev. use of specific therapy, suppression of lepra Soc. Bras. Med. Trop. 36(3) (2003) reactions, prevention of physical incapac- 373Ð382. [Article in Portuguese] ity, and physical and psychosocial rehabili- tation. Chemotherapy with rifampin, dap- Leprosy or Hansen’s disease is a chronic sone, and clofazimine have produced very infectious disease caused by the Mycobac- good results and the control of the disease terium leprae. The skin and nervous mani- in Brazil in the foreseeable future is festations of the disease present a singular likely.—Author’s Abstract clinical picture that is easily recognized. After India, Brazil still is the second coun- try with the greatest number of cases in the Dionne, M. S., Ghori, N., and Schneider, world. Around 94% of the known cases D. S. Drosophila melanogaster is a ge- and 94% of the new cases reported in netically tractable model host for Myco- America, come from Brazil. The disease bacterium marinum. Infect. Immun. presents itself in two well-defined stable 71(6) (2003) 3540Ð3550. and opposite poles (lepromatous and tuber- culoid) and two unstable groups (indeter- Mycobacterium marinum is a pathogenic minate and dimorphic). The spectrum of mycobacterial species that is closely re- presentation of the disease may also be lated to Mycobacterium tuberculosis and classified as: tuberculoid tuberculoid (TT), causes tuberculosis-like disease in fish and borderline tuberculoid (BT), borderline frogs. We infected the fruit fly Drosophila borderline (BB), borderline lepromatous melanogaster with M. marinum. This bac- (BL) and lepromatous lepromatous (LL). terium caused a lethal infection in the fly, The finding of acid fast bacillus in tissue is with a 50% lethal dose (LD(50)) of 5 CFU. the most useful method of diagnosis. The Death was accompanied by widespread tis- effective treatment of leprosy includes the sue damage. M. marinum initially prolifer-

383 384 International Journal of Leprosy 2003 ated inside the phagocytes of the fly; later identified by Ziehl-Neelsen staining, identi- in infection, bacteria were found both in- fication of MAP in human beings requires side and outside host cells. Intracellular M. culture or detection of MAP DNA or RNA. marinum blocked vacuolar acidification If infectious in origin, Crohn’s disease and failed to colocalize with dead Escheri- should be curable with appropriate antibi- chia coli, similar to infections of mouse otics. Many studies that argue against a macrophages. M. marinum lacking the causative role for MAP in Crohn’s disease mag24 gene were less virulent, as deter- have used antibiotics that are inactive mined both by LD(50) and by death kinet- against MAP. However, trials that include ics. Finally, in contrast to all other bacteria macrolide antibiotics indicate that a cure for examined, mycobacteria failed to elicit the Crohn’s disease is possible. The necessary production of antimicrobial peptides in length of therapy remains to be determined. DROSOPHILA: We believe that this sys- Mycobacterial diseases have protean clini- tem should be a useful genetically tractable cal manifestations, as does Crohn’s disease. model for mycobacterial infection.—Au- The necessity of stratifying Crohn’s disease thors’Abstract into two clinical manifestations (perforating and non-perforating) when interpreting the results of antibiotic therapy is discussed. Greenstein, R. J. Is Crohn’s disease caused Rational studies to evaluate appropriate by a mycobacterium? Comparisons with therapies to cure Crohn’s disease are pro- leprosy, tuberculosis, and Johne’s disease. posed.—Author’s Abstract Lancet Infect. Dis. 3(8) (2003) 507Ð514.

Although Crohn’s disease is considered Hernandez, A., Martro, E., Matas, L., to be autoimmune in origin, there is in- and Ausina, V. In-vitro evaluation of creasing evidence that it may have an infec- Perasafe compared with 2% alkaline glu- tious cause. The most plausible candidate is taraldehyde against Mycobacterium spp. Mycobacterium avium subspecies paratu- J. Hosp. Infect. 54(1) (2003) 52Ð56. berculosis (MAP). Intriguingly, Koch’s postulates may have been fulfilled for MAP Quantitative suspension and carrier tests and Crohn’s disease, even though they still were used to compare the activity of Perasafe have not been met for Mycobacterium lep- and Cidex against Mycobacterium tubercu- rae and leprosy. In animals MAP causes losis, Mycobacterium avium-intracellulare, Johne’s disease, a chronic wasting intestinal Mycobacterium fortuitum, and Mycobacte- diarrhoeal disease evocative of Crohn’s dis- rium chelonae. The interference of an or- ease. Johne’s disease occurs in wild and do- ganic load, and of hard water was also con- mesticated animals, including dairy herds. sidered. Both agents achieved reductions Viable MAP is found in human and cow exceeding 10(5)-fold within 20 and 30 min milk, and is not reliably killed by standard for all the strains tested. Perasafe is thus pasteurisation. MAP is ubiquitous in the en- mycobactericidal and a viable alternative to vironment including in potable water. Since Cidex for intermediate or high-level disin- cell-wall-deficient MAP usually cannot be fection.—Authors’Abstract

Chemotherapy

Babaoglu, K., Page, M. A., Jones, V. C., The emergence of multi-drug resistant tu- McNeil, M. R., Dong, C., Naismith, J. berculosis, coupled with the increasing over- H., and Lee, R. E. Novel inhibitors of lap of the AIDS and tuberculosis pandemics an emerging target in Mycobacterium tu- has brought tuberculosis to the forefront as a berculosis; substituted thiazolidinones as major worldwide health concern. In an at- inhibitors of dTDP-rhamnose synthesis. tempt to find new inhibitors of the enzymes Bioorg Med. Chem. Lett. 13(19) (2003) in the essential rhamnose biosynthetic path- 3227Ð3230. way, a virtual library of 2,3,5 trisubstituted- 71, 4 Current Literature, Chemotherapy 385

4-thiazolidinones was created. These com- berculosis. Expression profiles of M. tuber- pounds were then docked into the active site culosis treated with compounds that inhibit cavity of 6′hydroxyl; dTDP-6-deoxy-D- key metabolic pathways are required as ref- xylo-4-hexulose 3,5-epimerase (RmlC) from erences for the assessment of novel antimy- Mycobacterium tuberculosis. The resulting cobacterial agents. We have studied the re- docked conformations were consensus sponse of M. tuberculosis to treatment with scored and the top 5% were slated for syn- the mycolic acid biosynthesis inhibitors iso- thesis. Thus far, 94 compounds have been niazid, thiolactomycin, and triclosan. Thio- successfully synthesized and initially tested. lactomycin targets the beta-ketoacyl-acyl Of those, 30 (32%) have ≥50% inhibitory ac- carrier protein (ACP) synthases KasA and tivity (at 20 microM) in the coupled rham- KasB, while triclosan inhibits the enoyl- nose synthetic assay with seven of the 30 ACP reductase InhA. However, controversy also having modest activity against whole- surrounds the precise mode of action of iso- cell M.tuberculosis.—Authors’Abstract niazid, with both InhA and KasA having been proposed as the primary target. We have shown that although the global re- Bermudez, L. E., Reynolds, R., Kolo- sponse profiles of isoniazid and thiolacto- noski, P., Aralar, P., Inderlied, C. B., mycin are more closely related to each other and Young, L. S. Thiosemicarbazole than to that of triclosan, there are differences (thiacetazone-like) compound with ac- that distinguish the mode of action of these tivity against Mycobacterium avium in two drugs. In addition, we have identified mice. Antimicrob. Agents Chemother. two groups of genes, possibly forming ef- 47(8) (2003) 2685Ð2687. flux and detoxification systems, through which M. tuberculosis may limit the effects In vitro screening of thiacetazone deriva- of triclosan. We have developed a mathe- tives indicated that two derivatives, SRI- matical model, based on the expression of 286 and SRI-224, inhibited a panel of 25 21 genes, which is able to perfectly discrim- Mycobacterium avium complex (MAC) iso- inate between isoniazid-, thiolactomycin-, lates at concentrations of 2 micro g/ml or or triclosan-treated M. tuberculosis. This lower. In mice, SRI-224 and thiacetazone model is likely to prove invaluable as a tool had no significant activity against the MAC to improve the efficiency of our drug devel- in livers and spleens, but treatment with opment programs by providing a means to SRI-286 resulted in significant reduction of rapidly confirm the mode of action of thio- bacterial loads in livers and spleens. A com- lactomycin analogues or novel InhA in- bination of SRI-286 and moxifloxacin was hibitors as well as helping to translate en- significantly more active than single drug zyme activity into whole-cell activity.— regimens in liver and spleen.—Authors’ Authors’Abstract Abstract Ciccone, R., Mariani, F., Cavone, A., Persi- Betts, J. C., McLaren, A., Lennon, M. G., chini, T., Venturini, G., Ongini, E., Col- Kelly, F. M., Lukey, P. T., Blakemore, S. izzi, V., and Colasanti, M. Inhibitory ef- J., and Duncan, K. Signature gene ex- fect of NO-releasing ciprofloxacin (NCX pression profiles discriminate between 976) on Mycobacterium tuberculosis sur- isoniazid-, thiolactomycin-, and triclosan- vival. Antimicrob. Agents Chemother. treated Mycobacterium tuberculosis. An- 47(7) (2003) 2299Ð2302. timicrob. Agents Chemother. 47(9) (2003) 2903Ð2913. Here, we report the antimycobacterial ac- tivity of NCX 976, a new molecule ob- Genomic technologies have the potential tained adding a NO moiety to the fluoro- to greatly increase the efficiency of the drug quinolone ciprofloxacin, on Mycobacterium development process. As part of our tuber- tuberculosis H37Rv strain, both in a cell- culosis drug discovery program, we used free model and in infected human macro- DNA microarray technology to profile phages. Unlike unaltered ciprofloxacin, drug-induced effects in Mycobacterium tu- NCX976 displayed a marked activity also 386 International Journal of Leprosy 2003 at low-nanomolar concentrations.—Authors’ tivity, is in the process of being character- Abstract ized in laboratory studies. This review describes the emerging immunological properties of thalidomide, from a historical Cynamon, M. H. and Sklaney, M. Gati- context to present-day clinical applications, floxacin and ethionamide as the founda- most notably in multiple myeloma but also tion for therapy of tuberculosis. Antimi- in other cancers, inflammatory disease, and crob. Agents Chemother. 47(8) (2003) HIV. We also describe the laboratory stud- 2442Ð2444. ies that have led to the characterization and development of SelCIDs and IMiDs into The use of gatifloxacin (GAT) in combi- potentially clinically relevant drugs. Early nation with ethionamide (ETA) with or trial data suggest that these novel im- without pyrazinamide (PZA) for a 12-week munomodulatory compounds may su- treatment period followed by an 8-week ob- percede thalidomide to become established servation period was evaluated in a model therapies, particularly in certain cancers. of tuberculosis in mice. Mice treated with Further evidence is required, however, to GAT at 300 mg/kg of body weight in com- correlate the clinical efficacy of these com- bination with ETA (25 mg/kg) for 5 days pounds with their known immunomodula- per week had sterile lungs, whereas mice tory, antiangiogenic, and antitumor proper- treated with GAT (100 mg/kg) and ETA (25 ties.—Authors’Abstract mg/kg) had about 10 CFU/lung; however, there was regrowth of the organisms in both groups at the end of the observation period. Forslow, U., Geborek, A., Hjelte, L., When PZA (450 mg/kg 5 days per week) Petrini, B., and Heurlin, N. Early was added to the high-dose GAT-ETA regi- chemotherapy for non-tuberculous my- men, no viable mycobacteria were present cobacterial infections in patients with after the 8-week observation period. GAT in cystic fibrosis. Acta Paediatr. 92(8) combination with ETA and PZA has great (2003) 910Ð915. promise for the treatment of tuberculosis.— Authors’Abstract AIM: To evaluate the response rate to antimycobacterial drug therapy in patients with cystic fibrosis (CF) suffering from in- Dredge, K., Marriott, J. B., and Dal- fection by non-tuberculous mycobacteria gleish, A. G. Immunological effects of (NTM). METHODS: Ten patients, aged thalidomide and its chemical and func- 10Ð34 yrs, out of 180 CF patients, were di- tional analogs. Crit. Rev. Immunol. agnosed with NTM disease. They had 22(5Ð6) (2002) 425Ð437. been regularly checked and examined for pulmonary symptoms, and had had chest Thalidomide has recently shown consid- X-rays and sputum cultures (including for erable promise in the treatment of a number mycobacteria) performed. One additional of conditions, such as leprosy and cancer. 36-yr-old female received her CF diagnosis Its effectiveness in the clinic has been as- soon after the NTM diagnosis. RESULTS: cribed to wide-ranging properties, including Mycobacterium avium-intracellulare com- anti-TNF-alpha, T-cell costimulatory and plex (MAC) was found in 10 out of 11 antiangiogenic activity. Novel compounds patients and M. kansasii in 1 patient. with improved immunomodulatory activity Treatment with antimycobacterial drugs and side effect profiles are also being eval- resulted in clinical improvement (weight uated. These include selective cytokine in- gain or stabilization of weight and/or im- hibitory drugs (SelCIDs), with greatly im- proved or stabilized lung function in 8 out proved TNF-alpha inhibitory activity, and of 11 patients) and mycobacterial culture immunomodulatory drugs (IMiDs) that are turned negative in 10 out of 1. CONCLU- structural analogs of thalidomide, with SION: Promising results may be associ- improved properties. A third group recently ated with early intervention with anti- identified within the SelCID group, with mycobacterial therapy in CF patients.— phosphodiesterase type 4-independent ac- Authors’ Abstract 71, 4 Current Literature, Chemotherapy 387

Gomez-Reino, J. J., Carmona, L., sociated with an increased risk of active Valverde, V. R., Mola, E. M., Montero, TB. Proper measures are needed to prevent M. D., and BIOBADASER Group. and manage this adverse event.—Authors’ Treatment of rheumatoid arthritis with tu- Abstract mor necrosis factor inhibitors may predis- pose to significant increase in tuberculosis risk: a multicenter active-surveillance re- Luzzio, F. A., Mayorov, A. V., Ng, S. port. Arthritis Rheum. 48(8) (2003) S., Kruger, E. A., and Figg, W. D. 2122Ð2127. Thalidomide metabolites and analogues. 3. Synthesis and antiangiogenic activ- OBJECTIVE: The long-term safety of ity of the teratogenic and TNFalpha- therapeutic agents that neutralize tumor modulatory thalidomide analogue 2-(2,6- necrosis factor (TNF) is uncertain. Recent dioxopiperidine-3-yl)phthalimidine. J. evidence based on spontaneous reporting Med. Chem. 46(18) (2003) 3793Ð3799. shows an association with active tuberculo- sis (TB). We undertook this study to deter- Versatile synthesis of the teratogenic, mine and describe the long-term safety of 2 TNFalpha-modulatory, and antiangiogenic of these agents, infliximab and etanercept, thalidomide analogue 2-(2,6-dioxopiperidine- in rheumatic diseases based on a national 3-yl)phthalimidine (1) and its direct antian- active-surveillance system following the giogenic properties are described. With commercialization of the drugs. METH- thalidomide or thalidomide derivatives as ODS: We analyzed the safety data actively precursors, the synthesis involved either collected in the BIOBADASER (Base de carbonyl reduction/thiation-desulfurization Datos de Productos Biologicos de la So- or carbonyl reduction/acyliminium ion reduc- ciedad Espanola de Reumatologia) data- tion protocols. Compared to earlier studies base, which was launched in February 2000 with thalidomide, which was only active by the Spanish Society of Rheumatology. with microsomal treatment, 1 exhibited For the estimation of TB risk, the annual in- marginal inhibitory activity in the rat aortic cidence rate in patients treated with these ring assay, thereby demonstrating the re- agents was compared with the background quirement for metabolic activation.—Au- rate and with the rate in a cohort of patients thors’Abstract with rheumatoid arthritis (RA) assembled before the era of anti-TNF treatment. RE- SULTS: Seventy-one participating centers Marriott, J. B., Dredge, K., and Dal- sent data on 1578 treatments with inflix- gleish, A. G. Thalidomide derived im- imab (86%) or etanercept (14%) in 1540 munomodulatory drugs (IMiDs) as po- patients. Drug survival rates (reported as tential therapeutic agents. Curr. Drug the cumulative percentage of patients still Targets Immune Endocr. Metabol. Dis- receiving medication) for infliximab and ord. 3(3) (2003) 181Ð186. etanercept pooled together were 85% and 81% at 1 year and 2 years, respectively. In- Thalidomide is known to be effective in stances of discontinuation were essentially the treatment of a number of conditions, in- due to adverse events. Seventeen cases of cluding leprosy and various cancers. The TB were found in patients treated with in- exact mechanisms of action remain unclear fliximab. The estimated incidence of TB as- although these are known to include anti- sociated with infliximab in RA patients was tumour necrosis factor (TNF)-alpha, T cell 1893 per 100,000 in the year 2000 and 1113 costimulatory, anti-angiogenic and anti- per 100,000 in the year 2001. These find- tumour activities. However, thalidomide is ings represent a significant increased risk being superceded by novel structural deriv- compared with background rates. In the atives which have been designed to have first 5 months of 2002, after official guide- improved immunomodulatory activity and lines were established for TB prevention in side effect profiles. These are currently be- patients treated with biologics, only 1 new ing characterised and some are entering the TB case was registered (in January). CON- clinic in phase I/II studies. One novel group CLUSION: Therapy with infliximab is as- of structural analogues are classified as the 388 International Journal of Leprosy 2003

Immunomodulatory Drugs (IMiDs). This (1966ÐJanuary 2003) and on the Cochrane review describes the emerging immunolog- Clinical Trials Register (January 2003). In- ical, anti-angiogenic and direct anti-tumour formation available from meetings’ abstract properties of thalidomide and the charac- books, Internet, or pharmaceutical compa- terisation and clinical application of its nies was also considered. STUDY SELEC- IMiD analogues. We describe the labora- TION AND DATA EXTRACTION: All ar- tory studies which have led to the charac- ticles identified as relevant, including those terisation and development of IMiDs into from non-English literature, were consid- potentially clinically relevant drugs. Early ered in an attempt to provide to the reader trial data suggests that these compounds both the theoretical basis and practical may themselves become established thera- guidelines for thalidomide pharmacother- pies, particularly in certain cancers. Fur- apy. DATA SYNTHESIS: Thalidomide has thermore, ongoing studies will determine hypnosedative, antiangiogenic, antiinflam- how best to apply these compounds to the matory, and immunomodulatory properties. appropriate clinical settings. We will de- Moreover, it has been shown to selectively scribe the various clinical studies of lead inhibit the production of tumor necrosis compounds that are in progress and specu- factor-alpha and reduce the expression of late as to the potential and future develop- various integrin receptors on the membrane ment of these exciting compounds.—Au- of leukocytes and other cell types in a dose- thors’Abstract dependent fashion. Controlled trials dem- onstrated the efficacy of thalidomide in a number of diseases, including erythema no- Nasca, M. R., Micali, G., Cheigh, N. H., dosum leprosum, lupus erythematosus, aph- West, L. E., and West, D. P. Dermato- thosis, graft-versus-host disease, prurigo logic and nondermatologic uses of thalido- nodularis, and actinic prurigo. Single case mide. Ann. Pharmacother. 37(9) (2003) reports or studies in small series have also 1307Ð1320. suggested a possible role for thalidomide in numerous other dermatologic and nonder- OBJECTIVE: To review published data matologic disorders. Possibly severe and on thalidomide, with emphasis on current sometimes irreversible risks related to the knowledge about mechanism of action, new clinical use of thalidomide include terato- and/or potential dermatologic and non- genicity and neurotoxicity. CONCLU- dermatologic therapeutic applications, well- SIONS: Although teratogenicity and neuro- known and emerging adverse effects, and toxicity are significant adverse effects re- current indications for its safe use. DATA quiring cautious use, thalidomide is an SOURCES: Review articles, in vitro re- effective therapeutic modality in a variety search studies, references from retrieved of difficult-to-treat disorders and, providing articles, case reports, and clinical trials careful selection of patients, should offer an were identified from a computerized litera- acceptable risk-to-benefit ratio.—Authors’ ture search using MEDLINE and OVID Abstract Clinical Sciences

Abad, S., Gyan, E., Moachon, L., Bous- fection (BCGitis) after administration of cary, D., Sicard, D., Dreyfus, F., and alemtuzumab (Campath-1H).—Authors’ Blanche, P. Tuberculosis due to Myco- Abstract bacterium bovis after alemtuzumab ad- ministration. Clin. Infect. Dis. 37(2) (2003) e27Ðe28. Ang, P., Tay, Y. K., Ng, S. K., and Seow, C. S. Fatal Lucio’s phenomenon in 2 pa- We describe a patient with relapsing B tients with previously undiagnosed lep- chronic lymphocytic leukemia who devel- rosy. J. Am. Acad. Dermatol. 48(6) oped systemic bacille Calmette-Guerin in- (2003) 958Ð961. 71, 4 Current Literature, Clinical Sciences 389

We report 2 cases of Lucio’s phenomenon, agranulocytosis in an Indian mid- a rare, aggressive, occasionally fatal type 2 borderline leprosy patient. Lepr. Rev. reaction occurring in the diffuse nonnodular 74(2) (2003) 167Ð170. type of lepromatous leprosy. The clinical di- agnosis of Lucio’s phenomenon is difficult, Fatal agranulocytosis in an Indian male and there are no known predictive or prog- receiving 100 mg of dapsone daily, hospi- nostic factors. Despite institution of aggres- talized for mid-borderline leprosy in type I sive treatment after diagnosis, our 2 cases reaction with triple nerve paralysis is re- had fatal outcomes.—Authors’Abstract ported. Various case reports concerning dapsone-induced agranulocytosis are re- viewed.—Authors’Abstract Baker, B., Evans, M., DeCastro, F., and Schosser, R. Leprosy in a Mexican immi- grant. J. Ky. Med. Assoc. 101(7) (2003) Daines, B. S., Vroman, D. T., Sandoval, 289Ð294. H. P., Steed, L. L., and Solomon, K. D. Rapid diagnosis and treatment of myco- A new diagnosis of borderline leproma- bacterial keratitis after laser in situ ker- tous leprosy was established in a man who atomileusis. J. Cataract Refract. Surg. had immigrated to Kentucky from Mexico. 29(5) (2003) 1014Ð1018. He was placed on a World Health Organiza- tion treatment regimen consisting of dap- We report the results of laser in situ ker- sone, clofazimine, and rifampin. The biol- atomileusis (LASIK) in a 51-year-old ogy of leprosy, its diagnosis, treatment, and woman with subsequent mycobacterial ker- worldwide impact are reviewed. Because of atitis diagnosed by staining with acid-fast the potential for highly mobile populations and fluorochrome methods, a technique to export endemic diseases, Kentucky known to have good sensitivity and speci- physicians must expand their lists of differ- ficity for mycobacteria. A rapid diagnosis ential diagnoses.—Authors’Abstract was made without waiting for cultures, and treatment was instituted, including tapering of topical steroids and appropriate antibi- Bandoh, S., Fujita, J., Ueda, Y., Tojo, Y., otic therapy. The result was preservation of Ishii, T., Kubo, A., Yamamoto, Y., Ni- the LASIK flap and a favorable visual out- shiyama, Y., and Ishida, T. Uptake of come at 6 months.—Authors’Abstract fluorine-18-fluorodeoxyglucose in pul- monary Mycobacterium avium complex infection. Intern. Med. 42(8) (2003) Daniel, E., Koshy, S., Joseph, G. A., and 726Ð729. Rao, P. S. Ocular complications in inci- dent relapsed borderline lepromatous Two patients showing abnormal fluorine- and lepromatous leprosy patients in 18-fluorodeoxyglucose (FDG) uptake due south India. Indian J. Ophthalmol. 51(2) to Mycobacterium avium complex (MAC) (2003) 155Ð159. infection are presented. Intense focal FDG uptake in the lung field could have been PURPOSE: To determine the magnitude caused by an infectious disease such as of ocular complications that present in inci- MAC. This should be considered as a possi- dent cases of relapsed borderline leproma- bility when FDG whole-body scans of pa- tous (BL) and lepromatous leprosy (LL) tients with pulmonary nodules are inter- patients. METHOD: From 1991 to 1997, preted. To our knowledge, this is the first all new BL and LL patients who had re- description of an FDG-positron emission lapsed from an earlier disease, detected by tomography (FDG-PET) image of MAC in- active case finding in the geographically fection of the lung.—Authors’Abstract defined area of Gudiyattam taluk, were in- vited for ocular examination after their lep- rosy status was confirmed clinically and Bhat, R. M. and Radhakrishnan, K. A histopathologically. RESULTS: Sixty re- case report of fatal dapsone-induced lapsed lepromatous patients, 45 male and 390 International Journal of Leprosy 2003

15 females, were examined. Fifty-two pa- of the T-helper 1 cell response. Clin. In- tients had relapsed after receiving only fect. Dis. 37(5) (2003) 628Ð633. dapsone mono-therapy, 4 after receiving paucibacillary multi-drug therapy (PB- Cutaneous leishmaniasis, leprosy, and tu- MDT) preceded by dapsone mono-therapy berculosis are caused by intracellular patho- and 4 after only PB-MDT. Three (5%) pa- gens whose development depends on im- tients had lagophthalmos, 1 (1.6%) patients paired cell-mediated immunity. We report an each had ectropion and trichiasis, 32 (53%) exceptional triple association of American patients had impaired corneal sensation in cutaneous leishmaniasis, lepromatous lep- both eyes, 2 (3.3%) patients each had rosy, and pulmonary tuberculosis in a man corneal opacity (associated with reduced with no recognized immunodeficiency. vision), corneal nerve beading, punctate Normal immunological assessment of the keratitis, keratic precipitates, and iris atro- interferon-gamma pathway does not sup- phy, 4 (6.6%) patients had cataract associ- port the hypothesis of a genetic defect in ated with decreased vision, 1 (1.6%) pa- any of the genes involved in the T helper tient had blocked naso-lacrimal duct and (Th)-1 cytokine cascade in this patient. Un- 13 (21.7%) patients had pterygium. Seven responsiveness to interleukin (IL)-12 of his (12%) patients had a visual acuity of 6/18 T cells after stimulation with Leishmania or less, 4 (6.7%) patients had 6/60 or less guyanensis, Mycobacterium bovis bacille and one patients had vision below 3/60. Calmette-Guerin, and Mycobacterium lep- General ocular complications rather than rae antigens suggested the inability to leprosy-related ocular complications were mount an appropriate Th cell response to responsible for reduced vision. Lagoph- upregulate the IL-12 receptor expression.— thalmos was associated with increased du- Authors’Abstract ration of the disease (p = 0.009), Grade II deformity (p = 0.001), punctate keratitis (p <0.001) and cataract (p <0.001). Beaded Holzer, M. P., Solomon, K. D., Sandoval, corneal nerves were associated with lepro- H. P., and Auffarth. G. U. [Diagnosis matous leprosy (p <0.001) and high myco- and treatment of mycobacterial keratitis bacterial infection (p = 0.05). Patients following LASIK. Case report and review whose initial disease was categorised as BL of the literature.] Ophthalmologe 100(7) and LL had greater impairment of vision (p (2003) 550Ð553. [Article in German]. = 0.037), more iris atrophy (p = 0.013), in- creased keratic precipitates (p = 0.013) and BACKGROUND: Mycobacterial kerati- more corneal nerve beading (p = 0.013), tis is a rare complication following LASIK when compared with the group comprising but can lead to an extremely unfavourable Tuberculoid-tuberculoid (TT), Borderline- outcome. The diagnosis and treatment is of- tuberculoid (BT) and Intermediate (IND). ten delayed due to confusion with other en- CONCLUSION: This first report on ocular tities including diffuse lamellar keratitis and complications in relapsed lepromatous pa- poor clinical outcomes with flap amputation tients demonstrates that general and leprosy- and/or keratoplasty are often the case. PA- related ocular complications occur in these TIENT AND METHODS: We report the re- patients. However, they are not in excess of sults of LASIK in a 51-year-old woman those reported in other leprosy groups. with subsequent early-diagnosed mycobac- Borderline and lepromatous leprosy pa- terial keratitis and compared this case to tients tend to have had more ocular compli- treatments and outcomes reported in the lit- cations than patients with tuberculoid lep- erature. RESULTS: The patient presented rosy.—Authors’Abstract 10 days following LASIK with a white fo- cal infiltrate in the stromal interface. The flap was lifted and cultures from the stromal Delobel, P., Launois, P., Djossou, F., bed and the reverse of the flap were ob- Sainte-Marie, D., and Pradinaud, R. tained and the interface irrigated. The pa- American cutaneous leishmaniasis, lep- tient was treated with topical antibiotics romatous leprosy, and pulmonary tuber- (ciprofloxacin 0.3%, amikacin 2.5%, clar- culosis coinfection with downregulation ithromycin 40 mg/ml and tobramycin 15 71, 4 Current Literature, Immuno-Pathology 391

mg/ml) for 8 weeks and at the most recent and the pig in the island.—Authors’ Ab- follow-up she had a visual acuity of 1.25. stract CONCLUSION: In a large number of pub- lished cases in the literature the flap had to be amputated and/or corneal transplants Nakayama, S., Uesaka, Y., Kunimoto, were necessary. Early diagnosis and treat- M., Mikata, T., Shimizu, J., and Ishii, ment however, are essential to successfully N. [The painful multiple mononeuropa- treat post-LASIK keratitis. Therefore the thy of acute onset in the left arm which patients should be followed up carefully in was diagnosed as leprous neuropathy]. the early postoperative period.—Authors’ Rinsho Shinkeigaku. 43(5) (2003) Abstract 265Ð269. [Article in Japanese]

A 31-year-old man from Myanmar with Hong, S. T., Hong, S. J., Lee, S. H., Kim, I. leprous neuropathy was reported. The S., and Shin, J. S. [A Study On The In- progress of the disease was subacute but the testinal Helminths Of The Patients In A painful symptom at the time of the onset Leprosarium In Korea.] Kisaengchunghak was acute. Multiple mononeuropathy was Chapchi. 21(1) (1983) 102Ð104. [Article diagnosed by the biopsy findings of the left in Korean] superficial radial nerve. He was admitted to our hospital with the complaint of the A total of 2026 leprosy patients of the weakness of his left hand and fingers which National Sorokdo Hospital was examined were very painful and got worse in several their intestinal parasites by cellophane weeks. Motor palsy was observed in his left thick smear method in January 1983. The ulnar, median, and radial nerves, and there egg positive cases of Taenia spp. were was the hypesthesia or anesthesia in his left treated with bithionol and the segments of hand, forearm and the medial side of his left Taenia were collected for species identifi- upper arm. On nerve conduction studies, the cation. The results were as follows: 1. To- amplitudes of CMAP and SNAP severely tal egg positive rate of any kind helminth diminished or not detected. The pattern was was 78.2% and cumulative total was compatible with multiple mononeuropathy. 85.2%. The egg positive rate for each The biopsy of the left superficial radial helminth was as follow; Taenia spp. 3.4%, nerve was performed. The pathological find- Ascaris lumbricoides 4.5%, Trichuris ings were the destruction of nerve fascicles, trichiura 72.l%, Clonorchis sinensis 2.8% replacement of nerve fibers with inflamma- and other 0.05%. 2. A total of 66 Taenia tory cells, and Mycobacterium leprae was egg positive cases was treated; out of them found with the specific stain. These findings proglottids of Taenia were collected from confirmed the diagnosis of the leprous neu- 26 cases. All of the collected worms were ropathy. Leprous neuropathy is one of the identified as T. saginata. The results re- commonest causes of infectious neuropathy vealed significantly high egg positive rate in the world, especially in Southeast Asia. of T. trichiura. However, A. lumbricoides These days many foreign workers from that was found to be controlled considerably area are staying in Japan, and the chances to by repeated chemotherapy during past 3 see the disease are increasing. We have to years. If chemotherapeutic agent is re- recognize leprous neuropathy as a candidate placed with oxantel-pyrantel tablet, better for the multiple mononeuropathy of acute result is expected. No clue was found for onset with painful dysesthesia similar to prevalence of T. solium from both human vascular neuropathy.—Authors’Abstract Immuno-Pathology

Anes, E., Kuhnel, M. P., Bos, E., Moniz- some actin assembly and maturation re- Pereira, J., Habermann, A., and Grif- sulting in killing of pathogenic mycobac- fiths, G. Selected lipids activate phago- teria. Nat Cell Biol. 5(9) (2003) 793Ð802. 392 International Journal of Leprosy 2003

Pathogenic mycobacteria such as Myco- hanced HIV-1 gag/env mRNA and p24 pro- bacterium tuberculosis and Mycobacterium tein synthesis exhibited by wild-type Tg an- avium facilitate disease by surviving intra- imals. Together, these results argue that cellularly within a potentially hostile envi- TLR2 plays a crucial role in the activation ronment: the macrophage phagosome. They of HIV-1 expression by mycobacterial coin- inhibit phagosome maturation processes, fections.—Authors’Abstract including fusion with lysosomes, acidifica- tion and, as shown here, membrane actin assembly. An in vitro assay developed for Black, G. F., Weir, R. E., Chaguluka, S. D., latex bead phagosomes (LBPs) provided in- Warndorff, D., Crampin, A. C., Mwaun- sights into membrane signalling events that gulu, L., Sichali, L., Floyd, S., Bliss, L., regulate phagosome actin assembly, a Jarman, E., Donovan, L., Andersen, P., process linked to membrane fusion. Differ- Britton, W., Hewinson, G., Huygen, K., ent lipids were found to stimulate or inhibit Paulsen, J., Singh, M., Prestidge, R., actin assembly by LBPs and mycobacterial Fine, P. E., and Dockrell, H. M. Gamma phagosomes in vitro. In addition, selected interferon responses induced by a panel of lipids activated actin assembly and phago- recombinant and purified mycobacterial some maturation in infected macrophages, antigens in healthy, non-Mycobacterium resulting in a significant killing of M. tuber- bovis BCG-vaccinated Malawian young culosis and M. avium. In contrast, the poly- adults. Clin. Diagn. Lab. Immunol. 10(4) unsaturated sigma-3 lipids behaved differ- (2003) 602Ð611. ently and stimulated pathogen growth. Thus, lipids can be involved in both stimulatory We have previously shown that young and inhibitory signalling networks in the adults living in a rural area of northern phagosomal membrane.—Authors’Abstract Malawi showed greater gamma interferon (IFN-gamma) responses to purified protein derivatives (PPD) prepared from environ- Bafica, A., Scanga, C. A., Schito, M. L., mental mycobacteria than to PPD from My- Hieny, S., and Sher, A. Cutting edge: in cobacterium tuberculosis. In order to define vivo induction of integrated HIV-1 ex- the mycobacterial species to which individ- pression by mycobacteria is critically de- uals living in a rural African population pendent on Toll-like receptor 2. J. Im- have been exposed and sensitized, we munol. 171(3) (2003) 1123Ð1127. tested T-cell recognition of recombinant and purified antigens from M. tuberculosis Mycobacterial infection has been impli- (38 kDa, MPT64, and ESAT-6), M. bovis cated as a possible factor in AIDS progres- (MPB70), M. bovis BCG (Ag85), and M. sion in populations where HIV-1 and Myco- leprae (65 kDa, 35 kDa, and 18 kDa) bacterium tuberculosis are coendemic. In in >600 non-M. bovis BCG-vaccinated support of this concept, we have previously young adults in the Karonga District of shown that HIV-1-transgenic (Tg) mice in- northern Malawi. IFN-gamma was mea- fected with mycobacteria display enhanced sured by enzyme-linked immunosorbent viral gene and protein expression. In this assay (ELISA) in day 6 supernatants of di- study, we demonstrate that the induction of luted whole-blood cultures. The recombi- HIV-1 observed in this model is dependent nant M. leprae 35-kDa and 18-kDa and pu- on Toll-like receptor 2 (TLR2), a pattern rified native M. bovis BCG Ag85 antigens recognition receptor known to be involved induced the highest percentages of respon- in mycobacteria-host interaction. Spleen ders, though both leprosy and bovine tuber- cells from HIV-1-Tg mice deficient in culosis are now rare in this population. The TLR2 (Tg/TLR2(Ð/Ð)) were found to be M. tuberculosis antigens ESAT-6 and completely defective in p24 production in- MPT64 and the M. bovis antigen MPB70 in- duced in response to live M. tuberculosis or duced the lowest percentages of responders. Mycobacterium avium as well as certain One of the subjects subsequently developed mycobacterial products. Importantly, fol- extrapulmonary tuberculosis; this individual lowing in vivo mycobacterial infection, had a 15-mm-diameter reaction to the Man- Tg/TLR2(Ð/Ð) mice failed to display the en- toux test and responded to M. tuberculosis 71, 4 Current Literature, Immuno-Pathology 393

PPD, Ag85, MPT64, and ESAT-6 but not to p132Ð151, p206Ð224 and p267Ð286), which any of the leprosy antigens. We conclude were the most permissive from each region that in this rural African population, expo- and recognized by non-contacts with signifi- sure to M. tuberculosis or M. bovis is much cantly lower frequencies than other subject less frequent than exposure to environmen- groups, were identified. From this prelimi- tal mycobacteria such as M. avium, which nary result, we conclude that these four pep- have antigens homologous to the M. leprae tides were likely to be M. leprae-specific.— 35-kDa and 18-kDa antigens. M. tuberculo- Authors’Abstract sis ESAT-6 showed the strongest association with the size of the Mantoux skin test in- duration, suggesting that among the three M. Dannenberg, A. M. Jr. Macrophage turn- tuberculosis antigens tested it provided the over, division and activation within best indication of exposure to, or infection developing, peak and “healed” tubercu- with, M. tuberculosis.—Authors’Abstract lous lesions produced in rabbits by BCG. Tuberculosis (Edinb). 83(4) (2003) 251Ð260. Chua-Intra, B., Wattanapokayakit, S., Srisungngam, S., Srisungngam, T., This review is a synthesis and analysis of Mahotarn, K., Brennan, P. J., and our nine experimental pathology papers on Ivanyi, J. T-cell recognition of peptides macrophage kinetics in dermal tuberculous from the Mycobacterium leprae 35 kDa lesions produced in rabbits by BCG. It is protein in Thai leprosy patients, healthy presented at this time to summarize the contacts, and non-contacts. Immunol. macrophage kinetics in both active and es- Lett. 88(1) (2003) 71Ð76. sentially healed tuberculous lesions and to suggest that the bacilli frequently multiply The objective of the study was to identify and are destroyed in the viable granulation Mycobacterium leprae-specific immuno- tissue of many small arrested tuberculous genic peptides for the development of a skin lesions.The turnover of mononuclear cells test reagent. Such a reagent is required for (MN)—which were mostly macrophages the detection of M. leprae infection and pos- with some medium and large lympho- sibly for the diagnosis of patients with active cytes—was most rapid in BCG lesions at leprosy. For this purpose, we analyzed the in 2Ð3 weeks (when tuberculin sensitivity and vitro responses of human peripheral blood acquired cellular resistance were at their mononuclear cell (PBMCs) to peptides from peaks). At this time, more macrophages en- the 35 kDa protein of M. leprae. This protein tered, more died or left, more remained at is of interest since it has no homologue the site, and more became activated than within the Mycobacterium tuberculosis com- before or afterwards. Before this time, the plex, although it has a homologue in Myco- host had no delayed-type hypersensitivity bacterium avium. The subjects enrolled in (DTH) and cell-mediated immunity (CMI), the study were paucibacillary (PB) and so that no antigen-specific enhancement of multibacillary (MB) leprosy patients, healthy the inflammatory response occurred. After contacts, and non-contacts. Seventy-three this time, the bacilli and their antigenic PB and 124 MB leprosy patients were re- products had decreased, so that the stimuli cruited from four leprosy clinics in Thailand. for cell infiltration and activation were re- Fifty-seven healthy contacts were household duced. In “healed” lesions, the MN turnover contacts. Twenty non-leprosy contacts had still occurred, but was decreased.The con- no family history of or exposure to leprosy. tinuous entry of live non-activated macro- PBMCs from individuals were tested for phages into the viable parts of tuberculous stimulation with 12 overlapping peptides lesions provides fresh intracellular sites from the M. leprae 35 kDa protein using the where tubercle bacilli may multiply before lymphocyte proliferation assay. These pep- they are again inhibited by the DTH and tides were located in four areas containing CMI of the host. In tuberculosis, bacillary three to six residues which were distinct for dormancy of long duration may only be the M. leprae product in comparison to that present in caseous necrotic tissue where no from M. avium. Four peptides (p60Ð76, live host cells exist.—Author’s Abstract 394 International Journal of Leprosy 2003

Florido, M., Correia-Neves, M., Cooper, bial products. To delineate the role of TLR A. M., and Appelberg, R. The cytolytic proteins in the development of host immune activity of natural killer cells is not in- responses against mycobacteria, wild-type volved in the restriction of Mycobacte- and TLR-deficient mice were infected with rium avium growth. Int. Immunol. 15(8) nonpathogenic Mycobacterium bovis bacil- (2003) 895Ð901. lus Calmette-Guerin (BCG). Two weeks af- ter intraperitoneal challenge with BCG, few Severe combined immunodeficiency bacilli were present in the lungs of wild- (SCID) mice were used to analyze the role type and TLR4(Ð/Ð) mice, whereas bacterial of NK cells in resistance to Mycobacterium loads were tenfold higher in the lungs of in- avium. The neutralization of IFN-gamma in fected TLR2(Ð/Ð) mice. BCG challenge in these animals led to an exacerbation of the vitro strongly induced proinflammatory infection associated with a reduction in cytokine secretion by macrophages from macrophage activation, suggesting a role wild-type and TLR4(Ð/Ð) mice but not by for NK cells in innate immunity to myco- TLR2(Ð/Ð) macrophages. In contrast, in- bacteria. In contrast, administration of anti- tracellular uptake, intracellular bacterial asialo-GM(1) polyclonal serum or mAb growth, and suppression of intracellular specific for Thy1.2 did not affect mycobac- bacterial growth in vitro by interferon- terial growth or macrophage activation de- gamma (IFN-gamma) were similar in spite causing the almost complete abroga- macrophages from all three mouse strains, tion of the natural cytolysis of a tumor cell suggesting that BCG growth in the lungs of target. Treatment with anti-asialo-GM(1)- TLR2(Ð/Ð) mice was a consequence of de- specific serum depleted only two-thirds of fective adaptive immunity. Antigenic stim- the Thy1.2+ spleen cells, and anti-Thy1.2 ulation of splenocytes from infected wild- treatment allowed for the persistence of a type and TLR4(Ð/Ð) mice induced T cell small number of cells still exhibiting an NK proliferation in vitro, whereas T cells from cell marker recognized by mAb DX5 and TLR2(Ð/Ð) mice failed to proliferate. Unex- able to express IFN-gamma as analyzed by pectedly, activated CD4(+) T cells from flow cytometry. In vivo treatment of both TLR-deficient mouse strains secreted B6.SCID mice with anti-NK1.1 mAb again little IFN-gamma in vitro compared with failed to affect resistance to infection and control T cells. A role for TLR4 in the con- allowed for the persistence of 2Ð8% of IFN- trol of bacterial growth and IFN-gamma gamma-producing cells, many of them still production in vivo was observed only when expressing the DX5 marker. In vitro deple- mice were infected with higher numbers of tion studies showed that removal of IFN- BCG. Thus, TLR2 and TLR4 appear to reg- gamma-expressing cells required the com- ulate distinct aspects of the host immune re- bined action of anti-Thy1.2, anti-Ly49C sponse against BCG.—Authors’Abstract and DX5 antibodies in the presence of com- plement. Our data show that resistance to M. avium mediated by NK cells is indepen- Lopez, J. P., Clark, E., and Shepherd, V. dent of their cytolytic activity, and that L. Surfactant protein A enhances Myco- there is a marked phenotypic and functional bacterium avium ingestion but not heterogeneity of the NK cell lineage in vivo killing by rat macrophages. J. Leukoc. during infection.—Authors’Abstract Biol. 74(4) (2003) 523Ð530.

Mycobacterium avium complex (MAC) Heldwein, K. A., Liang, M. D., Andresen, is a significant cause of opportunistic infec- T. K., Thomas, K. E., Marty, A. M., tion in patients with acquired immunodefi- Cuesta, N., Vogel, S. N., and Fenton, ciency syndrome. Although the major route M. J. TLR2 and TLR4 serve distinct of entry of MAC is via the gastrointestinal roles in the host immune response tract, MAC can infect humans through the against Mycobacterium bovis BCG. J. respiratory tract and eventually encounter Leukoc. Biol. 74(2) (2003) 277Ð286. alveolar macrophages within the lung. Once in the lung, MAC can potentially in- Toll-like receptor (TLR) proteins mediate teract with surfactant protein A (SP-A), an cellular activation by microbes and micro- important component of the pulmonary 71, 4 Current Literature, Immuno-Pathology (Leprosy) 395 innate-immune response. Previous work alpha. However, intracellular survival of on other pulmonary pathogens including MAC was not altered by preopsonization Mycobacterium bovis Bacillus Calmette- with SP-A. In addition, inhibitors of in- Guerin (BCG) suggests that SP-A partici- ducible NO synthase did not alter MAC pates in promoting efficient clearance of clearance. These results suggest that SP-A these organisms by alveolar macrophages. can bind to and enhance the uptake of MAC In the present study, we investigated the by alveolar macrophages, similar to previ- role of SP-A in clearance of MAC by cul- ous findings with BCG and Mycobacterium tured rat macrophages. SP-A bound to tuberculosis.However, unlike BCG and MAC organisms and enhanced the inges- other pulmonary pathogens that are cleared tion of the mycobacteria by macrophages. effectively in the presence of SP-A via a Infection of macrophages with SP-A-MAC NO-dependent pathway, macrophage- complexes induced the production of nitric mediated clearance of MAC is not en- oxide (NO) and tumor necrosis factor- hanced by SP-A.—Authors’Abstract

Immuno-Pathology (Leprosy)

Antunes, S. L., Liang, Y., Neri, J. A., detected. In conclusion, the number of Haak-Frendscho, M., and Johansson, PGP- and NGFr-positive fibers were not O. The expression of NGFr and PGP 9.5 significantly different in the reactional and in leprosy reactional cutaneous lesions: post-reactional biopsies in the present an assessment of the nerve fiber status study. NGFr-staining of the nerve fibers is using immunostaining. Arq. Neurop- different from their PGP-imunoreactivity siquiatr. 61(2B) (2003) 346Ð352. and the evaluation of the nerve fiber status on an innervated target organ should be car- The effects of reactional episodes on the ried out choosing markers for both compo- cutaneous nerve fibers of leprosy patients nents of nerve fibers (Schwann cells and ax- was assessed in six patients (three with re- ons).—Authors’Abstract versal reactions and three with erythema nodosum leprosum). Cryosections of cuta- neous biopsy of reactional lesions taken Antunes, S. L., Liang, Y., Neri, J. A., during the episode and of another sample Sarno, E. N., Haak-Frendscho, M., during the remission period were immunos- and Johansson, O. Mast cell subsets tained with anti-NGFr and anti-PGP 9.5 (in- and neuropeptides in leprosy reactions. direct immunofluorescence). We found no Arq Neuropsiquiatr. 61(2A) (2003) significant statistical difference in the num- 208Ð219. ber of NGFr- and PGP 9.5-positive fibers between the reactional and post-reactional The immunohistochemical identification groups. A significant difference was de- of neuropeptides (calcitonin gene-related tected between the number of NGFr and peptide, vasoactive intestinal polypeptide, PGP 9.5-stained fibers inside of the reac- substance P, alpha-melanocyte stimulating tional group of biopsy cryosections but this hormone and gamma-melanocyte stim- difference was ascribed to the distinct ulating hormone) quantification of mast aspects of the nerve fibers displayed cells and their subsets (tryptase/chymase- whether stained with anti-NGFr or with immunoreactive mast cells = TCMC and anti-PGP 9.5; NGFr-positive branches tryptase-immunoreactive mast cells = TMC) looked larger and so interpreted as contain- were determined in biopsies of six patients ing more fibers. In addition, a substantial with leprosy reactions (three patients with number NGFr-positive fibers were PGP type I reaction and three with type II). 9.5-negative. No differences in the number Biopsies were compared with those taken of stained fibers among the distinct cu- from the same body site in the remission taneous regions examined (epidermis + stage of the same patient. We found a rela- upper dermis, mid and deep dermis) was tive increase of TMC in the inflammatory 396 International Journal of Leprosy 2003 infiltrate of the reactional biopsies com- found regarding neuroptide expression in pared to the post-reactional biopsy. Also, the reactional and post-reactional biopsies. the total number of mast cells and the The relative increase of TMC in the reac- TMC/TCMC ratio in the inflammatory tional infiltrates could implicate this mast infiltrate was significantly higher than in the cell subset in the reported increase of the intervening dermis of the biopsies of both immune response in leprosy reactions.— periods. No significant difference was Authors’Abstract

Immuno-Pathology (Tuberculosis)

Boitel, B., Ortiz-Lombardia, M., Duran, be brought down to the basal level. These R., Pompeo, F., Cole, S. T., Cervenan- results clearly suggest that cytokines, par- sky, C., and Alzari, P. M. PknB kinase ticularly IFN-gamma, induced NO release activity is regulated by phosphorylation and its reactive product with oxygen radi- in two Thr residues and dephosphoryla- cal, peroxynitrite, could play an important tion by PstP, the cognate phospho- role in the killing of M. tuberculosis by hu- Ser/Thr phosphatase, in Mycobacterium man mononuclear phagocytes. A significant tuberculosis. Mol. Microbiol. 49(6) production of interleukin-4 and interleukin- (2003) 1493Ð1508. 10, by the ex-vivo matured, untreated mac- rophages from the active tuberculosis pa- See Current Literature, Molecular and tients indicate that regulation of cytokine Genetic Studies, p. 422 network to encourage in situ/local produc- tion of nitric oxide may be useful in the management of pulmonary tuberculosis.— Bose, M., Farnia, P., Sharma, S., Chat- Authors’Abstract topadhya, D., and Saha, K. Nitric oxide dependent killing of Mycobacterium tu- berculosis by human mononuclear Botha, T. and Ryffel, B. Reactivation of phagocytes from patients with active tu- latent tuberculosis infection in TNF- berculosis. Int. J. Immunopathol. Phar- deficient mice. J. Immunol. 171(6) (2003) macol. 12(2) (1999) 69Ð79. 3110Ð3118.

In this study we have demonstrated that TNF-deficient mice are highly suscepti- nitric oxide, the product of the arginine de- ble to Mycobacterium tuberculosis H37Rv pendent pathway of human mononuclear infection. Here we asked whether TNF is phagocytes effectively kills the M. tubercu- required for postinfectious immunity in losis in-vitro. The release of reactive nitro- aerosol-infected mice. Chemotherapy for 4 gen intermediates was triggered by incuba- wk commencing 2 wk postinfection re- tion with various proinflammatory cytokines duced CFU to undetectable levels. While namely IFN gamma,TNF-alpha and IL-1R. wild-type mice had a slight rise in CFU, but We have earlier shown that human mononu- controlled infection upon cessation of clear phagocytes can be induced to release chemotherapy, TNF-deficient mice devel- nitric, oxide (NO) radicals which can kill tu- oped reactivation of infection with high mour cells. In the present communication, bacterial loads in lungs, spleen, and liver, by using colony forming assays we demon- which was fatal within 13Ð18 wk. The in- strated that human mononuclear phagocytes creased susceptibility of TNF-deficient can effectively kill M. tuberculosis by using mice was accompanied by diminished re- a NO dependent pathway. Treatment of cruitment and activation of T cells and mononuclear phagocytes with L-arginine macrophages into the lung, with defective resulted in markedly increased killing ac- granuloma formation and reduced inducible tivity whereas, by using NGMMA, an ana- NO synthase expression. Reduced chemo- logue of L-arginine, the cidal activity could kine production in the lung might explain 71, 4 Current Literature, Immuno-Pathology (Tuberculosis) 397 suboptimal recruitment and activation of T pattern. The ability of peptide 91Ð110 to cells and uncontrolled infection. Therefore, bind a wide range of HLA-DR molecules, despite a massive reduction of the myco- and to stimulate a Th1-type interferon bacterial load by chemotherapy, TNF- (IFN)-gamma response more readily, en- deficient mice were unable to compensate courage the use of this peptide as a subunit and mount a protective immune response. vaccine component.—Authors’Abstract In conclusion, endogenous TNF is critical to maintain latent tuberculosis infection, and in its absence no specific immunity is Danelishvili, L., McGarvey, J., Li, Y. J., generated.—Authors’Abstract and Bermudez, L. E. Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in Caccamo, N., Barera, A., Di Sano, C., human macrophages and alveolar epithe- Meraviglia, S., Ivanyi, J., Hudecz, F., lial cells. Cell. Microbiol. 5(9) (2003) Bosze, S., Dieli, F., and Salerno, A. Cy- 649Ð660. tokine profile, HLA restriction and TCR sequence analysis of human CD4+ T Mycobacterium tuberculosis interacts clones specific for an immunodominant with macrophages and epithelial cells in the epitope of Mycobacterium tuberculosis alveolar space of the lung, where it is able 16-kDa protein. Clin. Exp. Immunol. to invade and replicate in both cell types. 133(2) (2003) 260Ð266. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but The identification of immunodominant the mechanisms of host cell death are not and universal mycobacterial peptides could well understood. We examined the induc- be applied to vaccine design and have an tion of apoptosis and necrosis of human employment as diagnostic reagents. In this macrophages (U937) and type II alveolar paper we have investigated the fine speci- epithelial cells (A549) by virulent (H37Rv) ficity, clonal composition and HLA class II and attenuated (H37Ra) M. tuberculosis restriction of CD4+ T cell clones specific strains. Apoptosis was determined by both for an immunodominant epitope spanning enzyme-linked immunosorbent assay amino acids 91Ð110 of the 16-kDa protein (ELISA) and TdT-mediated dUTP nick end of Mycobacterium tuberculosis. Twenty- labelling (TUNEL) assay, whereas necrosis one of the tested 28 clones had a Th1 pro- was evaluated by the release of lactate de- file, while seven clones had a Th0 profile. hydrogenase (LDH). Both virulent and at- None of the clones had a Th2 profile. While tenuated M. tuberculosis induced apoptosis the TCR AV gene usage of the clones was in macrophages; however, the attenuated heterogeneous, a dominant TCR BV2 gene strain resulted in significantly more apopto- family was used by 18 of the 28 clones. The sis than the virulent strain after 5 days of in- CDR3 regions of BV2+ T cell clones fection. In contrast, cytotoxicity of alveolar showed variation in lengths, but a putative cells was the result of necrosis, but not common motif R-L/V-G/S-Y/W-E/D was apoptosis. Although infection with M. tu- detected in 13 of the 18 clones. Moreover, berculosis strains resulted in apoptosis of the last two to three residues of the putative 14% of the cells on the monolayer, cell CDR3 loops, encoded by conserved BJ se- death associated with necrosis was ob- quences, could also play a role in peptide served in 59% of alveolar epithelial cells af- recognition. Antibody blockade and fine re- ter 5 days of infection. Infection with M. tu- striction analysis using HLA-DR homozy- berculosis suppressed apoptosis of alveolar gous antigen-presenting cells established epithelial cells induced by the kinase in- that 16 of 18 BV2+ peptide-specific clones hibitor, staurosporine. Because our findings were DR restricted and two clones were suggest that M. tuberculosis can modulate DR-DQ and DR-DP restricted. Additionally, the apoptotic response of macrophages and five of the 18 TCRBV2+ clones recognized epithelial cells, we carried out an apoptosis peptide 91Ð110 in association with both pathway-specific cDNA microarray analy- parental and diverse HLA-DR molecules, sis of human macrophages and alveolar ep- indicating their promiscuous recognition ithelial cells. Whereas the inhibitors of 398 International Journal of Leprosy 2003 apoptosis, bcl-2 and Rb, were upregulated sis patients through the Fas-FasL and per- over 2.5-fold in infected (48 hr) alveolar forin pathways. Mtb-induced CD4(+) CTL epithelial cells, the proapoptotic genes, bad from tuberculosis patients and N controls and bax, were downregulated. The opposite preferentially employed the Fas-FasL mech- was observed when U937 macrophages anism. Mtb-induced CD8(+) CTL effector were infected with M. tuberculosis. Upon cells from patients used the perforin-based infection of alveolar epithelial cells with M. mechanism while cells from N controls also tuberculosis, the generation of apoptosis, as used the Fas-FasL pathway. While Mtb- determined by the expression of caspase-1, induced gammadelta CTL from patients caspase-3 and caspase-10, was inhibited. and PPD(Ð)N employed the latter mecha- Inhibition of replication of intracellular nism cells from PPD(+)N individuals also bacteria resulted in an increase in apoptosis used the perforin pathway. It can be con- in both cell types. Our results showed that cluded that shifts in the CTL response and the differential induction of apoptosis be- the cytolytic mechanisms take place as the tween macrophages and alveolar epithelial pulmonary involvement becomes more se- cells represents specific strategies of M. tu- vere.—Authors’Abstract berculosis for survival in the host.—Au- thors’Abstract Dieli, F., Taniguchi, M., Kronenberg, M., Sidobre, S., Ivanyi, J., Fattorini, L., De La Barrera, S. S., Finiasz, M., Frias, Iona, E., Orefici, G., De Leo, G., A., Aleman, M., Barrionuevo, P., Fink, Russo, D., Caccamo, N., Sireci, G., Di S., Franco, M. C., Abbate, E., and del Sano, C., and Salerno, A. An anti- C Sasiain, M. Specific lytic activity inflammatory role for Valpha14 NK T against mycobacterial antigens is in- cells in Mycobacterium bovis bacillus versely correlated with the severity of tu- Calmette-Guerin-infected mice. J. Im- berculosis. Clin. Exp. Immunol. 132(3) munol. 171(4) (2003) 1961Ð1968. (2003) 450Ð461. The possible contribution of NKT cells to The ability of peripheral blood mononu- resistance to Mycobacterium tuberculosis clear cells (PBMC) from patients with ac- infection remains unclear. In this paper we tive tuberculosis to display cytotoxic re- characterized the Valpha14 NKT cell popu- sponses against autologous Mycobacterium lation following infection with Mycobacte- tuberculosis (Mtb)-pulsed macrophages rium bovis bacillus Calmette-Guerin was evaluated. Non-MHC restricted cell- (BCG). BCG infection determined an early dependent lytic activity was observed in ex expansion of Valpha14 NKT cells in liver, vivo effector cells from tuberculosis pa- lungs, and spleen, which peaked on day 8 tients and was mediated mainly by and was sustained until day 30. However, CD3(+)gammadelta TCR(+) T (gam- an NK1.1(+) Valpha14 NKT population madelta T) cells bearing CD56 and/or preferentially producing IFN-gamma pre- CD16 molecules. MHC-restricted and non- dominated at an early stage (day 8), which MHC restricted cytotoxic T cells (CTL) was substituted by an NK1.1(Ð) population were differentially expanded upon stimula- preferentially producing IL-4 at later stages tion with Mtb in tuberculosis patients and (day 30). Despite the fact that Valpha14 normal controls (N). Class-I restricted NKT cell-deficient mice eliminated BCG as CD8(+) CTL and class-II restricted CD4(+) did control mice, they had significantly CTL were generated in PPD(+)N and to a higher numbers of granulomas in liver and lesser extent in PPD(Ð)N. Mtb-stimulated lungs. Additionally, while control mice de- effector cells from tuberculosis patients be- veloped organized small granulomas, those came progressively non-MHC restricted in Valpha14 NKT-deficient mice had signs CD4(Ð)CD8(Ð)gammadelta T cells, while of caseation, large cellular infiltrates, and lytic activity of CD4(+) and CD8(+)CTL some multinucleated macrophages, sug- decreased gradually as the disease became gesting that Valpha14 NKT cells may actu- more severe. On the other hand, target cells ally work as anti-inflammatory cells by lim- were lysed by ex vivo cells from tuberculo- iting excessive lymphocyte influx and tis- 71, 4 Current Literature, Immuno-Pathology (Tuberculosis) 399 sue pathology. In agreement, we found an Garcia-Perez, B. E., Mondragon-Flores, increased spontaneous production and R., and Luna-Herrera, J. Internalization mRNA expression of TNF-alpha in liver of Mycobacterium tuberculosis by and lungs of Valpha14 NKT-deficient mice, macropinocytosis in non-phagocytic cells. whose neutralization in vivo by anti-TNF- Microb. Pathog. 35(2) (2003) 49Ð55. alpha mAbs consistently reduced the num- ber of granulomas in liver and lungs. To- Mycobacterium tuberculosis (MTB) is an gether, our results support a regulatory role intracellular pathogen that initially invades for Valpha14 NKT cells in the course of the alveolar macrophages of infected indi- BCG infection through their ability to limit viduals. MTB is also known to invade res- the extent of inflammatory response and piratory epithelial cells. To understand the point to an important role for this cell sub- mechanism of epithelial invasion, we in- set as a regulator of the balance between vestigated the interaction of MTB (H37Rv protective responses and immunopathol- strain) with non-phagocytic type-II (A549) ogy.—Authors’Abstract human pneumocytes. The internalization of the organism was analyzed through optical, fluorescent and electron (transmission and Dubnau, E. and Smith, I. Mycobacterium scanning) microscopy. Infection of A549 tuberculosis gene expression in macro- cells with MTB showed intracellular mul- phages. Microbes Infect. 5(7) (2003) tiplication of the organism. Microscopy 629Ð637. revealed the formation of membrane ruffles totally or partially surrounding the surface This review provides a discussion on adherent mycobacteria. Fluorescent mi- the current information about the response croscopy showed that MTB induced of Mycobacterium tuberculosis to the en- changes in the distribution of actin fila- vironment encountered in the macro- ments. Since heat killed MTB failed to phage. We focus on the types of genes induce actin mobilization, perhaps, inter- shown to be upregulated when the patho- nalization process is mediated by the solu- gen grows in macrophages and discuss the ble products of the metabolically active possible roles of these genes in adaptation mycobacterium. Overall, these findings to the conditions in the eukaryotic cell, in suggest that internalization of MTB by non- the context of enhancing the survival of phagocytic cells might be through a mac- the pathogen during infection.—Authors’ ropinocytosis or induced-phagocytosis Abstract processes, and possibly some bacterial se- cretory product is responsible for triggering this phenomenon.—Authors’Abstract Flynn, J. L. and Chan, J. Immune evasion by Mycobacterium tuberculosis: living with the enemy. Curr. Opin. Immunol. Gehring, A. J., Rojas, R. E., Canaday, D. 15(4) (2003) 450Ð455. H., Lakey, D. L., Harding, C. V., and Boom, W. H. The Mycobacterium tuber- Mycobacterium tuberculosis is successful culosis 19-kilodalton lipoprotein inhibits as a pathogen because of its ability to per- gamma interferon-regulated HLA-DR sist in an immunocompetent host. This bac- and Fc gamma R1 on human macro- terium lives within the macrophage, a cell phages through Toll-like receptor 2. In- whose function is the elimination of mi- fect. Immun. 71(8) (2003) 4487Ð4497. crobes. Recent advances have improved our understanding of how M. tuberculosis Mycobacterium tuberculosis survives in evades two major antimicrobial mechan- macrophages in the face of acquired isms of macrophages: phagolysosome fu- CD4(+) T-cell immunity, which controls sion and the production of toxic reactive ni- but does not eliminate the organism. trogen intermediates. M. tuberculosis also Gamma interferon (IFN-gamma) has a cen- modulates antigen presentation to prevent tral role in host defenses against M. tuber- the detection of infected macrophages by culosis by activating macrophages and reg- CD4(+) T cells.—Authors’Abstract ulating major histocompatibility complex 400 International Journal of Leprosy 2003 class II (MHC-II) antigen (Ag) processing. tions (1Ð20 microg/ml) of anti mycobacte- M. tuberculosis interferes with IFN-gamma rial drugs (isoniazid, rifampin, thiaceta- receptor (IFN-gamma R) signaling in zone, streptomycin, and ethambutol). Inhi- macrophages, but the molecules responsi- bition of the intracellular growth of M. tu- ble for this inhibition are poorly defined. berculosis by all drugs studied/correlated This study determined that the 19-kDa with inhibition of permeability transition lipoprotein from M. tuberculosis inhibits (PT) alterations; TNFalpha, IL-10, and ni- IFN-gamma-regulated HLA-DR protein tric oxide production, and caspase-1 activa- and mRNA expression in human macro- tion. However, these drugs did not affect phages. Inhibition of HLA-DR expression PPD-induced apoptosis or its associated was associated with decreased processing events, suggesting that the ability of an- and presentation of soluble protein Ags and timycobacterial drugs to block macrophage M. tuberculosis bacilli to MHC-II-restricted apoptosis could be explained by their ef- T cells. Inhibition of HLA-DR required fects on the metabolic activities of Mtb. All prolonged exposure to 19-kDa lipoprotein drugs, except isoniazid, at higher concen- and was blocked with a monoclonal anti- trations, induced PT alterations in nonin- body specific for Toll-like receptor 2 (TLR- fected macrophages in a way that appears to 2). The 19-kDa lipoprotein also inhibited be dependent of calcium, since a calcium IFN-gamma-induced expression of Fc chelator prevented it. The results presented gamma RI. Thus, M. tuberculosis, through herein suggest that the pharmacological 19-kDa lipoprotein activation of TLR-2, in- manipulation of pathways associated with hibits IFN-gamma R signaling in human macrophage apoptosis may affect the intra- macrophages, resulting in decreased MHC- cellular growth of Mtb.—Authors’Abstract II Ag processing and recognition by MHC- II-restricted CD4 T cells. These findings provide a mechanism for M. tuberculosis Hanekom, W. A., Mendillo, M., Manca, persistence in macrophages.—Authors’Ab- C., Haslett, P. A., Siddiqui, M. R., stract Barry, C. 3rd, and Kaplan, G. Myco- bacterium tuberculosis inhibits matura- tion of human monocyte-derived den- Gil, D., Garcia, L. F., and Rojas, M. Mod- dritic cells in vitro. J. Infect. Dis. 188(2) ulation of macrophage apoptosis by an- (2003) 257Ð266. timycobacterial therapy: physiological role of apoptosis in the control of Myco- To induce effector immunity, dendritic bacterium tuberculosis. Toxicol. Appl. cells (DCs) must differentiate into fully ma- Pharmacol. 190(2) (2003) 111Ð119. ture cells. We show that, after human monocyte-derived DCs were infected with Apoptosis is a form of cell death that virulent Mycobacterium tuberculosis, up- avoids inflammatory responses. We had regulation of cellular-surface maturation previously reported that Mycobacterium tu- markers was minimal and reversible. In the berculosis (Mtb) and Purified Protein De- presence of a potent stimulus for maturation rivative (PPD) induce apoptosis in murine (tumor necrosis factor [TNF]-alpha, inter- macrophages. The production of TNFalpha leukin [IL]-1beta, and prostaglandin E2 and IL-10 in response to Mtb infection [PGE2]), M. tuberculosis inhibited phe- modulates apoptosis by controlling nitric notypic DC maturation. M. tuberculosis- oxide production and caspase activation. To infected DCs had an impaired ability to in- further explore the role of macrophage duce allogeneic lymphoproliferation and apoptosis in tuberculosis, we studied the ca- activated autologous memory CD4+ and pacity of standard antimycobacterial drugs CD8+ T cells optimally only in the pres- to modulate different events associated with ence of TNF-alpha, IL-1beta, and PGE2. the induction of apoptosis. The B10R Thus, virulent M. tuberculosis inhibits phe- murine macrophage line was infected or not notypic and functional maturation of human with Mtb (5:1 bacteria to macrophage ratio) monocyte-derived DCs. This mechanism, or exposed to PPD (10 microg/ml), in the which has been described elsewhere for var- presence or absence of varying concentra- ious viruses and for the virulent mycobacte- 71, 4 Current Literature, Immuno-Pathology (Tuberculosis) 401 rium M. leprae, may be a novel mechanism tuberculosis recombinant 27-kilodalton that this pathogen uses to evade the host’s lipoprotein induces a strong Th1-type im- immune response.—Authors’Abstract mune response deleterious to protection. Infect. Immun. 71(6) (2003) 3146Ð3154.

Hohn, H., Kortsik, C., Tully, G., Nilges, Th1 immune response is essential in the K., Necker, A., Freitag, K., Neukirch, protection against mycobacterial intracellu- C., Galle, P., Lohr, H., and Maeurer, lar pathogens. Lipoproteins trigger both hu- M. J. Longitudinal analysis of Mycobac- moral and cellular immune responses and terium tuberculosis 19-kDa antigen- may be candidate protective antigens. We specific T cells in patients with pul- studied in BALB/c mice the immunogenic- monary tuberculosis: association with ity and the protection offered by the recom- disease activity and cross-reactivity to a binant 27-kDa Mycobacterium tuberculosis peptide from HIVenv gp120. Eur. J. Im- lipoprotein and the corresponding DNA munol. 33(6) (2003) 1613Ð1623. vaccine. Immunization with the 27-kDa antigen resulted in high titers of im- CD8(+) T cells play a central role in munoglobulin G1 (IgG1) and IgG2a with a immune protection against infection with typical Th1 profile and a strong delayed hy- Mycobacterium tuberculosis. One of the persensitivity response. A strong prolifera- target epitopes for anti-M. tuberculosis tion response was observed in splenocytes, directed CD8(+) T cells is the HLA-A2- and significant nitric oxide production and restricted 19-kDa lipoprotein peptide gamma interferon secretion but not inter- VLTDGNPPEV. T cell clones directed leukin 10 secretion were measured. Based against this epitope recognized not only the on these criteria, the 27-kDa antigen in- nominal peptide ligand, but also a closely duced a typical Th1-type immune response related peptide (VPTDPNPPEV) from the thought to be necessary for protection. Sur- HIV envelope gp120 (HIV(env) gp120) prisingly, in 27-kDa-vaccinated mice (pro- protein characterized by IFN-gamma re- tein or DNA vaccines) challenged by M. tu- lease. This cross-reactivity was confirmed in berculosis H37Rv or BCG strains, there ex vivo in M. tuberculosis 19-kDa tetramer- was a significant increase in the numbers of sorted T cells from patients with tuberculo- CFU in the spleen compared to that for con- sis and in HIVgp120 tetramer-reactive T trol groups. Furthermore, the protection cells sorted from HIV(+) patients. M. tuber- provided by BCG or other mycobacterial culosis 19-kDa antigen-reactive T cells were antigens was completely abolished once the present in HLA-A2(+) patients (10/10) with 27-kDa antigen was added to the vaccine HIV infection with no evidence of M. tuber- preparations. This study indicates that the culosis infection, but they are absent in pe- 27-kDa antigen has an adverse effect on the ripheral blood lymphocytes from healthy protection afforded by recognized vaccines. HLA-A2(+) individuals (10/10). M. tuber- We are currently studying how the 27-kDa culosis 19-kDa antigen-reactive T cells were antigen modulates the mouse immune re- elevated in acute pulmonary tuberculosis, sponse.—Authors’Abstract declined with response to therapy (7/10 pa- tients) and resided in the terminally differ- entiated CD8(+) T cell subset. CD8(+) Jo, E. K., Park, J. K., and Dockrell, H. cross-reactive T cells recognizing HIV(env) M. Dynamics of cytokine generation in or M. tuberculosis 19-kDa antigens may patients with active pulmonary tubercu- contribute to pathogenesis in individuals co- losis. Curr. Opin. Infect. Dis. 16(3) infected with both pathogens and may also (2003) 205Ð210. present a marker for active tuberculosis.— Authors’Abstract PURPOSE OF REVIEW: Cytokines have been implicated in the protective im- munity, pathophysiology and development Hovav, A. H., Mullerad, J., Davidovitch, of tuberculosis. Most people who become L., Fishman, Y., Bigi, F., Cataldi, A., infected with Mycobacterium tuberculosis and Bercovier, H. The Mycobacterium mount an effective protective immune re- 402 International Journal of Leprosy 2003 sponse, but 5Ð10% develop disease. Active whether Toll-like receptor 4 (TLR4) was pulmonary tuberculosis can be considered critical in resistance to M. tuberculosis in- to reflect an ineffective immune response fection, we compared the morbidity and against mycobacterial infection. A better mortality of TLR4-defective C3H/HeJ mice understanding of how cytokine production to those of TLR4-sufficient C3H mouse sub- contributes to immunity and pathology strains. TLR4-defective C3H/HeJ mice and would aid the development of new vaccines TLR4-sufficient C3H/HeSnJ, C3HeB/FeJ, and therapeutic strategies. RECENT FIND- and C3H/HeOuJ mice were infected by the INGS: At the time of diagnosis, production aerosol route with M. tuberculosis. TLR4- of M. tuberculosis or mycobacterial antigen- defective C3H/HeJ mice had levels of cy- induced interferon-gamma by peripheral tokines in their bronchoalveolar lavage fluids blood mononuclear cells from tuberculosis and in vitro mycobacterial antigen-specific patients is usually depressed, compared recall responses similar to those of other with that of healthy control subjects, C3H mouse substrains. In addition, bacterial whereas cytokine production at the site of replication and long-term survival of mice disease is elevated. In most patients, de- following infection appeared to be indepen- pressed interferon-gamma production by dent of TLR4. Interestingly, C3HeB/FeJ peripheral blood mononuclear cells seems mice were significantly more susceptible to to be a transient response because it is sig- M. tuberculosis infection, indicating that nificantly increased in most active tubercu- genetic heterogeneity among inbred C3H losis patients during and following success- mouse substrains modifies resistance to in- ful antituberculous therapy. However, some fection. Therefore, cautious interpretation is patients remain anergic in vivo and in vitro required when the C3H/HeJ strain is used after chemotherapy, and the underlying bio- as a model of a TLR4-defective mouse chemical mechanisms for T cell anergy in strain, as there are significant allelic differ- modulating protection or pathology in tu- ences between C3H/HeJ and other C3H berculosis needs further clarification. mouse substrains in response to M. tubercu- Among the cytokines contributing to pro- losis infection. With this caveat, our data in- tective immunity, interleukins 12 and 18, dicate that TLR4 may not be required for and tumour necrosis factor-alpha are impor- optimal immunity of mice to M. tuberculo- tant, the basis of recent studies with tuber- sis.—Authors’Abstract culosis patients. SUMMARY: A more com- plete understanding of cytokine dynamics in individual cells in active pulmonary tu- Mukae, H., Ashitani. J., Tokojima, M., berculosis patients will provide further Ihi, T., Kohno, S., and Matsukura, S. knowledge about immunopathogenesis and Elevated levels of circulating adhesion protective immunity in human tuberculosis. molecules in patients with active pul- This should ultimately enhance develop- monary tuberculosis. Respirology. 8(3) ment of preventive and therapeutic strate- (2003) 326Ð331. gies against this enormously successful in- tracellular pathogen.—Authors’Abstract Elevated levels of circulating adhesion molecules in patients with active pulmonary tuberculosis MUKAE H, ASHITANI J-I, Kamath, A. B., Alt, J., Debbabi, H., and Be- TOKOJIMA M, IHI T, KOHNO S, MAT- har. S. M. Toll-like receptor 4-defective SUKURA S. Respirology 2003; 8: 326Ð331 C3H/HeJ mice are not more susceptible OBJECTIVE: Recent studies have indicated than other C3H substrains to infection the importance of cell adhesion molecules with Mycobacterium tuberculosis. Infect. in the pathogenesis of various inflammatory Immun. 71(7) (2003) 4112Ð4118. lung diseases. Our study was designed to determine whether five soluble adhesion Mycobacterium tuberculosis produces a molecules including soluble L-, E- and variety of molecules capable of activating P-selectin (sL-, sE- and sP-selectin), in- Toll-like receptors, a family of pattern recog- tercellular adhesion molecule-1 (sICAM-1), nition receptors expressed by macrophages and vascular cell adhesion molecule-1 and a variety of other cells. To determine (sVCAM-1) in serum reflect the severity of 71, 4 Current Literature, Microbiology 403 active pulmonary tuberculosis (TB), and the number of mycobacteria bacilli present) whether there is a distinct profile of these and all of the above adhesion molecules, soluble molecules in this disease. except for sL-selectin. Serum levels of sE- METHODOLOGY: Using enzyme-linked selectin, sL-selectin and sICAM-1 also cor- immunosorbent assays, we measured the related with the CXR radiological score. serum levels of these five soluble adhesion Higher levels of sL-selectin and sICAM-1 molecules in 31 patients with active TB and were detected in the serum of patients with 11 healthy volunteers. RESULTS: Serum radiological cavity formation compared to levels of sE-selectin, sP-selectin and those without. The ESR, C-reactive protein sICAM-1, but not sL-selectin or sVCAM-1, and circulating neutrophil counts all corre- were significantly higher in patients with lated significantly with sE-selectin, sP-se- active TB than in the control subjects (p lectin, sICAM-1 and sVCAM-1. CONCLU- <0.001, each). Significant correlations were SION: The results suggest that there is a dis- detected only between serum levels of sE- tinct profile of soluble adhesion molecules in selectin and sP-selectin, sE-selectin and active pulmonary TB and that sE-selectin, sICAM-1, and sP-selectin and sICAM-1. sP-selectin, and especially sICAM-1 appear There was a significant correlation between to be the most sensitive clinical measures of the Gaffky scale result (a scale assessing disease severity.—Authors’Abstract

Microbiology

Agarwal, N. and Tyagi, A. K. Role of 5′- tween MAA and MAP in the number of tan- TGN-3′ motif in the interaction of myco- dem repeat motifs occurring at each MIRU bacterial RNA polymerase with a pro- locus. Locus specific PCR at 4 of these loci moter of ‘extended-10′ class. FEMS segregated MAP into two major groups, Microbiol. Lett. 225(1) (2003) 75Ð83. which could be differentiated from ovine- pigmented strains of MAP and the MAP See Current Literature, Molecular and vaccine strain 316F. The same PCR differ- Genetic Studies, p. 420 entiated MAA into five MIRU profiles. PCR at either MIRU locus 1 or MIRU locus 4 distinguished between MAP and all other Bull, T. J., Sidi-Boumedine, K., McMinn, M. avium complex (MAC) tested. PCR at E. J., Stevenson, K., Pickup, R., and both loci 1 and 4 also distinguished MAP Hermon-Taylor, J. Mycobacterial inter- from Mycobacterium intracellulare. MIRU spersed repetitive units (MIRU) differen- typing may provide an additional simple tiate Mycobacterium avium subspecies and rapid procedure for the differentiation paratuberculosis from other species of the between MAP and other MAC.—Authors’ Mycobacterium avium complex. Mol. Abstract Cell. Probes. 17(4) (2003) 157Ð164.

Mycobacterial interspersed repetitive Consaul, S. A., Jacobs, W. R. Jr., and units (MIRU) comprise short tandem repeat Pavelka, M. S. Jr. Extragenic suppres- structures found at multiple loci throughout sion of the requirement for diamino- the Mycobacterium tuberculosis genome pimelate in diaminopimelate auxotrophs and have been used for typing these of Mycobacterium smegmatis. FEMS pathogens. We have identified MIRU at 18 Microbiol. Lett. 225(1) (2003) 131Ð135. conserved loci throughout the common por- tions of the Mycobacterium avium sub- Mycobacteria, like many prokaryotes, species paratuberculosis (MAP) and M. have a peptidoglycan with peptides com- avium subspecies avium (MAA) genomes. posed of L-alanine (or glycine), D-iso- Six of these loci were found to differ be- glutamine, meso-diaminopimelate, and 404 International Journal of Leprosy 2003

D-alanine. We sought to study mycobacterial Garnier, T., Eiglmeier, K., Camus, J. C., peptidoglycan biosynthesis by constructing Medina, N., Mansoor, H., Pryor, M., diaminopimelate (DAP) auxotrophs of My- Duthoy, S., Grondin, S., Lacroix, C., cobacterium smegmatis and then isolating Monsempe, C., Simon, S., Harris, B., spontaneous mutants of these auxotrophs Atkin, R., Doggett, J., Mayes, R., that can grow in the absence of DAP. Here Keating, L., Wheeler, P. R., Parkhill, we report the isolation and characterization J., Barrell, B. G., Cole, S. T., Gordon, of seven classes of spontaneous M. smegma- S. V., and Hewinson, R. G. The com- tis mutants with extragenic mutations that plete genome sequence of Mycobacte- can suppress the DAP requirement of DAP rium bovis. Proc. Natl. Acad. Sci. U.S.A. auxotrophs.—Authors’Abstract 100(13) (2003) 7877Ð7882.

Mycobacterium bovis is the causative Falkinham, J. O. 3rd. Factors influencing agent of tuberculosis in a range of animal the chlorine susceptibility of Mycobacte- species and man, with worldwide annual rium avium, Mycobacterium intracellu- losses to agriculture of $3 billion. The hu- lare, and Mycobacterium scrofulaceum. man burden of tuberculosis caused by the Appl. Environ. Microbiol. 69(9) (2003) bovine tubercle bacillus is still largely un- 5685Ð5689. known. M. bovis was also the progenitor for the M. bovis bacillus Calmette-Guerin vac- The susceptibility of representative cine strain, the most widely used human strains of Mycobacterium avium, Mycobac- vaccine. Here we describe the 4,345,492-bp terium intracellulare, and Mycobacterium genome sequence of M. bovis AF2122/97 scrofulaceum (the MAIS group) to chlorine and its comparison with the genomes of was studied to identify factors related to Mycobacterium tuberculosis and Mycobac- culture conditions and growth phase that in- terium leprae. Strikingly, the genome se- fluenced susceptibility. M. avium and M. in- quence of M. bovis is >99.95% identical to tracellulare strains were more resistant to that of M. tuberculosis, but deletion of ge- chlorine than were strains of M. scrofu- netic information has led to a reduced laceum. Transparent and unpigmented genome size. Comparison with M. leprae colony variants were more resistant to chlo- reveals a number of common gene losses, rine than were their isogenic opaque and suggesting the removal of functional redun- pigmented variants (respectively). Depend- dancy. Cell wall components and secreted ing on growth stage and growth rate, MAIS proteins show the greatest variation, indi- strains differed in their chlorine susceptibil- cating their potential role in host-bacillus ities. Cells from strains of all three species interactions or immune evasion. Further- growing in early log phase at the highest more, there are no genes unique to M. bo- growth rates were more susceptible than vis, implying that differential gene expres- cells in log and stationary phase. Rapidly sion may be the key to the host tropisms of growing cells were more susceptible to human and bovine bacilli. The genome se- chlorine than slowly growing cells. The quence therefore offers major insight on the chlorine susceptibility of M. avium cells evolution, host preference, and pathobiol- grown at 30 degrees C was increased when ogy of M. bovis.—Authors’Abstract cells were exposed to chlorine at 40 degrees C compared to susceptibility after exposure at 30 degrees C. Cells of M. avium grown in Guerardel, Y., Maes, E., Briken, V., Chi- 6% oxygen were significantly more chlo- rat, F., Leroy, Y., Locht, C., Strecker, rine susceptible than cells grown in air. G., and Kremer, L. Lipomannan and Chlorine-resistant MAIS strains were more lipoarabinomannan from a clinical isolate hydrophobic and resistant to Tween 80, of Mycobacterium kansasii: novel struc- para-nitrobenzoate, hydroxylamine, and ni- tural features and apoptosis-inducing trite than were the chlorine-sensitive properties. J. Biol. Chem. 278(38) strains.—Author’s Abstract (2003) 36637Ð36651. 71, 4 Current Literature, Microbiology (Leprosy) 405

Although Mycobacterium kansasii has contrasting with the single mannopyranosyl emerged as an important pathogen fre- residues substituting the mannan core in all quently encountered in immunocompro- the other structures reported so far; and 2) mised patients, little is known about the 5-methylthiopentose residues that were de- mechanisms of M. kansasii pathogenicity. scribed to substitute the arabinan moiety Lipoarabinomannan (LAM), a major myco- from Mycobacterium tuberculosis LAM. bacterial cell wall lipoglycan, is an impor- With respect to the arabinan domain of Kan- tant virulence factor for many mycobacte- LAM, succinyl groups were found to substi- ria, as it modulates the host immune re- tute the C-3 position on 5-arabinofuranosyl sponse. Therefore, the detailed structures of residues, reported to be linked to the C-2 of the of M. kansasii LAM (KanLAM), as the 3,5-arabinofuranose in Mycobacterium well as of its biosynthetic precursor lipo- bovis bacillus calmette-guerin LAM. Be- mannan (KanLM), were determined in a cause M. kansasii has been reported to in- clinical strain isolated from a human im- duce apoptosis, we examined the possibility munodeficiency virus-positive patient. of the M. kansasii lipoglycans to induce Structural analyses revealed that these lipo- apoptosis of THP-1 cells. Our results indi- glycans possess important differences as cate that, in contrast to KanLAM, KanLM compared with those from other mycobacte- was a potent apoptosis-inducing factor. This rial species. KanLAM carries a man- work underlines the diversity of LAM nooligosaccharide cap but is devoid of the structures among various pathogenic myco- inositol phosphate cap present in Mycobac- bacterial species and also provides evidence terium smegmatis. Characterization of the of LM being a potential virulence factor in mannan core of KanLM and KanLAM M. kansasii infections by inducing apopto- demonstrated the following occurrences: 1) sis.—Authors’Abstract alpha1,2-oligo-mannopyranosyl side chains, Microbiology (Leprosy)

Amako, K., Takade, A., Umeda, A., Mat- with a membrane (stage 2), and a cell that suoka, M., Yoshida, S., and Naka- has lost its membrane (stage 3). The pepti- mura, M. Degradation process of Myco- doglycan layer was found to remain intact bacterium leprae cells in infected tissue in these cell groups.—Authors’Abstract examined by the freeze-substitution method in electron microscopy. Micro- biol Immunol. 47(6) (2003) 387Ð394. Kang, T. J., Kim, S. K., Lee, S. B., Chae, G. T., and Kim, J. P. Comparison of Mycobacterium leprae cells (strain Thai- two different PCR amplification prod- 53) harvested from infected mouse foot ucts (the 18-kDa protein gene vs. RLEP pads were examined by electron mi- repetitive sequence) in the diagnosis of croscopy using the freeze-substitution tech- Mycobacterium leprae. Clin. Exp. Der- nique. The population of M. leprae cells matol. 28(4) (2003) 420Ð424. from the infected tissue consisted of a large number of degraded cells and a few normal To determine the best molecular method cells. These thin sectioned cell profiles for diagnosing leprosy, two sets of Myco- could be categorized into four groups de- bacterium leprae-specific primers were pending on the alteration of the membrane compared. Fresh biopsies and slit skin structures, and the degradation process is smear samples were obtained from 67 lep- considered to occur in stages, namely from rosy patients and examined by touchdown stages 1 to 3. These are the normal cells (TD) PCR using primers amplifying either with an asymmetrical membrane, a seem- a 129-bp fragment of the RLEP repetitive ingly normal cell but with a symmetrical sequence or a 360-bp fragment of the 18- membrane (stage 1), a cell possessing con- kDa protein gene of M. leprae. Seventeen tracted and highly concentrated cytoplasm of 30 (56.7%) biopsy specimens and four of 406 International Journal of Leprosy 2003

37 (10.8%) slit skin smear specimens were sults demonstrate that detection of M. lep- positive using the primer for the 18-kDa rae using PCR with primers to a RLEP se- protein gene, whereas 24 of 30 (80%) quence is more sensitive and specific than biopsy and 27 of 37 (73%) slit skin smear PCR with the 18-kDa protein gene primers samples showed detectable PCR products and also slit smears with acid fast staining. in the RLEP repetitive sequence. Twenty- PCR of RLEP repetitive sequences is there- one of 31 cases (67.7%) with a bacterial in- fore a useful means of detecting M. leprae dex of zero were PCR positive for the DNA even when it is present at very low primer RLEP repetitive sequence. These re- levels.—Authors’Abstract

Microbiology (Tuberculosis)

Banaiee, N., Bobadilla-del-Valle, M., Riska, In total, 72 MTC cultures were tested. The P. F., Bardarov, S. Jr., Small, P. M., overall agreement between the LRPs and Ponce-de-Leon, A., Jacobs, W. R. Jr., BACTEC-460 was 98.6%. Four isolates Hatfull, G. F., and Sifuentes-Osornio, J. (5.6%) were falsely identified as ethambutol- Rapid identification and susceptibility resistant. The median turnaround time for testing of Mycobacterium tuberculosis susceptibility testing was 3 days (range from MGIT cultures with luciferase re- 3Ð57) with the LRPs and 9 days (range porter mycobacteriophages. J. Med. Mi- 7Ð29) with BACTEC-460. LRPs offer an crobiol. 52(Pt 7) (2003) 557Ð561. accurate and rapid approach for identifica- tion and susceptibility testing of M. tuber- In a prospective study conducted in a diag- culosis from MGIT-960 cultures.—Au- nostic laboratory in Mexico City, luciferase thors’Abstract reporter mycobacteriophages (LRPs) were evaluated for their utility and performance in identification and antibiotic-susceptibility Carvalho, W. S., Spindola de Miranda, S., testing of Mycobacterium tuberculosis Costa, K. M., Araujo, J. G., Augusto, complex (MTC) isolates from MGIT-960 C. J., Pesquero, J. B., Pesquero, J. L., cultures. Eighty-four consecutive MGIT and Gomes, M. A. Low-stringency cultures recovered from 54 patients were single-specific-primer PCR as a tool for included in this study. The LRPs confirmed detection of mutations in the rpoB gene mycobacterial growth in 79 (94%) of 84 of rifampin-resistant Mycobacterium tu- MGIT cultures. Failure to confirm growth berculosis. J. Clin. Microbiol. 41(7) was due to low inoculum (N = 1) or growth (2003) 3384Ð3386. with non-tuberculous mycobacteria (N = 4). The median time to confirmation of MGIT See Current Literature, Molecular and cultures was 1 day (range 1–55). Confirmed Genetic Studies, p. 422 cultures were identified with p-nitro-alpha- acetylamino-beta-hydroxypropiophenone (NAP), a selective inhibitor of MTC Dahl, J. L., Kraus, C. N., Boshoff, H. I., species, and results obtained with LRPs Doan, B., Foley, K., Avarbock, D., were compared with those obtained by Kaplan, G., Mizrahi, V., Rubin, H., and BACTEC-460. The sensitivity and speci- Barry, C. E. III. The role of RelMtb- ficity of the LRP NAP test were respec- mediated adaptation to stationary phase in tively 97 and 100%, and the median turn- long-term persistence of Mycobacterium around time for identification was 3 days tuberculosis in mice. Proc Natl. Acad. Sci. with both methods. The accuracy and speed U. S. A. 100(17) (2003) 10026Ð10031. of the LRPs for susceptibility testing with rifampicin, streptomycin, isoniazid and Long-term survival of nonreplicating My- ethambutol were compared with BACTEC- cobacterium tuberculosis (Mtb) is ensured 460 and discrepant results were tested by by the coordinated shutdown of active me- the conventional agar proportion method. tabolism through a broad transcriptional pro- 71, 4 Current Literature, Microbiology (Tuberculosis) 407 gram called the stringent response. In Mtb, in shallow standing versus shaking cultures, this response is initiated by the enzymatic in low- versus high-oxygen conditions, and action of RelMtb and deletion of relMtb pro- intracellularly within macrophages versus duces a strain (H37RvDeltarelMtb) severely extracellularly in tissue culture medium. compromised in the maintenance of long- However, there were quantitative differ- term viability. Although aerosol inoculation ences in expression among promoters and of mice with H37RvDeltarelMtb results in among conditions for each promoter. A normal initial bacterial growth and contain- conserved 18-bp palindromic sequence mo- ment, the ability of this strain to sustain tif was identified in all ACPs by Gibbs chronic infection is severely impaired. Sig- sampling-based computational analyses. nificant histopathologic differences were Two such motifs overlap regions in the acr noted in lungs and spleens of mice infected and acg promoters that were previously with H37RvDeltarelMtb compared with shown to be required for their expression. controls throughout the course of the infec- In addition, we found that 5% carbon diox- tion. Microarray analysis revealed that ide was required for growth of Mycobacte- H37RvDeltarelMtb suffers from a general- rium bovis BCG under microaerophilic ized alteration of the transcriptional appara- (1.3% O(2)) culture conditions and fully tus, as well as specific changes in the ex- prevented the growth cessation typically as- pression of virulence factors, cell-wall sociated with rapid removal of oxygen. biosynthetic enzymes, heat shock proteins, These findings are likely to be relevant to and secreted antigens that may alter im- the in vivo environment and will contribute mune recognition of the recombinant or- to our understanding of the pathogenesis of ganism. Thus, RelMtb is critical for the suc- tuberculosis infection.—Authors’Abstract cessful establishment of persistent infection in mice by altering the expression of anti- genic and enzymatic factors that may con- Fossati, G., Izzo, G., Rizzi, E., Gancia, E., tribute to successful latent infection.—Au- Modena, D., Moras, M. L., Niccolai, thors’Abstract N., Giannozzi, E., Spiga, O., Bono, L., Marone, P., Leone, E., Mangili, F., Harding, S., Errington, N., Walters, Florczyk, M. A., McCue, L. A., C., Henderson, B., Roberts, M. M., Purkayastha, A., Currenti, E., Wolin, Coates, A. R., Casetta, B., and M. J., and McDonough, K. A. A family Mascagni, P. Mycobacterium tuberculo- of acr-coregulated Mycobacterium tu- sis chaperonin 10 is secreted in the berculosis genes shares a common DNA macrophage phagosome: is secretion due motif and requires Rv3133c (dosR or to dissociation and adoption of a par- devR) for expression. Infect. Immun. tially helical structure at the membrane? 71(9) (2003) 5332Ð5343. J. Bacteriol. 185(14) (2003) 4256Ð4267.

Previous work has shown that the diver- To confirm that Mycobacterium tubercu- gently transcribed Mycobacterium tubercu- losis chaperonin 10 (Cpn10) is secreted out- losis genes acr (hspX, Rv2031c) and acg side the live bacillus, infected macrophages (Rv2032) are induced under conditions of were examined by electron microscopy. shallow standing culture and low oxygen This revealed that the mycobacterial protein and intracellularly within macrophages. We accumulates both in the wall of the bacte- used a combination of computational and rium and in the matrix of the phagosomes experimental methods to identify promoters in which ingested mycobacteria survive for eight additional genes that are regulated within infected macrophages. To under- in a similar manner and that comprise an stand the structural implications underlying acr-coregulated promoter (ACP) family. this secretion, a structural study of M. tu- Transcriptional regulation of these ACP berculosis Cpn10 was performed under family members was evaluated by using a conditions that are generally believed to plasmid-based promoter-green fluorescent mimic the membrane environment. It was protein fusion system and flow cytometry. found that in buffer-organic solvent mix- All promoters showed increased expression tures, the mycobacterial protein forms two 408 International Journal of Leprosy 2003 main species, namely, a partially helical B gene (kasB) grew poorly in macrophages, monomer that prevails in dilute solutions at although growth in vitro was unaffected. room temperature and a dimer that folds Detailed analyses by thin-layer chromatog- into a beta-sheet-dominated structure and raphy, nuclear magnetic resonance (NMR), prevails in either concentrated protein solu- matrix-assisted laser desorption/ionization tions at room temperature or in dilute solu- time-of-flight mass spectrometry, infrared tions at low temperature. A partially helical spectroscopy, and chemical degradations monomer was also found and was com- showed that the kasB mutants synthesize pletely associated with negatively charged mycolic acids that are 2Ð4 carbons shorter detergents in a micelle-bound state. Re- than wild type; the defect was localized to markably, zwitterionic lipids had no effect the proximal portion of the meromycolate on the protein structure. By using N- and chain. In addition, these mutants showed a C-truncated forms of the protein, the C- and significant (approximately 30%) reduction N-terminal sequences were identified as in the abundance of keto-mycolates, with a possessing an amphiphilic helical character slight compensatory increase of both alpha- and as selectively associating with acidic and methoxy-mycolates. Despite these detergent micelles. When the study was ex- small changes in mycolate length and com- tended to other chaperonins, it was found position, the kasB mutants exhibited strik- that human Cpn10 is also monomeric and ingly altered cell wall permeability, leading partially helical in dilute organic solvent- to a marked increase in susceptibility to buffer mixtures. In contrast, Escherichia lipophilic antibiotics and the host antimi- coli Cpn10 is mostly dimeric and predomi- crobial molecules defensin and lysozyme. nately beta-sheet in both dilute and concen- The abnormalities of the kasB mutants were trated solutions. Interestingly, human fully complemented by expressing M. tu- Cpn10 also crosses biological membranes, berculosis kasB, but not by the closely re- whereas the E. coli homologue is strictly lated gene kasA. These studies identify cytosolic. These results suggest that disso- kasB as a novel target for therapeutic inter- ciation to partially helical monomers and vention in mycobacterial diseases.—Au- interaction with acidic lipids may be two thors’Abstract important steps in the mechanism of secre- tion of M. tuberculosis Cpn10 to the exter- nal environment.—Authors’Abstract Gao, L. Y., Laval, F., Lawson, E. H., Groger, R. K., Woodruff, A., Morisaki, J. H., Cox, J. S., Daffe, M., and Brown, Gao, L. Y., Laval, F., Lawson, E. H., E. J. Requirement for kasB in Mycobac- Groger, R. K., Woodruff, A., Morisaki, terium mycolic acid biosynthesis, cell J. H., Cox, J. S., Daffe, M., and Brown, wall impermeability and intracellular E. J. Requirement for kasB in Mycobac- survival: implications for therapy. Mol. terium mycolic acid biosynthesis, cell Microbiol. 49(6) (2003) 1547Ð1563. wall impermeability and intracellular survival: implications for therapy. Mol. Mycobacterium tuberculosis infects one- Microbiol. 49(6) (2003) 1547Ð1563. third of the world’s population and causes two million deaths annually. The unusually Mycobacterium tuberculosis infects one- low permeability of its cell wall contributes third of the world’s population and causes to the ability of M. tuberculosis to grow two million deaths annually. The unusually within host macrophages, a property re- low permeability of its cell wall contributes quired for pathogenesis of infection. Myco- to the ability of M. tuberculosis to grow bacterium marinum is an established model within host macrophages, a property re- for discovering genes involved in mycobac- quired for pathogenesis of infection. Myco- terial infection. Mycobacterium marinum bacterium marinum is an established model mutants with transposon insertions in the for discovering genes involved in mycobac- beta-ketoacyl-acyl carrier protein synthase terial infection. Mycobacterium marinum B gene (kasB) grew poorly in macrophages, mutants with transposon insertions in the although growth in vitro was unaffected. beta-ketoacyl-acyl carrier protein synthase Detailed analyses by thin-layer chromatog- 71, 4 Current Literature, Microbiology (Tuberculosis) 409 raphy, nuclear magnetic resonance (NMR), conformation polymorphism (PCR-SSCP), matrix-assisted laser desorption/ionization and one phenotypic assay, phage amplified time-of-flight mass spectrometry, infrared biological assay (PhaB) were standardised spectroscopy, and chemical degradations in-house and performed on coded 101 showed that the kasB mutants synthesize rifampicin-resistant and 100 rifampicin- mycolic acids that are 2Ð4 carbons shorter sensitive M. tuberculosis clinical isolates than wild type; the defect was localized to for the identification of rifampicin resis- the proximal portion of the meromycolate tance. RESULTS AND CONCLUSION: chain. In addition, these mutants showed a The results obtained using the three assays significant (approximately 30%) reduction were compared with those from the conven- in the abundance of keto-mycolates, with a tional indirect sensitivity test. The sensitiv- slight compensatory increase of both alpha- ities and specificities of DNA sequencing, and methoxy-mycolates. Despite these PCR-SSCP and PhaB were 97% and 100%, small changes in mycolate length and com- 76% and 100%, and 97% and 84%, respec- position, the kasB mutants exhibited strik- tively. DNA sequencing was found to be ingly altered cell wall permeability, leading more sensitive and specific than the other to a marked increase in susceptibility to tests.—Authors’Abstract lipophilic antibiotics and the host antimi- crobial molecules defensin and lysozyme. The abnormalities of the kasB mutants were Murray, S. J., Barrett, A., Magee, J. G., fully complemented by expressing M. tu- and Freeman, R. Optimisation of acid berculosis kasB, but not by the closely re- fast smears for the direct detection of lated gene kasA. These studies identify mycobacteria in clinical samples. J. Clin. kasB as a novel target for therapeutic inter- Pathol. 56(8) (2003) 613Ð615. vention in mycobacterial diseases.—Au- thors’Abstract AIMS: Despite its long history, the acid fast smear remains unstandardised. Techni- cal variations in both the preparation of He, X. Y., Zhuang, Y. H., Zhang, X. G., clinical material and subsequent staining and Li, G. L. Comparative proteome mean that smear sensitivity relative to cul- analysis of culture supernatant proteins ture may vary from 50% to over 80%. This of Mycobacterium tuberculosis H37Rv study assessed the sensitivity of acid fast and H37Ra. Microbes Infect. 5(10) microscopy at each of five stages of sample (2003) 851Ð856. preparation and by both commonly used staining methods. METHODS: Sputum See Current Literature, Molecular and samples thought for varying reasons to be Genetic Studies, p. 425 highly likely to be culture positive were used to prepare a series of smears in which the effects of digestion (liquefaction), Mani, C., Selvakumar, N., Kumar, V., concentration (centrifugation), and decon- Narayanan, S., and Narayanan, P. R. tamination (sodium hydroxide) could be comparison of DNA sequencing, PCR- assessed, together with a comparison of SSCP and PhaB assays with indirect sen- staining by the auramine/phenol and Ziehl- sitivity testing for detection of rifampicin Neelsen techniques. RESULTS: The most resistance in Mycobacterium tuberculo- effective method for the demonstration of sis. Int. J. Tuberc. Lung Dis. 7(7) (2003) acid fast organisms in sputum was found to 652Ð659. be an auramine phenol stain applied to a liquefied, concentrated sample and exam- SETTING: Tuberculosis Research Cen- ined before the decontamination process. tre, Chennai, India. OBJECTIVE: To CONCLUSIONS: The auramine phenol rapidly identify multidrug-resistant Myco- stain applied to a liquefied, concentrated bacterium tuberculosis using phenotypic sample and examined before the decontam- and genotypic methods. DESIGN: Two ination process is the most effective method genotypic assays, DNA sequencing and for the demonstration of acid fast organisms polymerase chain reaction single strand in sputum.—Authors’Abstract 410 International Journal of Leprosy 2003 Experimental Infections

Baek, K. M., Ko, S. Y., Lee, M., Lee, J. S., molecules in humans. These lipid-specific T Kim, J. O., Ko, H. J., Lee, J. W., Lee, S. cells are cytolytic, secrete pro-inflammatory H., Cho, S. N., and Kang, C. Y. Compar- cytokines and have bactericidal activity. ative analysis of effects of cytokine gene Here, we describe studies in which lipids adjuvants on DNA vaccination against from M. tuberculosis were incorporated Mycobacterium tuberculosis heat shock into liposomes with adjuvant and tested as protein 65. Vaccine 21(25Ð26) (2003) vaccines in a guinea pig aerosol tuberculo- 3684Ð3689. sis challenge model. Animals vaccinated with mycobacterial lipids showed reduced DNA-based vaccines generate potent cel- bacterial burdens in the lung and spleen at 4 lular immunity as well as humoral immu- weeks after infection. In addition, the lungs nity. It seems evident that cytokines play a of lipid-vaccinated animals also had signif- crucial role in generation of effector T cell icantly less pathology, with granulomatous subsets and in determining the magnitude lesions being smaller and more lympho- of the response by DNA vaccines. In this cytic. In contrast, animals receiving only study, we compared the effects of several vehicle control immunizations had granulo- TH1 cytokine genes as adjuvant in DNA matous lesions that were larger and often vaccination using mycobacterial Hsp65 as a contained caseous necrotic centers. Quan- model antigen. Our results demonstrated tification of histopathology by morpho- that although the overall immune response metric analysis revealed that the overall to Hsp65 was enhanced by co-injection of percentage of lung occupied by diseased Hsp65 DNA with cytokine genes, each cy- tissue was significantly smaller in lipid- tokine gene was shown to affect different vaccinated animals as compared to vehicle immune response elements. Co-injection of control animals. In addition, the mean area of Hsp65 DNA with IL-12 or GM-CSF led to individual granulomatous lesions was found an increase in IFN-gamma production and to be significantly smaller in both lipid- and represented potent protections against My- bacillus Calmette-Guerin-vaccinated guinea cobacterium tuberculosis challenge, while pigs. These data support an important role that with Eta-1, IL-12 or IL-18 gene led to for lipid antigens in the immune response to an elevated IgG2a/IgG1 ratio. Interestingly, M. tuberculosis infection, potentially co-administration of Flt3L gene was shown through the generation of CD1-restricted T to enhance the Ag-specific CTL response. cells. Immunogenic lipids thus represent a These results show that the direction and novel class of antigens that might be in- magnitude of immune response in DNA cluded to enhance the protective effects of vaccination against Hsp65 of M. tuberculo- subunit vaccine formulations.—Authors’ sis could be modulated in different ways by Abstract co-injection of an appropriate cytokine gene as adjuvant.—Authors’Abstract Gonzalez-Juarrero, M., Shim, T. S., Kip- nis, A., Junqueira-Kipnis, A. P., and Dascher, C. C., Hiromatsu, K., Xiong, X., Orme, I. M. Dynamics of macrophage Morehouse, C., Watts, G., Liu, G., Mc- cell populations during murine pul- Murray, D. N., LeClair, K. P., Porcelli, monary tuberculosis. J. Immunol. 171(6) S. A., and Brenner, M. B. Immunization (2003) 3128Ð3135. with a mycobacterial lipid vaccine im- proves pulmonary pathology in the The influx of macrophages into the lungs guinea pig model of tuberculosis. Int. is the major component of the granuloma- Immunol. 15(8) (2003) 915Ð925. tous response to infection with Mycobacte- rium tuberculosis. In this investigation we Lipids and glycolipid molecules derived used flow cytometric analysis to define from Mycobacterium tuberculosis can be macrophage populations entering the air- presented to T cells by CD1 cell-surface ways and lung tissues of infected mice. We 71, 4 Current Literature, Experimental Infections 411 demonstrate that by the judicious use of lymph nodes, and these responses were fur- cell surface markers, especially CD11b and ther boosted by i.n. delivery of M.85A. Fol- CD11c, several cell populations can be dis- lowing aerosol challenge with M. tubercu- tinguished, allowing cell sorting and mor- losis, i.n. boosting of BCG with either BCG phological definition. Primary populations or M.85A afforded unprecedented levels of of CD11b(Ð)/CD11c(+/high) were defined protection in both the lungs (2.5 log) and as alveolar macrophages, CD11b(high)/ spleens (1.5 log) compared with naive con- CD11c(+/high) as dendritic cells, and trols. Protection in the lung correlated with CD11b(+/mid)/CD11c(+/mid) as small mac- the induction of Ag 85A-specific, IFN- rophages or monocytes, and changes in the gamma-secreting T cells in lung lymph activation phenotype of these populations nodes. These findings support further eval- were followed over the early course of the uation of mucosally targeted prime-boost infection. In further studies, these cell vaccination approaches for tuberculosis.— populations were compared with cells har- Authors’Abstract vested during the chronic stage of the dis- ease. During the chronic stage of infection, Ag-presenting class II molecules and acti- Ha, S. J., Jeon, B. Y., Kim, S. .C., Kim, D. vation markers were poorly expressed on J., Song, M. K., Sung, Y. C., and Cho, dendritic, small macrophage, and mono- S. N. Therapeutic effect of DNA vac- cyte cell populations, which may have im- cines combined with chemotherapy in a portant implications for the breakdown of latent infection model after aerosol in- the lesions during reactivation disease. fection of mice with Mycobacterium tu- This analytical approach may facilitate the berculosis. Gene Ther. 10(18) (2003) further characterization of macrophage 1592Ð1599. populations entering into the lung tissues and their relative contributions to host re- The prevention of Mycobacterium tuber- sistance to tuberculosis infection.—Au- culosis (M. tuberculosis) reactivation would thors’Abstract greatly reduce the incidence of the disease, particularly among the elderly. Here, we evaluated the efficacy of DNA vaccine in Goonetilleke, N. P., McShane, H., Hannan, combination with a conventional TB C. M., Anderson, R. J., Brookes, R. H., chemotherapy on the prevention of M. tu- and Hill, A. V. Enhanced immunogenicity berculosis reactivation. Mice were treated and protective efficacy against Mycobac- with isoniazid and pyrazinamide for 3 terium tuberculosis of bacille Calmette- months from 4 weeks after aerosol infection Guerin vaccine using mucosal administra- with M. tuberculosis H37Rv. During this tion and boosting with a recombinant period of chemotherapy, DNA immuniza- modified vaccinia virus Ankara. J. Im- tion was performed three times monthly munol. 171(3) (2003) 1602Ð1609. with an antigen 85A (Ag85A) DNA or an IL-12 mutant (IL-12N220L) DNA, which is Heterologous prime-boost immunization known to lead to a reduction in the secre- strategies can evoke powerful T cell im- tion of the p40 subunit, but not of a bioac- mune responses and may be of value in tive IL-12p70. The reactivation of M. tuber- developing an improved tuberculosis vac- culosis was dramatically reduced in mice cine. We show that recombinant modified treated with either Ag85A DNA (p <0.01) vaccinia virus Ankara, expressing Myco- or IL-12N220L DNA (p <0.05) in combina- bacterium tuberculosis Ag 85A (M.85A), tion with chemotherapy, compared with strongly boosts bacille Calmette-Guerin control mice receiving only chemotherapy. (BCG)-induced Ag 85A specific CD4(+) Ag85A DNA vaccine showed higher IFN- and CD8(+) T cell responses in mice. A gamma responses to Ag85A protein, but a comparison of intranasal (i.n.) and par- lower response to culture filtrate than IL- enteral immunization of BCG showed that 12N220L DNA vaccine. In addition, while both routes elicited comparable T cell Ag85A DNA vaccine prevented the reacti- responses in the spleen, only i.n. delivery vation of M. tuberculosis more efficiently elicited specific T cell responses in the lung than IL-12N220L DNA vaccine, indicating 412 International Journal of Leprosy 2003 that Ag85A-specific IFN-gamma response H37Rv resulted in increased survival and might correlate with M. tuberculosis control. reduced pathology in lungs and spleens This study suggests that immunotherapy us- relative to non-immunized animals.—Au- ing Ag85A or IL-12N220L DNA vaccine thors’Abstract combined with conventional chemotherapy might be effective clinically for the preven- tion of tuberculosis reactivation and may Lai, X., Shen, Y., Zhou, D., Sehgal, P., offer a more effective cure for humans than Shen, L., Simon, M., Qiu, L., Letvin, chemotherapy alone.—Authors’Abstract N. L., and Chen, Z. W. Immune biology of macaque lymphocyte populations dur- ing mycobacterial infection. Clin. Exp. Hamasur, B., Haile, M., Pawlowski, A., Immunol. 133(2) (2003) 182Ð192. Schroder, U., Williams, A., Hatch, G., Hall, G., Marsh, P., Kallenius, G., and Immune responses of lymphocyte popu- Svenson, S. B. Mycobacterium tubercu- lations during early phases of mycobacte- losis arabinomannan-protein conjugates rial infection and reinfection have not been protect against tuberculosis. Vaccine well characterized in humans. A non-human 21(25Ð26) (2003) 4081Ð4093. primate model of Mycobacterium bovis bacille Calmette-Guerin (BCG) infection Lipoarabinomannan (LAM) is a major was employed to characterize optimally the structural surface component of mycobacte- immune responses of mycobacteria-specific ria. Arabinomannan (AM) oligosaccharides T cells. Primary BCG infection induced derived from LAM of Mycobacterium tu- biphasic immune responses, characterized berculosis H37Rv were isolated and cova- by initial lymphocytopenia and subsequent lently conjugated to tetanus toxoid (TT) or expansion of CD4+, CD8+ and gammadelta to short-term culture filtrate proteins (anti- T cell populations in the blood, lymph gen 85B (Ag85B) or a 75kDa protein) from nodes and the pulmonary compartment. M. tuberculosis strain Harlingen. The dif- The potency of detectable T cell immune ferent AM oligosaccharide (AMOs)-protein responses appears to be influenced by the conjugate vaccine candidates proved to be timing and route of infection as well as highly immunogenic, inducing boosterable challenge doses of BCG organisms. Sys- IgG responses against the AMOs portion of temic BCG infection introduced by intra- the conjugates in rabbits and guinea-pigs. venous challenge induced a dose-dependent Proliferation of T-cells from C57BL/6 mice expansion of circulating CD4+, CD8+ and immunized with the conjugates was seen gammadelta T cells whereas, in the pul- upon in vitro stimulation with PPD. In monary compartment, the systemic infec- C57BL/6 mice subcutaneous immunization tion resulted in a predominant increase in with the AMOs-antigen 85B conjugate in numbers of gammadelta T cells. In contrast, alum provided significant protection com- pulmonary exposure to BCG through the pared to sham (alum only) immunized mice bronchial route induced detectable expan- (p <0.021) as estimated by long term sur- sions of CD4+, CD8+ and gammadelta T vival against intravenous challenge with cell populations in only the lung but not in 10(5) M. tuberculosis H37Rv. Subcuta- the blood. A rapid recall expansion of these neous immunization followed by nasal T cell populations was seen in the boost with an AMOs-TT conjugate in Eu- macaques reinfected intravenously and rocine L3 adjuvant provided high (p bronchially with BCG. The expanded al- <0.025) protection as determined by long phabeta and gammadelta T cell populations term survival after intranasal challenge with exhibited their antigen specificity for myco- 10(5) virulent M. tuberculosis strain Harlin- bacterial peptides and non-peptide phos- gen. This level of protection was compara- pholigands, respectively. Finally, the major ble to that obtained with the conventional expansion of T cells was associated with a live attenuated BCG vaccine. In guinea- resolution of active BCG infection and rein- pigs, immunization with AMOs-Ag85B in fection. The patterns and kinetics of CD4+, Eurocine L3 adjuvant followed by aero- CD8+ and gammadelta T cell immune re- genic challenge with M. tuberculosis sponses during BCG infection might con- 71, 4 Current Literature, Epidemiology and Prevention 413 tribute to characterizing immune protection munity (CMI) causing activation or apopto- against tuberculosis and testing new tuber- sis of infected cells. The phenomenon of culosis vaccines in primates.—Authors’ apoptosis is associated with Mycobacterium Abstract tuberculosis survival. In this study, using frozen lung sections (N = 33), our results showed increased CD40, IL-12 and TGF- Mogga, S. J., Mustafa, T., Sviland, L., beta1 expression in macrophages with pro- and Nilsen, R. In situ expression of gression of disease. High percentages of CD40, CD40L (CD154), IL-12, TNF-al- mycobacterial antigens (M.Ags), CD40L pha, IFN-gamma and TGF-beta1 in and IFN-gamma expression were main- murine lungs during slowly progressive tained throughout infection, and TNF- primary tuberculosis. Scand. J. Immunol. alpha-expressing cells were decreased. In 58(3) (2003) 327Ð334. lymphocytes, the percentage of IFN- gamma-positive cells was increased, but The distribution and expression of CD40, CD40L and IL-12 were maintained with the its ligand CD40L (154) and related cytokines progression of disease. M.Ags, CD40 and interleukin-12 (IL-12), tumour necrosis CD40L were expressed in the same areas of factor-alpha (TNF-alpha), interferon-gamma the lesions. We conclude that changes in the (IFN-gamma) and transforming growth expression of CD40-CD40L and cytokines factor-beta1 (TGF-beta1) were studied in associated with M. tuberculosis infection the lungs of B6D2F1 hybrid mice during favour the hypothesis that M. tuberculosis slowly progressive primary tuberculosis causes resistance of host cells to apoptosis (TB) by immunohistochemistry. CD40 and causing perpetuation of infection.—Au- CD40L are implicated in cell-mediated im- thors’Abstract

Epidemiology and Prevention

Figueiredo, I. A. and da Silva, A. A. [In- Hatta, M. Epidemiology of leprosy. crease in leprosy detection rates in Sao Molecular, biological, and immunologi- Luis, Maranhao, Brazil, from 1993 to cal approach. Adv. Exp. Med. Biol. 531 1998: is the endemic expanding?] Cad (2003) 269Ð278. Saude Publica. 19(2) (2003) 439Ð445. [Article in Portuguese] Leprosy is an infectious disease for which humans are considered the only A descriptive epidemiologic study on source of infection. The major hindrance in the detection of new leprosy cases was leprosy control and thus in reaching the conducted in Sao Luis, Maranhao, Brazil, elimination goal is that numerous leprosy from 1993 to 1998. A database was created cases remain undetected for a long time. for the purpose, covering 2796 reported Many of these patients are a continuous cases. General detection rates were calcu- source of infection and, and hence perpetu- lated, as well as specific rates by gender, ate transmission. The goal of the World clinical type, and age group. Linear, expo- Health Organization (WHO) is to eliminate nential, geometric, and log adjustment leprosy as a public problem by the year models were performed to analyze time 2000; that is, to reach as a global preva- trends in the disease. An increase in detec- lence of <1 per 10,000 people. The epi- tion was observed, involving mostly fe- demiological data generated routinely by male and paucibacillary cases, mainly of health services are greatly influenced by tuberculoid leprosy. The increase in detec- their policies and activities. The data do tion was most evident in the 15 to 19 year- not, however necessarily reflect the true old population. The percentage of detec- situation in the field. Information on the tion under 15 indicated the need for active magnitude of the leprosy problem in any case search in this group.—Authors’ Ab- one area is important for the health services stract with regard to their planning, monitoring 414 International Journal of Leprosy 2003 and evaluation of leprosy control activities. India, to evaluate the prevalence of leprosy Our studies have suggested that the high in the area. A total of 60,179 persons from prevalence of antibodies in children may be more than 120 smaller localities in both indicative of the active transmission of M. semi-urban and slum areas were examined. leprae in their surroundings. The prevalence Chi-square test (χ2) was used to determine of these antibodies may also be important for the prevalence, while logistic regression leprosy control programs in order to detect was used to compute for adjusted odd ra- new patients as early as possible and in an ef- tios. The overall prevalence of leprosy was fective and sustainable manner. Based on 33.9 per 10,000 population (range, 9.7Ð40.7), PCR data, it seems that the environment also whereas the new case detection rate was 28.2 plays an important role in the transmission of per 10,000 (range, 9.7Ð30.7). Among chil- leprosy in endemic areas. The results of our dren <15 years, the leprosy prevalence was study show that contact with a leprosy patient 4.4 per 10,000. Adult males aged >15 years is the major determinant in the incidence of had a significantly higher prevalence rate leprosy and that this concept shows similari- than females of similar age group (92.0 ver- ties with the “stone-in-the-pond” principle of sus 41.6 per 10,000). It was noted that the tuberculosis transmission in concentric circle prevalence rates increased with increasing around patients.—Author’s Abstract age (p <0.0001). Moreover, workers en- gaged in manual work were found to have significantly higher prevalence rates than Kumar, A., Girdhar, A., Girdhar, B. K. other workers (94.9 versus 21.3, p <0.0001). Epidemiology of leprosy in urban Agra. Of the 204 leprosy cases detected, 84.2% Lepr. Rev. 74(1) (2003) 31–34.—Tropi- were new cases. Of all the cases, 37.3% cal Disease Bulletin were of the multibacillary type. Disabilities of Grade II or higher were observed in During May 2000ÐJune 2001, a survey 12.7% of all cases, of which 9.4% were was carried out in the urban areas of Agra, new cases.—Tropical Disease Bulletin

Rehabilitation and Social Concerns

Brandsma, J. W. 26th Kellersberger new field in medicine and not (well) devel- Memorial Lecture. Lessons from leprosy oped in many developing countries. Reha- rehabilitation for general rehabilitation. bilitation requires an integrated approach Ethiop. Med. J. 41(1) (2003) 77Ð87. from different disciplines and professionals. As for other medical specialty fields, reha- Leprosy is primarily a disease of skin and bilitation demands evidence based prac- peripheral nerves. Because of nerve func- tice.—Author’s Abstract tion impairment, leprosy patients may de- velop primary nerve related impairments such as, loss of sensation and weakness or Kalk, A. [Cooperation between an NGO paralysis. These primary impairments may and “host” states in the control of leprosy lead to secondary impairments such as ul- in Latin America] Cad Saude Publica. ceration and contractures. Many other dis- 19(2) (2003) 663Ð666. [Article in Por- eases and disorders present with similar im- tuguese] pairments as seen in leprosy e.g., diabetes and peripheral nerve injuries. Nerve func- The proliferation of nongovernmental or- tion assessment and ulcer prevention and ganizations (NGOs) can be considered the treatment are areas that have been re- result of the inability of the current demo- searched in leprosy but these research find- cratic system to perform all the tasks de- ings are not yet commonly known and sired by its citizens. Although NGOs often adopted in diseases and disorders that ‘re- do quite positive work, they tend to dimin- late’ to leprosy. Rehabilitation is a relatively ish governmental power and are capable of 71, 4 Current Literature, Other Mycobacterial Diseases 415 interfering in the internal affairs of other Brazil, where the government prioritizes countries. In this context, there are efforts the control of Hansen disease and commu- to control their activities, and this control nity participation in the political process— can produce both negative effects (blocking NGOs generally work “in harmony” with the defense of human rights) and positive national authorities. The most useful contri- ones (correcting the lack of coordination in bution to state efforts has been the technical the work by NGOs). NGOs working with and financial support for training health the control of leprosy have a long history of personnel, supervision, and awareness- cooperation with “host” states in Latin raising campaigns. Thus, the NGO becomes America. In the worst cases they work in a “quasi-governmental” in performing its vacuum left by the state. In a country like tasks.—Author’s Abstract

Other Mycobacterial Diseases

Albright, J. T. and Pransky, S. M. Nontu- and rifampicin. The current literature on the berculous mycobacterial infections of epidemiology, clinical characteristics and the head and neck. Pediatr. Clin. North management of Mycobacterium marinum Am. 50(2) (2003) 503Ð514. infections is reviewed.—Authors’Abstract

Nontuberculous mycobacteria are ubiqui- tous in the environment. Immunocompetent Amrami, K. K., Sundaram, M., Shin, A. Y., children are commonly infected by these re- and Bishop, A. T. Mycobacterium mar- silient organisms. Cervical lymphadenitis, inum infections of the distal upper extrem- the most frequent head and neck manifesta- ities: clinical course and imaging findings tion of NTM infection, often presents as in two cases with delayed diagnosis. Skele- chronic, unilateral lymphadenopathy with tal. Radiol. 32(9) (2003) 546Ð549. characteristic violaceous overlying skin changes. Diagnosis is ultimately dependent Mycobacterium marinum infections cause on culture or histopathologic examination tenosynovitis of the distal upper extremities of specimen obtained through excisional and develop as a consequence of skin abra- lymph node biopsy or FNA. The principal sions acquired in contaminated water. We treatment of NTM infection remains the sur- report on two patients whose MR imaging gical excision of diseased tissue. Antibiotics studies showed tenosynovitis of the distal augment surgical therapy and their potential upper extremity secondary to M. marinum. role as a single-modality therapy continues In one patient sequential MR imaging to be investigated.—Authors’Abstract showed development of bony erosions. Ap- propriate treatment was delayed in both pa- tients because the diagnosis was not consid- Amoateng-Adjepong, Y., Salloum, A., St ered. We report on and discuss the clinical Martin, D., and Coles, M. J. Mycobac- course and MR imaging findings in two pa- terium marinum infection in Connecti- tients with M. marinum infection.—Au- cut: report of four cases. Conn. Med. thors’Abstract 67(6) (2003) 333Ð335.

Mycobacterium marinum is emerging as Ara, M., de Santamaria, C. S., Zaballos, an important human pathogen in the United P., Yus, C., and Lezcano, M. A. Myco- States. We report four cases incidentally di- bacterium chelonae infection with multi- agnosed from culture of biopsy specimens of ple cutaneous lesions after treatment wrist lesions at a Connecticut inner city hos- with acupuncture. Int. J. Dermatol. 42(8) pital between 1996 and 1999. There was no (2003) 642Ð644. clear association with aquatic exposure and only one patient recalled prior trauma. All A 58-year-old woman was first seen in were successfully treated with ethambutol November 1999 with a 4-week history of 416 International Journal of Leprosy 2003 several tender, deep red or purple, suppurat- cell sonicate and MMP-specific antibodies ing subcutaneous nodules on the skin of the were purified from these sera by affinity abdomen, suggestive of a panniculitis. She chromatography. MMP was localized to the had no history of systemic immunosup- surface of M. paratuberculosis by immuno- pression. Three months prior to examina- electron microscopy and by immunoblot tion, the patient had treated with acupunc- analysis of fractionated protein lysates. ture for obesity. Two biopsy specimens of Both anti-MMP antibodies and MBP-MMP the nodules were taken and sent for culture protein inhibited M. paratuberculosis inva- and histologic examination. Histology sion of cultured Madin-Darby bovine kid- showed a pattren of panniculitis with chronic ney cells by 30%. In similar invasion exper- inflammatory cells mixed with areas of iments with M. paratuberculosis incubated polymorphonuclear abscesses and necrosis. in low oxygen tension, these antibodies and Culture of the biopsy specimen grew acid- protein decreased invasion by 60%. Collec- fast bacilli within 4 days, later identified tively, these data show that the 35 kDa with biochemical and molecular tests as MMP is a surface exposed protein that Mycobacterium chelonae (subspecies che- plays a role in invasion of epithelial cells. lonae). Polymerase chain reaction-restriction The authors suggest that the MMP is a viru- enzyme pattern analysis (PRA) was used ofr lence factor of M. paratuberculosis that molecular identification of mycobacteria. In may be important in the initiation of infec- vitro sensitivity tests showed sensitivity to tion in vivo.—Authors’Abstract clarithromycin, amikacin, tobramycin, doxy- cycline, and erythromycin, and resistance to ciprofloxacin, ofloxacin, trimethoprim- Bull, T. J., McMinn, E. J., Sidi-Boumedine, sulfamethoxazole, imipenem, and cefoxitin. K., Skull, A., Durkin, D., Neild, P., Oral clarithromycin (500 mg b.d.) was Rhodes, G., Pickup, R., and Hermon- started and after 3 months of therapy, the le- Taylor, J. Detection and verification of sions had cleared completely.—Authors’ Mycobacterium avium subsp. paratuber- Abstract culosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn’s disease. J. Clin. Mi- Bannantine, J. P., Huntley, J. F., Miltner, crobiol. 41(7) (2003) 2915Ð2923. E., Stabel, J. R., and Bermudez, L. E. The Mycobacterium avium subsp. paratu- Mycobacterium avium subsp. paratuber- berculosis 35 kDa protein plays a role in culosis is a robust and phenotypically ver- invasion of bovine epithelial cells. Mi- satile pathogen which causes chronic in- crobiology 149(Pt 8) (2003) 2061Ð2069. flammation of the intestine in many species, including primates. M. avium subsp. paratu- Mycobacterium avium subsp. paratuber- berculosis infection is widespread in do- culosis (M. paratuberculosis) enters intesti- mestic livestock and is present in retail pas- nal epithelial cells of cattle and other rumi- teurized cows’ milk in the United Kingdom nants via a mechanism that remains to be and, potentially, elsewhere. Water supplies fully elucidated. This study showed that a are also at risk. The involvement of M. gene encoding the M. paratuberculosis 35 avium subsp. paratuberculosis in Crohn’s kDa major membrane protein (MMP) is ex- disease (CD) in humans has been uncertain pressed at a higher level in low-oxygen and because of the substantial difficulties in de- high-osmolarity conditions that are similar tecting this pathogen. In its Ziehl-Neelsen to the environment of the intestine. In addi- staining-negative form, M. avium subsp. tion, cattle with Johne’s disease produced paratuberculosis is highly resistant to chem- antibodies against MMP, suggesting that ical and enzymatic lysis. The present study the protein is present during infection. The describes the development of optimized gene encoding MMP was cloned and ex- sample processing and DNA extraction pro- pressed as a fusion protein with the cedures with fresh human intestinal mu- maltose-binding protein (MBP-MMP) in cosal biopsy specimens which ensure ac- Escherichia coli. Rabbit antisera were cess to M. avium subsp. paratuberculosis raised against a M. paratuberculosis whole- DNA and maximize detection of these low- 71, 4 Current Literature, Other Mycobacterial Diseases 417 abundance pathogens. Also described are T., Matsushima, T., Fukunaga, H., and two nested PCR methodologies targeted at Ishida, T. Pathological findings of bron- IS900, designated IS900[L/AV] and chiectases caused by Mycobacterium IS900[TJ1-4], which are uniquely specific avium intracellulare complex. Respir. for IS900. Detection of M. avium subsp. Med. 97(8) (2003) 933Ð938. paratuberculosis in mucosal biopsy speci- mens was also evaluated by using myco- It has been argued whether bronchiecta- bacterial growth indicator tube (MGIT) cul- sis is truly caused by MAC infection or tures (Becton Dickinson). IS900[L/AV] just a predisposed condition in which PCR detected M. avium subsp. paratubercu- MAC colonizes. Our present study was de- losis in 34 of 37 (92%) patients with CD signed to evaluate the pathological find- and in 9 of 34 (26%) controls without CD ings of bronchiectases caused by Myco- (noninflammatory bowel disease [nIBD] bacterium avium intracellulare complex controls) (p = 0.0002; odds ratio = 3.47). M. (MAC) lung infection and to demonstrate avium subsp. paratuberculosis was detected MAC in the lesion of bronchiectases. A by IS900[L/AV] PCR in MGIT cultures af- retrospective study was performed in nine ter 14 to 88 weeks of incubation in 14 of 33 cases with positive cultures for MAC in (42%) CD patients and 3 of 33 (9%) nIBD whom lung resections were performed. A controls (p = 0.0019; odds ratio = 4.66). determination of whether or not MAC Nine of 15 (60%) MGIT cultures of speci- caused pulmonary disease was made using mens from CD patients incubated for more the 1997 criteria required by the American than 38 weeks were positive for M. avium Thoracic Society. In addition, MAC were subsp. paratuberculosis. In each case the cultured from all nine lung specimens. identity of IS900 from M. avium subsp. Pathological findings of bronchiectases paratuberculosis was verified by amplicon were evaluated in these nine patients. De- sequencing. The rate of detection of M. struction of bronchial cartilage and smooth avium subsp. paratuberculosis in individu- muscles layer, obstruction of airway by als with CD is highly significant and impli- granulomas, and ulceration of bronchial cates this chronic enteric pathogen in dis- mucosa were frequently observed. Our ease causation.—Authors’Abstract present study demonstrates that destruc- tion of fundamental bronchial structure due to extensive granuloma formation Cynamon, M. H., Elliott, S. A., DeSte- throughout the airways was likely the fano, M. S., and Yeo, A. E. Activity of main cause of bronchiectases in MAC in- clarithromycin alone and in combination fection.—Authors’ Abstract in a murine model of Mycobacterium kansasii infection. J. Antimicrob. Chemother. 52(2) (2003) 306Ð307. Garofalo, R., Chadwick, E. G., and Yo- gev, R. Mycobacterium gordonae bac- Activities of clarithromycin alone and in teremia in an asymptomatic adolescent combination with rifampicin, gatifloxacin with acquired immunodeficiency syn- or linezolid were evaluated against Myco- drome. Pediatr. Infect. Dis. J. 22(6) bacterium kansasii in a murine infection (2003) 569Ð570. model. Clarithromycin was the most active single agent. Rifampicin and gatifloxacin Mycobacterium gordonae is historically had similar activities, but were less active viewed as an organism with low pathogenic than clarithromycin. Clarithromycin in potential, but it has increasingly become combination with rifampicin was the most implicated in clinical disease in immuno- active combination therapy.—Authors’ Ab- compromised hosts. Illness related to M. stract gordonae infection ranges from localized infections to rare cases of disseminated dis- ease. This report describes treatment of the Fujita, J., Ohtsuki, Y., Shigeto, E., first case of occult M. gordonae bacteremia Suemitsu, I., Yamadori, I., Bandoh, S., in an adolescent with AIDS.—Authors’Ab- Shiode, M., Nishimura, K., Hirayama, stract 418 International Journal of Leprosy 2003

Guarner, J., Bartlett, J., Whitney, E. A., inantly the M. fortuitum-M. abscessus-M. Raghunathan, P. L., Stienstra, Y., chelonae group were the most common soft Asamoa, K., Etuaful, S., Klutse, E., tissue infections. Atypical mycobacteria Quarshie, E., van der Werf, T. S., van were isolated from significant numbers of der Graaf, W. T., King, C. H., and sputum ‘smear positive’ patients, requiring Ashford, D. A. Histopathologic features further tests to exclude M. tuberculosis. of Mycobacterium ulcerans infection. Geographical differences were observed for Emerg. Infect. Dis. 9(6) (2003) 651Ð656. some Mycobacterium species, notably the isolation of M. haemophilum from Western Because of the emergence of Buruli ulcer Australia, and M. ulcerans from Victoria disease, the World Health Organization and Queensland. Newer molecular tech- launched a Global Buruli Ulcer Initiative in niques, while improving precision and ac- 1998. This indolent skin infection is caused curacy of identification, raise additional by Mycobacterium ulcerans. During a study questions about the ecology of the atypical of risk factors for the disease in Ghana, ade- mycobacteria and their role in disease.— quate excisional skin-biopsy specimens were Author’s Abstract obtained from 124 clinically suspicious le- sions. Buruli ulcer disease was diagnosed in 78 lesions since acid-fast bacilli (AFB) were Horwitz, M. E., Uzel, G., Linton, G. F., found by histopathologic examination. Le- Miller, J. A., Brown, M. R., Malech, H. sions with other diagnoses included filariasis L., and Holland, S. M. Persistent Myco- (3 cases), zygomycosis (2 cases), ulcerative bacterium avium infection following squamous cell carcinomas (2 cases), keratin nonmyeloablative allogeneic peripheral cyst (1 case), and lymph node (1 case). blood stem cell transplantation for Thirty-seven specimens that did not show interferon-gamma receptor-1 deficiency. AFB were considered suspected Buruli ul- Blood 102(7) (2003) 2692Ð2694. cer disease cases. Necrosis of subcutaneous tissues and dermal collagen were found Interferon-gamma receptor-1 (IFNgam- more frequently in AFB-positive specimens maR1) deficiency is a rare inherited immun- compared with specimens from suspected odeficiency. We performed a nonmyeloabla- case-patients (p <0.001). Defining histo- tive allogeneic stem cell transplantation on a logic criteria for a diagnosis of Buruli ulcer boy with complete IFNgammaR1 deficiency disease is of clinical and public health im- and refractory disseminated Mycobacterium portance since it would allow earlier treat- avium infection. Despite the patient’s pro- ment, leading to less deforming sequelae.— found immune defect, early donor stem cell Authors’Abstract engraftment was low. Full donor engraft- ment was accomplished only following multiple donor lymphocyte infusions. De- Haverkort, F. Australian Mycobacterium tection of IFNgammaR1 expression on pe- Reference Laboratory Network; Special ripheral blood monocytes and neutrophils Interest Group in Mycobacteria within corresponded with establishment of stable, the Australian Society for Microbiology. complete donor hematopoietic chimerism. National atypical mycobacteria survey, However, expression of, and signaling 2000. Commun. Dis. Intell. 27(2) (2003) through IFNgammaR1 disappeared shortly 180Ð189. thereafter. Disseminated Mycobacterium avium infection persisted and the patient Infections with atypical mycobacteria in died. Coculture of Mycobacterium avium Australia during 2000 occurred at a rate of with normal myeloid cells resulted in an 1.8 cases per 100,000 population. The main IFNgamma signaling defect similar to that sites of disease were the respiratory tract, observed in vivo. Active disseminated My- soft tissue, and the lymphatics. The Myco- cobacterium avium infection may signifi- bacterium avium complex was the most cantly compromise normal immune recon- common group of mycobacteria isolated stitution following allogeneic stem cell from respiratory, lymphatic sites, and blood. transplantation. Patients with IFNgam- The rapidly growing mycobacteria, predom- maR1 deficiency should receive transplants 71, 4 Current Literature, Other Mycobacterial Diseases 419 before developing refractory mycobacterial Liou, J. H., Huang, P. Y., Hung, C. C., infections.—Authors’Abstract and Hsiao, C. H. Mycobacterial spindle cell pseudotumor of skin. J. Formos. Med. Assoc. 102(5) (2003) 342Ð345. Kullavanijaya, P., Rattana-Apiromyakij, N., Sukonthapirom-Napattalung, P., Spindle cell pseudotumors may occur Sirimachand, S., and Duangdeeden, I. due to mycobacterial infection in immuno- Disseminated Mycobacterium chelonae compromised hosts, particularly those with cutaneous infection: recalcitrant to com- acquired immunodeficiency syndrome bined antibiotic therapy. J. Dermatol. (AIDS). Most of the reported mycobacterial 30(6) (2003) 485Ð491. spindle cell pseudotumors were found in the lymph nodes. We report a case of spin- A 25-year-old Thai housewife had a his- dle cell pseudotumor in a 37-year-old man tory of tuberculosis of the lymph nodes for with AIDS who presented with a firm nod- six years that had been successfully treated ule over his right arm. Histologically, the with a course of anti-TB drugs. She devel- tumor was composed of proliferative spin- oped several red, circumscribed, infiltrative dle cells admixed with histiocytes and in- plaques composed of umbilicated papules flammatory cells. Ziehl-Neelsen stain re- and pustules on her face and upper part of vealed many acid-fast bacilli in the spindle the body with cervical lymphadenopathy cells and histiocytes. The acid-fast bacilli six months later. A pus smear from the le- were shown to be Mycobacterium avium in- sion grew acid fast bacilli (AFB). tracellulare by culture and sequencing of Histopathological examination showed a the polymerase chain reaction product of mixed cell granuloma suggestive of infec- mycobacterial 65-kDa heat shock protein tion. A T cell study showed a low CD4 gene. Immunohistochemically, the spindle count, and multi skin tests indicated cuta- cells were reactive to CD68, suggesting neous anergy. Culture from a biopsy speci- macrophage differentiation of these cells. It men taken from the skin lesion grew M. is important for pathologists to recognize chelonae; the cultures from blood, urine, this unusual manifestation of mycobacterial and bone marrow. The lesions were not infection in immunocompromised patients responsive to an anti TB drug given for 2 and avoid mistaking the lesion for a mes- months based upon the results of the AFB enchymal neoplasm.—Authors’Abstract positive pus smear before the culture and sensitivity reports were obtained. Since then the patient was treated with antibiotics Mauriello, J. A. Jr. and Atypical Myco- according to the results of the sensitivity bacterial Study Group. Atypical myco- tests. A combination of amikacin and bacterial infection of the periocular re- clarithromycin was started and hyper- gion after periocular and facial surgery. thermic therapy was later added with a Ophthal. Plast. Reconstr. Surg. 19(3) partial response. Based upon the sensitivity (2003) 182Ð188. test, kanamycin was introduced but had to be stopped because of ototoxicity. PURPOSE: To delineate the clinico- Sparfloxacin was used with an effective re- pathologic features of patients who have sult but was discontinued for economic rea- atypical mycobacterial infections of the pe- sons. Finally, clarithromycin in combina- riorbital region after periocular and facial tion with clofazimine and cryotherapy were surgery and to define the sequelae after given for a year before the lesions healed treatment and their management. METH- completely. It took a three years duration ODS: A case series of patients from 7 prac- for the total course of treatment for this pa- tices of ophthalmic plastic and reconstruc- tient. She is still in remission after two tive surgeons was analyzed retrospectively. years of follow-up period. This extensive RESULTS: Thirteen patients had infection cutaneous M. chelonae infection needed a in the following clinical settings: 8 patients prolonged combination of antibiotics with had infections after blepharoplasty, 2 pa- the addition of cryotherapy for the non-re- tients had infections that involved the sponsive lesions.—Authors’Abstract anophthalmic socket, 1 patient had orbital 420 International Journal of Leprosy 2003 cellulitis after orbital fracture repair with a chalazion, develop in a sutured incision, an alloplastic implant, and 2 patients had or occur without any inflammatory signs. infections involving the lacrimal system, Orbital abscess formation may occur in the one after silicone tube insertion and the setting of transconjunctival blepharo- other after dacryocystorhinostomy with plasty. Cultures for acid-fast bacilli and silicone tube intubation. Sequelae of infec- excisional biopsy of nodules with perfor- tion included eyelid retraction and ectro- mance of acid-fast stains may be necessary pion requiring surgical repair (two pa- for diagnosis. The selection of systemic tients) and enophthalmos (one patient). antibiotic therapy, usually clarithromycin, Twelve of 13 patients required extensive and the length of treatment should be antibiotic therapy. One infection resolved guided by results of culture and sensitivity after local excision of eyelid lesions. An- laboratory studies, biopsy results, and clin- other patient had recurrent infection after 4 ical response to treatment. Surgical re- weeks of antibiotic treatment. CONCLU- moval of any implanted foreign bodies SIONS: Delayed infection with erythema- should be performed expeditiously. Con- tous nodules, particularly when a foreign sultation with an infectious disease spe- body is implanted weeks after periocular cialist may be useful in selected cases. Se- surgery, should arouse suspicion of an quelae of infection may include eyelid atypical mycobacterial infection. Delayed scarring and retraction and enophthal- infection after blepharoplasty may mimic mos.—Authors’ Abstract

Molecular & Genetic Studies

Agarwal, N. and Tyagi, A. K. Role of 5′- in the loss of >75% of its activity. Binding TGN-3′ motif in the interaction of myco- of RNA polymerase with wild-type pro- bacterial RNA polymerase with a pro- moter as well as its TG- mutant revealed moter of ‘extended -10’ class. FEMS Mi- that the TGN motif is required for the transi- crobiol. Lett. 225(1) (2003) 75Ð83. tion from a close complex into an open com- plex. Further, it was observed that the pres- In a systematic approach to understand ence of the TGN motif reduces the thermal the transcriptional machinery of mycobac- energy required for the conversion of a close teria, we had previously isolated and charac- complex into an open complex, necessary terized mycobacterial promoter regions. In for initiation of transcription. this study, we have investigated molecular interactions between mycobacterial RNA polymerase holoenzyme, reconstituted with Ahmed, N., Alam, M., Abdul Majeed, A., different sigma subunits and the promoter el- Asad Rahman, S., Cataldi, A., Cousins, ement of the Mycobacterium tuberculosis D., and Hasnain, S. E. Genome se- gene pknH (Rv1266c), a representative of quence based, comparative analysis of promoters belonging to the ‘extended-10’ the fluorescent amplified fragment length class. In vitro transcription assays using the polymorphisms (FAFLP) of tubercle pknH promoter and reconstituted RNA poly- bacilli from seals provides molecular ev- merase holoenzyme demonstrated that tran- idence for a new species within the My- scription from the pknH promoter is specif- cobacterium tuberculosis complex. Infect ically initiated by sigmaA, the principal Genet Evol. 2(3) (2003) 193Ð199. sigma factor of mycobacteria. DNase I pro- tection assay and deletion studies with the Tuberculosis in seals is caused by a pknH promoter revealed that the minimal member of the Mycobacterium tuberculosis region required for optimal transcription complex referred to as the ‘seal bacillus.’ carries the sequence from position Ð37 to Fluorescent amplified-fragment length position +6. Moreover, mutation in the polymorphism (FAFLP) analysis was ap- TGN motif of the pknH promoter resulted plied to isolates from four Australian and 71, 4 Current Literature, Molecular and Genetic Studies 421 six Argentinean seals and compared with clinical isolates, the combination of these FAFLP pattern for standard strains belong- two assays has provided a promising dis- ing to the M. tuberculosis complex. The criminatory tool for the identification of FAFLP profiles derived from EcoRI/MseI commonly encountered clinical mycobacte- restricted fragments of blind coded DNA ria species.—Authors’Abstract samples differentiated the seal bacillus from other members of the M. tuberculosis complex. According to the phylogenetic Betts, J. C., McLaren, A., Lennon, M. G., analysis performed using FAFLP data, seal Kelly, F. M., Lukey, P. T., Blakemore, S. bacilli appear to have diverged significantly J., and Duncan, K. Signature gene ex- from other members of the M. tuberculosis pression profiles discriminate between complex. We describe the suitability of a isoniazid-, thiolactomycin-, and triclosan- panel of 19 highly polymorphic markers for treated Mycobacterium tuberculosis.An- rapid identification and comparative ge- timicrob. Agents Chemother. 47(9) (2003) nomic analyses of the seal bacillus strains. 2903Ð2913. It is likely that these bacilli got separated from the M. tuberculosis lineage as a result See Current Literature, Chemotherapy, p. of different insertion deletion events occur- 385 ring on a genome wide scale. Our analysis reveals that the seal bacillus and M. bovis are genetically related and therefore, might Blokpoel, M. C., O’Toole, R., Smeulders, have originated from a common ancestor. M. J., and Williams, H. D. Develop- Our data additionally support the hypothe- ment and application of unstable GFP sis that seal bacillus occupies a unique tax- variants to kinetic studies of mycobacte- onomic position within the M. tuberculosis rial gene expression. J. Microbiol. Meth- complex.—Authors’Abstract ods. 54(2) (2003) 203Ð211.

Unstable variants of green fluorescent Bamaga, M. S., Wright, D. J., and protein (GFP) tagged with C-terminal ex- Zhang, H. An assessment of a multiplex tensions, which are targets for a tail specific PCR assay for differentiating clinically protease, have been described in Escheri- important mycobacteria based on pncA chia coli and Pseudomonas putida [Appl. gene variation. Mol. Cell. Probes. 17(2Ð3) Envir. Microbiol. 64 (1998) 2240]. We in- (2003) 69Ð75. vestigated whether similar modifications to flow cytometer optimized GFP (GFPmut2) The pncA genes in mycobacteria are could be used to generate unstable variants responsible for the production of pyrazin- of GFP for gene expression studies in myco- amidase (PZase). In Mycobacterium tuber- bacteria. We constructed GFP variants in a culosis, PZase hydrolyses pyrazinamide mycobacterial shuttle vector under the con- (PZA) to pyrazonic acid, a compound that trol of the regulatory region of the inducible possesses bactericidal activity against tuber- Mycobacterium smegmatis acetamidase cle bacilli. Nucleotide sequences of pncA gene. GFP expression was induced by the genes found within mycobacteria where addition of acetamide and the stability of the aligned in an effort to ascertain the signifi- GFP variants in M. smegmatis, following the cance of any variability in sequence. Three removal of the inducer to switch off their ex- sets of primers (one degenerate and five con- pression, was determined using spectrofluo- sensus sequences) were designed and em- rometry and flow cytometry. We demon- ployed in a multiplex PCR assay to amplify strate that, compared to the GFPmut2 (half- the pncA region in seven clinically common lives >7 days), the modified GFP variants mycobacteria. The banding patterns gener- exhibit much lower half-lives (between 70 ated from each species in conjunction with and 165 min) in M. smegmatis. To investi- PZase activity tests demonstrated that the gate their utility in the measurement of my- mycobacterial species examined could be cobacterial gene expression, we cloned the clearly identified and differentiated from promoter region of a putative amino acid ef- one another. Although not yet tested with flux pump gene, lysE (Rv1986), from Myco- 422 International Journal of Leprosy 2003 bacterium tuberculosis together with the di- vides a regulation mechanism of mycobac- vergently transcribed, putative lysR-type terial kinases and strongly suggest that regulator gene (Rv1985c) upstream of one PknB and PstP could work as a functional of the unstable GFP variants. We found that pair in vivo to control mycobacterial cell the expression kinetics of the lysRE-gfp fu- growth.—Authors’Abstract sion were identical throughout the M. smeg- matis growth curve to those measured using a conventional lysRE-xylE reporter fusion, Carvalho, W. S., Spindola de Miranda, peaking upon entry into stationary phase. In S., Costa, K. M., Araujo, J. G., addition, it was established that the tagged Augusto, C. J., Pesquero, J. B., Pes- GFP variants were also unstable in Myco- quero, J. L., and Gomes, M. A. Low- bacterium bovis BCG. Thus, we have stringency single-specific-primer PCR as demonstrated that unstable GFP variants a tool for detection of mutations in the are suitable reporter genes for monitoring rpoB gene of rifampin-resistant Myco- transient gene expression in fast- and slow- bacterium tuberculosis. J. Clin. Micro- growing mycobacteria.—Authors’Abstract biol. 41(7) (2003) 3384Ð3386.

By the low-stringency single-specific- Boitel, B., Ortiz-Lombardia, M., Duran, primer PCR technique, a highly sensitive R., Pompeo, F., Cole, S. T., Cervenan- and rapid method for diagnosis of rifampin sky, C., and Alzari, P. M. PknB kinase resistance in Mycobacterium tuberculosis activity is regulated by phosphorylation was established. Seven rifampin-resistant in two Thr residues and dephosphoryla- and five rifampin-susceptible specimens tion by PstP, the cognate phospho- were analyzed. Rifampin resistance was de- Ser/Thr phosphatase, in Mycobacterium termined by MIC measurement. A complex tuberculosis. Mol. Microbiol. 49(6) electrophoretic pattern consisting of many (2003) 1493Ð1508. bands was obtained for both susceptible and rifampin-resistant isolates. The same pat- Bacterial genomics revealed the wide- tern was obtained for all of the susceptible spread presence of eukaryotic-like protein specimens, but differences between resis- kinases and phosphatases in prokaryotes, tant and susceptible isolates were found. but little is known on their biochemical DNA sequencing showed that a particular properties, regulation mechanisms and mutation produces a specific electro- physiological roles. Here we focus on the phoretic pattern.—Authors’Abstract catalytic domains of two trans-membrane enzymes, the Ser/Thr protein kinase PknB and the protein phosphatase PstP from My- Duan, H. F., Zhou, X. H., Ma, Y., Li, C. cobacterium tuberculosis. PstP was found Y., Chen, X. Y., Gao, W. W., Zheng, S. to specifically dephosphorylate model H. [A study on the association of 3′UTR phospho-Ser/Thr substrates in a Mn2+- polymorphisms of NRAMP1 gene with dependent manner. Autophosphorylated susceptibility to tuberculosis in Hans.] PknB was shown to be a substrate for Pstp Zhonghua Jie He He Hu Xi Za Zhi. 26(5) and its kinase activity was affected by PstP- (2003) 286Ð289. [Article in Chinese]. mediated dephosphorylation. Two threo- nine residues in the PknB activation loop, OBJECTIVE: To determine whether found to be mostly disordered in the crystal 3′UTR polymorphisms of the NRAMP1 structure of this kinase, namely Thr171 and gene are associated with tuberculosis in Thr173, were identified as the target for Hans. METHODS: 3′UTR polymorphisms PknB autophosphorylation and PstP de- of NRAMP1 gene were typed by PCR- phosphorylation. Replacement of these RFLP among 147 patients with active tu- threonine residues by alanine significantly berculosis and 145 healthy individuals. The decreased the kinase activity, confirming relationship between 3′UTR polymor- their direct regulatory role. These results in- phisms and susceptibility to tuberculosis dicate that, as for eukaryotic homologues, was studied, and cases were grouped ac- phosphorylation of the activation loop pro- cording to genotypes. RESULTS: In the tu- 71, 4 Current Literature, Molecular and Genetic Studies 423 berculosis patients, genotype TGTG/ which may be helpful in solving many TGTG, TGTG/TGTG deleted, and TGTG questions concerning the reservoirs, patho- deleted/TGTG deleted were observed in 95, genicities, and modes of transmission of 50 and 2 cases respectively, while the geno- these isolates.—Authors’Abstract types of the healthy controls were TGTG/ TGTG in 115, TGTG/TGTG deleted in 29 and TGTG deleted/TGTG deleted in 1 case. Ganesh, N. and Muniyappa, K. Charac- The frequency of the genotype TGTG/ terization of DNA strand transfer pro- TGTG was found more often among con- moted by Mycobacterium smegmatis trols than that in patients (chi(2) = 7.79, p RecA reveals functional diversity with <0.01). The frequency of allele TGTG and Mycobacterium tuberculosis RecA. Bio- the frequency of variant allele were 0.85 chemistry. 42(23) (2003) 7216Ð7225. and 0.15 respectively. CONCLUSIONS: 3′UTR polymorphisms of NRAMP1 gene The RecA-like proteins constitute a are associated with susceptibility to tuber- group of DNA strand transfer proteins ubiq- culosis in Hans. The variant allele observed uitous in eubacteria, eukarya, and archaea. in Hans is more common than that in Cau- However, the functional relationship among casians. These observations might explain RecA proteins is poorly understood. For in- in part why Hans have greater susceptibility stance, Mycobacterium tuberculosis RecA to tuberculosis than Caucasians.—Authors’ is synthesized as a large precursor, which Abstract undergoes an unusual protein-splicing reac- tion to generate an active form. Whereas the precursor was inactive, the active form pro- Dubnau, E. and Smith, I. Mycobacterium moted DNA strand transfer less efficiently tuberculosis gene expression in macro- compared to EcRecA. Furthermore, gene phages. Microbes Infect. 5(7) (2003) disruption studies have indicated that the 629Ð637. frequencies of allele exchange are relatively lower in Mycobacterium tuberculosis com- See Current Literature, Immuno-pathology, pared to Mycobacterium smegmatis. The p. 399 mechanistic basis and the factors that con- tribute to differences in allele exchange re- main to be understood. Here, we show that Gaafar, A., Unzaga, M. J., Cisterna, R., the extent of DNA strand transfer promoted Clavo, F. E., Urra, E., Ayarza, R., and by the M. smegmatis RecA in vitro differs Martin, G. Evaluation of a modified significantly from that of M. tuberculosis single-enzyme amplified-fragment length RecA. Importantly, M. smegmatis RecA by polymorphism technique for fingerprint- itself was unable to promote strand transfer, ing and differentiating of Mycobacterium but cognate or noncognate SSBs rendered it kansasii type I isolates. J. Clin. Micro- efficient even when added prior to RecA. In biol. 41(8) (2003) 3846Ð3850. the presence of SSB, MsRecA or MtRecA catalyzed strand transfer between ssDNA The usefulness of single-enzyme amplified- and varying lengths of linear duplex DNA fragment length polymorphism (AFLP) anal- with distinctly different pH profiles. The fac- ysis for the subtyping of Mycobacterium tors that were able to suppress the formation kansasii type I isolates was evaluated. This of DNA networks greatly stimulated strand simplified technique classified 253 type I transfer reactions promoted by MsRecA or strains into 12 distinct clusters. The dis- MtRecA. Although the rate and pH profiles criminating power of this technique was of dATP hydrolysis catalyzed by MtRecA high, and the technique easily distinguished and MsRecA were similar, only MsRecA between the epidemiologically unrelated was able to couple dATP hydrolysis to DNA control strains and our clinical isolates. strand transfer. Together, these results pro- Overall, the technique was relatively rapid vide insights into the functional diversity in and technically simple, yet it gave repro- DNA strand transfer promoted by RecA pro- ducible and discriminatory results. This teins of pathogenic and nonpathogenic technique provides a powerful typing tool species of mycobacteria.—Authors’Abstract 424 International Journal of Leprosy 2003

Garcia de Viedma, D. Rapid detection of a pattern recognition receptor involved in resistance in Mycobacterium tuberculo- innate immunity. Due to the biological im- sis: a review discussing molecular ap- portance of these molecules, the chemical proaches. Clin. Microbiol. Infect. 9(5) structure of PIM was revisited. The structure (2003) 349Ð359. of PIM2 was recently published (Gilleron, M., Ronet, C., Mempel, M., Monsarrat, B., The last few years have seen the develop- Gachelin, G., and Puzo, G. (2001) J. Biol. ment of several molecular designs to search Chem. 276, 34896Ð34904). Here we report for mutations encoding resistance to antitu- the purification and molecular characteriza- berculous drugs in Mycobacterium tubercu- tion of PIM6 in their native form. For the losis. Most of these are highly efficient for first time, four acyl forms of this molecule RIF-r detection and are well adapted to have been purified, using hydrophobic in- search for the most relevant INH-R muta- teraction chromatography. Mono- to tetra- tions. In this review, these new molecular acylated molecules were identified in M. approaches are explained and are presented bovis bacillus Calmette Guerin, M. tubercu- according to the molecular strategies on losis H37Rv, and M. smegmatis 607 using a which they are based. In this sense, tech- sophisticated combination of analytical niques based on DNA-sequencing, elec- tools, including matrix-assisted laser des- trophoresis and hybridization are reviewed orption/ionization-time of flight-mass spec- and the newer designs based on real-time trometry and two-dimensional homo- and PCR and microarrays are also included. heteronuclear NMR spectroscopy. These Molecular methods are sure to transform experiments revealed that the major acyl standard approaches to the issue of resis- forms are similar to the ones described for tance in the mycobacteriology laboratory. PIM2. Finally, we show that PIM6, like This will allow laboratories to speed up the PIM2, activate primary macrophages to se- performance of resistance assays and pro- crete TNF-alpha through TLR2, irrespec- vide access to essential information for tive of their acylation pattern, and that they highly refined detection, follow-up and signal through the adaptor MyD88.—Au- management of antibiotic resistance in M. thors’Abstract tuberculosis.—Author’s Abstract Hawkey, P. M., Smith, E. G., Evans, J. T., Gilleron, M., Quesniaux, V. F., and Puzo, Monk, P., Bryan, G., Mohamed, H. H., G. Acylation state of the phosphatidyl- Bardhan, M., and Pugh, R. N. Mycobac- inositol hexamannosides from Mycobac- terial interspersed repetitive unit typing of terium bovis bacillus Calmette Guerin Mycobacterium tuberculosis compared to and Mycobacterium tuberculosis H37Rv IS6110-based restriction fragment length and its implication in Toll-like receptor polymorphism analysis for investigation response. J. Biol. Chem. 278(32) (2003) of apparently clustered cases of tubercu- 29880Ð29889. losis. J. Clin. Microbiol. 41(8) (2003) 3514Ð3520. The dimannoside (PIM2) and hexaman- noside (PIM6) phosphatidyl-myo-inositol An evaluation of the utility of IS6110- mannosides are the two most abundant based restriction fragment length polymor- classes of PIM found in Mycobacterium bo- phism (RFLP) typing compared to a combi- vis bacillus Calmette Guerin, Mycobacte- nation of variable number tandem repeat rium tuberculosis H37Rv, and Mycobacte- (VNTR) typing and mycobacterial inter- rium smegmatis 607. Recently, these long spersed repetitive unit (MIRU) typing was known molecules received a renewed inter- undertaken. A total of 53 patient isolates of est due to the fact that PIM2 constitute the Mycobacterium tuberculosis from four pre- anchor motif of an important constituent of sumed episodes of cross-infection were ex- the mycobacterial cell wall, the lipoarabino- amined. Genomic DNA was extracted from mannans (LAM), and that both LAM the isolates by a cetyl trimethylammonium (phosphoinositol-capped LAM) and PIM bromide method. The number of copies of are agonists of Toll-like receptor 2 (TLR2), tandem repeats of the five loci ETR(A) to 71, 4 Current Literature, Molecular and Genetic Studies 425

ETR(E) and 12 MIRU loci was determined tuberculosis H37Ra. Protein spot no. 1 was by PCR amplification and agarose gel elec- identified as Rv2346c. Protein spot no. 2 trophoresis of the amplicons. VNTR typing was identified as Rv2347c, Rv1197, identified the major clusters of strains in the Rv1038c, and Rv3620c, which shared sig- three investigations in which they occurred nificant homology and had the same peptide (each representing a different evolutionary fingerprinting using tryptic digestion. No clade: 32333, 42235, and 32433). The ma- M. tuberculosis protein matched protein jority of unrelated isolates (by epidemiol- spot no. 3. Rv2346c, Rv2347c, Rv1038c, ogy and RFLP typing) were also identified and Rv3620c of M. tuberculosis H37Rv by VNTR typing. The concordance be- were located on the M. tuberculosis H37Ra tween the RFLP and MIRU typing was chromosome, and multiple mutations were complete, with the exception of two isolates observed in the corresponding areas of M. with RFLP patterns that differed by one tuberculosis H37Ra. Codon 59 (CAG, Gln) band each from the rest of the major epi- of Rv2347c and Rv3620c was replaced by demiologically linked groups of isolates in termination codon (TAG) in M. tuberculo- investigation A. All of these isolates had sis H37Ra, which probably terminated the identical MIRU and VNTR types. A further polypeptide elongation. These results pair of isolates differed in the number of demonstrate the importance of studying the tandem repeat copies at two MIRU alleles gene products of M. tuberculosis and show but had identical RFLP patterns. The speed that subtle differences in isogenic mutant of the combined VNTR and MIRU typing strains might play an important role in iden- approach enabled results for some of the in- tifying the attenuating mutations.—Au- vestigations to be supplied in “real time,” thors’Abstract influencing choices in contact tracing. The ease of comparison of results of MIRU and VNTR typing, which are recorded as single Iwamoto, T., Sonobe, T., and Hayashi, K. multidigit numbers, was also found to Loop-mediated isothermal amplification greatly facilitate investigation management for direct detection of Mycobacterium and the communication of results to health tuberculosis complex, M. avium, and M. care professionals.—Authors’Abstract intracellulare in sputum samples. J. Clin. Microbiol. 41(6) (2003) 2616Ð2622.

He, X. Y., Zhuang, Y. H., Zhang, X. G., Loop-mediated isothermal amplification and Li, G. L. Comparative proteome (LAMP) is a novel nucleic acid ampli- analysis of culture supernatant proteins fication method in which reagents react of Mycobacterium tuberculosis H37Rv under isothermal conditions with high and H37Ra. Microbes Infect. 5(10) specificity, efficiency, and rapidity. We (2003) 851Ð856. used LAMP for detection of Mycobacte- rium tuberculosis complex, Mycobacte- To examine the virulence factors of rium avium, and Mycobacterium intracel- Mycobacterium tuberculosis H37Rv, the lulare directly from sputum specimens as proteome was used to characterize the dif- well as for detection of culture isolates ferences in protein expression between vir- grown in a liquid medium (MGIT; Nippon ulent M. tuberculosis H37Rv and attenuated Becton Dickinson Co., Ltd., Tokyo, Japan) M. tuberculosis H37Ra. Two-dimensional or on a solid medium (Ogawa’s medium). gel electrophoresis was performed to sepa- Species-specific primers were designed by rate culture supernatant proteins extracted targeting the gyrB gene, and their specifici- from M. tuberculosis H37Rv and M. tuber- ties were validated on 24 mycobacterial culosis H37Ra. The protein spots of interest species and 7 nonmycobacterial species. were identified by mass spectrometry, and The whole procedure is quite simple, start- then the genes encoding the identified pro- ing with the mixing of all reagents in a teins were cloned and sequenced. Compari- single tube, followed by an isothermal re- son of silver-stained gels showed that three action during which the reaction mixture is well-resolved protein spots were present in held at 63 degrees C. The resulting ampli- M. tuberculosis H37Rv but absent from M. cons are visualized by adding SYBR Green I 426 International Journal of Leprosy 2003 to the reaction tube. The only equipment method of rapidly identifying bacterial needed for the amplification reaction is a species through the combined use of gyrB regular laboratory water bath or heat block genes and microarrays. Closely related bac- that furnishes a constant temperature of 63 teria were not identified at the species level degrees C. The assay had a detection limit using 16S rRNA sequence analysis, whereas of 5 to 50 copies of purified DNA with a they were identified at the species level 60-min incubation time. The reaction time based on the reaction patterns of oligonu- could be shortened to 35 min for the cleotides on our microarrays using gyrB species identification of M. tuberculosis genes.—Authors’Abstract complex, M. avium, and M. intracellulare from a solid-medium culture. Residual DNA lysates prepared for the Amplicor as- Kang, T. J., Kim, S. K., Lee, S. B., Chae, say (Roche Diagnostics GmbH) from 66 G. T., and Kim, J. P. Comparison of sputum specimens were tested in the two different PCR amplification prod- LAMP assay. Although the sample size ucts (the 18-kDa protein gene vs. RLEP used for the latter assay was small, 2.75 repetitive sequence) in the diagnosis of micro l of the DNA lysates, it showed a Mycobacterium leprae. Clin. Exp. Der- performance comparable with that of the matol. 28(4) (2003) 420Ð424. Amplicor assay, which required 50 micro l of the lysates. This LAMP-based assay is See Current Literature, Microbiology, simple, rapid, and sensitive; a result is Leprosy, p. 405 available in 35 min for a solid-medium cul- ture and in 60 min for a liquid-medium cul- ture or for a sputum specimen that contains Kurabachew, M., Sandaa, R. A., Enger, O., a corresponding amount of DNA available and Bjorvatn, B. Sequence analysis in for testing.—Authors’Abstract the 23S rDNA region of Mycobacterium tuberculosis and related species. J. Micro- biol. Methods. 54(3) (2003) 373Ð380. Kakinuma, K., Fukushima, M., and Kawaguchi, R. Detection and identifica- We sequenced a 516 base pair segment in tion of Escherichia coli, Shigella, and the 23S rRNA gene of 54 Mycobacterium Salmonella by microarrays using the tuberculosis isolates, 52 of which were clin- gyrB gene. Biotechnol. Bioeng. 83(6) ical isolates from Ethiopia. Sequence poly- (2003) 721Ð728. morphism was observed with 19 of the 54 strains; the polymorphic sites occurred in Commonly, 16S ribosome RNA (16S less than 2% (9/516) of the total sequence rRNA) sequence analysis has been used for positions. The sequence variations repre- identifying enteric bacteria. However, it sented base pair substitutions (14/23), inser- may not always be applicable for distin- tions (9/23) or both (1/23). Insertions oc- guishing closely related bacteria. Therefore, curred at one site only, whereas substitutions we selected gyrB genes that encode the sub- were observed in various regions of the unit B protein of DNA gyrase (a topo- gene. There was no relation between muta- isomerase type II protein) as target genes. tional sites and drug susceptibility. How- The molecular evolution rate of gyrB genes ever, using information from the GenBank is higher than that of 16S rRNA, and gyrB database, comparison between the 23S genes are distributed universally among rDNA sequences of M. tuberculosis and the bacterial species. Microarray technology in- corresponding sequences of other mycobac- cludes the methods of arraying cDNA or teria and of related non-mycobacterial oligonucleotides on substrates such as glass species revealed considerable variation, slides while acquiring a lot of information suggesting that this region may provide a simultaneously. Thus, it is possible to iden- target for rapid detection and identification tify the enteric bacteria easily using mi- of mycobacteria both at the genus or croarray technology. We devised a simple species level.—Authors’Abstract