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307 INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. (ISSN 0148-916X) INTERNATIONAL JOURNAL OF LEPROSY and Other Mycobacterial Diseases
VOLUME 71, NUMBER 4DECEMBER 2003
Long-term Efficacy of 2 Year WHO Multiple Drug Therapy (MDT) in Multibacillary (MB) Leprosy Patients1
Roland V. Cellona, Maria V. F. Balagon, Eduardo C. dela Cruz, Jasmin A. Burgos, Rodolfo M. Abalos, Gerald P. Walsh, Richard Topolski, Robert H. Gelber, and Douglas S. Walsh 2, 3 ABSTRACT Relapse rate estimates after 2 year WHO multiple drug therapy (MDT) in multi-bacillary (MB) leprosy vary. Between 1987 and 1994, 500 MB leprosy patients completing 2 year MDT were enrolled in a prospective relapse study. The majority of patients (N = 316) were treated and followed at the physician-staffed Cebu Skin Clinic (CSC), whereas others (N = 184) received therapy from government clinics and were followed by CSC technicians in the field. Relapse definition was an increased bacteriologic index (BI) and new skin lesions, sup- plemented with mouse footpad inoculations. Through 2002, follow-up was 5368 person- years, with a mean of 10.8 years per patient. The absolute relapse rate was 3% (15/498; 0.28/100 person-years), with a cumulative risk estimate of 3.9% at 15 yrs. For a subset of 217 patients followed for ≥12 yrs or until relapse, relapses occurred in 9% (13/142) attend- ing the CSC, versus 3% (2/75) assessed in the field (p = 0.09). The rate for patients followed at CSC for ≥12 yrs and a pre-treatment BI ≥2.7+ was 13% (13/98). All relapses were BL or LL, with pre-treatment BI’s of ≥2.7+. Relapses occurred long after completion of therapy, between 3 and 11 yrs from the midpoint of the examination without relapse to detection, or between 6 to 13 yrs to the actual year of detection, 7 occurring at ≥10 yrs. Lesion material from all relapses contained M. leprae that was rifampin and clofazimine sensitive, whereas 3 showed partial or full dapsone resistance. [Follow-up rigor and time], medical expertise, and pre-treatment bacterial load influence relapse rates after 2 yr MDT. RESUMÉ Les estimations des taux de rechutes des patients lépreux multibacillaires (MB), après deux années de polychimiothérapie (PCT) selon les recommendations de l’OMS, varient. Entre 1987 et 1994, 500 MB patients hanséniens ayant complété deux années de PCT furent
1Received for publication 9 December 2002. Accepted for publication 9 October 2003. 2 R. V. Cellona, M. V. F. Balagon, E. C. dela Cruz, J. A. Burgos, R. M. Abalos, G. P. Walsh, R. Topolski, R. H. Gelber, and D. S. Walsh, Leonard Wood Memorial Center for Leprosy Research, Cebu City, Philippines, De- partment of Psychology, Augusta State University, Augusta, Georgia, USA, and Dermatology Service, Dwight David Eisenhower Army Medical Center, Ft. Gordon, Georgia, U.S.A. Reprint requests: Roland V. Cellona, MD, DPH, Chief, Epidemiology Branch, Leonard Wood Memorial Cen- ter for Leprosy Research, P.O. Box 727, Cebu City 6000, Philippines. E-mail: [email protected] 3 The opinions or assertions presented herein are the private views of the authors (DSW) and are not to be con- strued as official or as reflecting the views of the U.S. Departments of the Army or Defense.
308 71, 4 Cellona, et al.: Relapse Rates in MB Leprosy after 2 Year WHO-MDT 309
enrôlés dans une étude prospective de rechute. La majorité des patients (N=316) furent traités et suivis par l’équipe de médecins de la Cinique des maladies de la peau de Cébu (CSC), tandis que les autres (N = 184) reçurent un traitement des cliniques gouvernemen- tales et furent suivis par des techniciens de la CSC sur le terrain. La définition de la rechute était une augmentation de l’index bactérioscopique (IB) et l’apparition de nouvelles lésions cutanées, complétées par des inoculations à la voute plantaire de souris. Le suivi de 2002 a été de 5368 personnes par année, avec une moyenne de suivi de 10,8 années par patient. Le taux absolu de rechute était de 3% (15/498; 0,28/100 personnes par années) avec une esti- mation de risque cumulé de 3,9% à 15 ans. Pour une sous-population de 217 patients suivie pendant plus de 12 ans ou bien jusqu’à la rechute, les rechutes représentaient 9% (13/142) des malades suivis pas le CSC, contre 3% (2/75) chez ceux suivis sur le terrain (p = 0,09). Le taux de rechute pour des patients suivis pendant plus de 12 ans et avec un BI avant le traitement de plus de 2,7+ était de 13% (13/98). Toutes les rechutes ont été observées chez des patients BL ou LL, avec un BI avant le traitement de plus de 2,7+. Les rechutes ont eu lieu bien après la fin du traitement, entre 3 et 11 années de la médiane d’examens sans rechute à la détection, soit entre 6 et 13 années après l’année réelle de détection, 7 rechute ayant apparu après 10 années. Toutes les biopsies des personnes ayant rechutées contenaient des M. leprae qui étaient toutes sensibles à la rifampicine et la clofasimine, bien que 3 mon- traient une résistance partielle ou totale à la dapsone. La rigueur et le temps du suivi, l’ex- pertise médicale et la charge bactérienne avant le traitement influencent les taux de rechute après deux années de PCT. RESUMEN La tasa de recaída después de 2 años de tratamiento con poliquimioterapia (PQT-OMS) en la lepra multibacilar (MB) varía debido a diferentes factores. Entre 1987 y 1994, 500 pa- cientes MB que completaron 2 años de PQT fueron enrolados en un estudio prospectivo so- bre recaídas. La mayoría de los pacientes (N = 316) fueron tratados y supervisados por el personal médico de la clínica de la piel de Cebu (CSC), mientras que otros (N = 184) reci- bieron la terapia en clínicas periféricas del gobierno y fueron supervisados por técnicos de la CSC en el campo. La definición de recaída fue un aumento en el índice bacteriológico (IB) y nuevas lesiones en la piel. La resistencia o susceptibilidad a drogas se probó por in- oculación en la almohadilla plantar del ratón. Durante el año 2002, el tiempo de seguimiento fue de 5368 persona-años, con una media de 10.8 años por paciente. La tasa absoluta de re- caída fue del 3% (15/498; 0.28/100 persona-años), con un riesgo acumulativo estimado de 3.9% a 15 años. Para un subconjunto de 217 pacientes supervisados por >12 años o hasta que se presentó la recaída, las recaídas ocurrieron en el 9% (13/142) de los pacientes aten- didos en la CSC, contra el 3% (2/75) de los pacientes atendidos en el campo (p = 0.09). La tasa para los pacientes atendidos en la CSC por >12 años y un IB pre-tratamiento de >2.7+ fue del 13% (13/98). Todas las recaídas fueron de casos BL o LL con un IB pre-tratamiento de >2.7+. Las recaídas ocurrieron mucho tiempo después de haberse concluido la terapia (entre 3 y 13 años), ocurriendo 7 casos a >10 años. El material de las lesiones de todas las recaídas contuvieron M. leprae que fue sensible a la rifampina y clofazimina, aunque 3 ais- lados mostraron resistencia parcial o total a la dapsona. El rigor en el seguimiento y el tiempo del mismo, la experiencia médica, y la carga bacteriana previa al tratamiento, in- fluyen en la tasa de recaída después de 2 años de PQT.
Dapsone monotherapy was used to treat therapy (MDT), a convenient, relatively patients with all forms of leprosy from the inexpensive regimen consisting of monthly 1940’s until the 1990’s. However, concerns rifampin and clofazimine, and daily dapsone about dapsone resistance and relapses upon and clofazimine administered for at least 2 discontinuation of therapy, especially in yrs in MB patients. By 1994, MDT was im- those with multi-bacillary (MB) disease, led plemented worldwide and the overall preva- to the development of rifampin-containing lence of leprosy, but not incidence, dropped bactericidal regimens in the 1970’s, mod- dramatically because patients completing eled after successful short-course rifampin- MDT were removed from prevalence lists based regimens for pulmonary tuberculosis (2, 17). Relapse rates after MDT were pur- (10). By 1982, the WHO abandoned dapsone ported to be low, but sufficiently discrepant monotherapy and introduced multiple-drug estimates, now coupled with evidence that 310 International Journal of Leprosy 2003 patients with high pre-treatment M. leprae staffed CSC, as well as those treated at lo- loads are at higher risk and that mean re- cal government clinics with a documented lapse incubation periods for rifampin- diagnosis of MB leprosy and satisfactory containing regimens are likely beyond 5 yrs, completion of 2 year WHO-MDT. MDT indicate a need for additional, rigorous, long- consisted of 24 observed doses of rifampin term studies to more clearly define the pro- (600 mg) and clofazimine (300 mg) given tective efficacy of 2 year MDT (1, 3, 9, 57, 58). at monthly intervals within a 24 to 30 With the extraordinary time and effort re- month period, and unobserved daily dap- quired to treat and then follow patients to ac- sone (100 mg) and clofazimine (50 mg), curately measure relapse, coupled with with no additional efforts to enhance self- widespread MDT implementation only since administration (24). the early 1990’s, prospective, rigorously con- Patients were excluded if they resided in ducted studies are scarce (1, 3, 4, 6, 9, 57). Indeed, a leprosaria or more than 100 kilometers many reports are retrospective analyses away from the CSC, or had tuberculosis, expressed in absolute numbers, and lack ad- malignancy, or other chronic systemic ill- equate follow-up time or sufficient partici- ness. Volunteers were advised on follow-up pants, or vary in the definition of relapse cri- requirements and potential benefits, and a teria, making comparisons across studies field team monitored some patients without difficult (6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 18). For prospec- transportation. All volunteers received vita- tive 2 year MDT relapse studies, overall re- mins and travel reimbursement. lapse rates range from 0% to 20%, likely re- To the greatest extent possible, clinical ex- flecting variability in study site, treatment aminations, lepra reaction monitoring, and compliance, relapse definition, frequency and slit skin smears for acid-fast bacilli (AFB), operational differences in follow-up, length of following a standard 6 site sampling proce- follow-up, and pre-treatment bacterial indices dure (25), were done annually. Unless other- (BI) (1, 3, 5, 6, 8, 9, 11, 12, 13, 14, 15, 16, 19, 20, 21, 22, 23, 57). wise specified, all BI values refer to the Jamet found that post-MDT relapse rates mean value obtained from the 6 sites. Gen- increased from 3% to 20% when mean fol- erally, patients treated at the CSC received low-up was extended from 3.5 to 6 yrs (9), follow-up by physician-leprologists every 1 and in 2 studies, the proportion of patients to 2 years, and those recruited from the lo- relapsing with high pre-treatment bacterial cal government clinics received follow-up indices (BI; ≥4+) was notably higher (3, 9). by a CSC field team every 2 to 3 years. In 1987, we started a prospective longitu- When more than 1 year had elapsed between dinal study to assess for relapses in MB pa- smears for a relapsed patient, time to relapse tients who had satisfactorily completed 2 was estimated by selecting the “midpoint” year MDT. Mean follow-up time was about year of the period between most recent 10 years per patient, longer than other sim- smear and the year of smear positivity (9). ilarly designed studies (1, 3, 9). Relapse was Relapse was defined as the appearance of defined by 2 field-expedient criteria, includ- new skin lesions consistent with leprosy ing new skin lesions consistent with leprosy and a BI increase of ≥2+ at any site (6, 9). and an increasing BI. Supplemental assays Lesion tissue was then harvested for his- included histology and mouse footpad as- tology and mouse footpad inoculation, and says, the latter assessing M. leprae viability the patient treated with ROM, consisting of and sensitivity to the individual compo- 12 monthly, supervised doses of rifampin nents of MDT. (600 mg), ofloxacin (400 mg), and minocy- cline (100 mg) (26). Lepra reactions, includ- METHODS AND MATERIALS ing reversal reaction (RR), characterized by Protocol. Enrollment was conducted swelling and erythema of existing leprosy from 1987 to 1994 at the Cebu Skin Clinic lesions and erythema nodosum leprosum (CSC), an established leprosy referral cen- (ENL), characterized by crops of tender ter for Cebu province. Any patient with papulo-nodules, fever, and malaise, were MB leprosy who had satisfactorily com- graded as mild, moderate or severe (27). pleted 2 years of WHO-MDT and was will- Reactions were treated with oral cortico- ing to comply with long term follow-up steroids until resolution. was eligible. The cohort included patients Histology and mouse footpad studies. treated and followed at the leprologist- For each relapse, mouse footpad tests were 71, 4 Cellona, et al.: Relapse Rates in MB Leprosy after 2 Year WHO-MDT 311
TABLE 1. Demographic characteristics of volunteers.
Demographics Enrolled 500 1987 79 1988 58 1989 85 1990 50 1991 71 1992 68 1993 86 1994 4 Male : Female 377:123 Mean age (yr), (range) 32.5 (10Ð69)
Mean BI Mean BI at MDT Clinical diagnosis pre-MDT Pre-MDT (range) completion (range) BT 24 1.3 (0.5Ð1.3) 0.2 (0Ð1.5) BB 16 2.1 (0.8Ð2.3) 0.1 (0Ð1) BL 253 2.9 (0.2Ð5) 0.2 (2.4) LL 207 4.1 (1.2Ð5.5) 1 (0Ð4.3) All All 3.34 (0.2Ð5.5) 0.5 (0Ð4.3)
BT = borderline tuberculoid, BB = borderline, BL = borderline lepromatous, LL = lepromatous
used to assess viability of M. leprae organ- SigmaStat (version 2.03, SPSS, UK), SPSS isms and characterize drug sensitivities as (UK), and Minitab (version 13). The pri- previously described (28, 29, 30). A skin punch mary outcome was relapse, and secondary biopsy from a clinically active lesion was outcomes were BI-relapse relationships, obtained and processed for inoculation into mouse footpad studies, and incidence of inbred CBA/J mice. Approximately 5 × 103 lepra reactions. Relapse rates were ex- acid-fast bacilli (AFB) were inoculated into pressed as the percentage of patients re- each hind footpad. One group of untreated lapsed, with incidence densities calculated mice was used to confirm M. leprae viabil- as the number of relapsed patients divided ity, whereas other groups of mice received by the total person-years of follow-up. The clofazimine or dapsone in the feed Kaplan-Meier method was used to estimate (0.0001%, 0.001% and 0.01%), or rifampin the cumulative risk of relapse during fol- twice weekly by gastric gavage (5 mg/kg, low-up (32, 33). Lepra reactions were re- 10 mg/kg and 20 mg/kg). For all experi- ported as proportions. ments, both hind footpads of each mouse were pooled, processed, stained (Fite- RESULTS Faraco), and counted (28, 31). M. leprae via- Volunteers. From 1987 to 1994, 500 pa- bility was determined by periodically sacri- tients were enrolled, including 184 govern- ficing 1 or 2 untreated mice, starting at 6 ment clinic referrals with documented satis- months. Upon viability confirmation, de- factory MDT completion. Recruitment and fined as ≥1.5 × 105 M. leprae/footpad, demographics are shown in Tables 1 and 2. treated mice were assessed for M. leprae Clinical classification was according to growth. Drug resistance was defined as a Ridley and Jopling (34). For bacterial loads, harvest of ≥105 M. leprae/footpad (≥20-fold 181 volunteers had a “high” BI (≥4.0+) increase in the inoculum). (36%) and 319 had a “low” BI (<4.0+) Data collection and analysis. Data were (64%). At MDT completion, 219 volunteers recorded on standardized case report forms, (44%) remained smear positive, with a entered into a computerized database (Ex- downward trend through year 6, when all cel 2000), and cross-checked for agree- smears were negative. In general, BI values ment. Graphical and statistical analyses fell about 1 log per year. Through 2002, 83 were done by Sigmaplot 8 (SPSS, UK), patients were no longer in follow-up due to 312 International Journal of Leprosy 2003
TABLE 2. Range of BI pre-MDT, and proportion of positive smears during and after MDT.*
Number Number at Number of smear positives yearly post-MDT BI range pre-MDT MDT completion Yr 1 Yr 2 Yr 3 Yr 4 Yr 5 5.0Ð6.0+ 63 000000 4.0Ð4.9+ 118 210000 3.0Ð3.9+ 142 11 1 0 0 0 0 2.0Ð2.9+ 119 30 4 2 2 0 0 1.0Ð1.9+ 48 72 7 11 0 0 0 0.1Ð0.9+ 10 104 29 16 15 5 1 Total 500 219/500 42/202 29/211 17/207 5/250 1/208 (43.8%) (20.8%) (13.7%) (8.1%) (2.0%) (0.48%)
*All smears negative at 6th year. relapse (N = 15), death unrelated to leprosy tween 6 and 13 yrs (7 relapses between 10 (N = 32), ingestion of anti-leprosy drugs (N and 12 yrs), with a mean relapse incubation = 3), and relocation (N = 33). Overall, 465 time 9.0 yrs (95% CI, 7.7 to 10.4 yrs). volunteers remained in follow-up for ≥5 All patients fulfilling the criteria for re- years or relapsed (Figure). lapse presented with new papules, nodules, Relapses. Two volunteers were excluded or plaques and a rising BI therein (5+ [N = from analysis because they failed to return 12], 4+ [N = 2], and 3+ [N = 1]). In all but a for any follow-up. Through 2002, follow- single relapse (R-341), the BI had become 0 up was 5368 patient-years, with a mean of at all 6 sites sampled on the examination 10.8 years per patient. Overall, the relapse prior to relapse detection. In 10 relapses, rate was 3.0% (15/498), with an incidence AFB were present in new skin lesions as density of 0.28/100 patient-years. A Kaplan- well as earlobes, 8 bilaterally, with BIs rang- Meier survival curve (Figure) estimated cu- ing from 1+ to 5+ (mean 3.3+), whereas all mulative risks of relapse at 5, 7, and 15 yrs other standard smear sites were negative for of 0.64% (95% confidence interval [CI], AFB. Overall, 5 relapsed volunteers had a 0.03%Ð1.25%), 1.7% (95% CI, 1.0%Ð BI of 0 on MDT completion, with all but one 2.4%), and 3.9% (95% CI, 2.2%Ð5.6%), re- attaining a BI of 0 prior to relapse detection. spectively. For those with a high pre-MDT BI, 2 of 6 at- All relapses were borderline lepromatous tained a BI of 0 at MDT completion. (BL) or polar lepromatous (LL) before MDT Post-MDT reversal reaction (RR) and ery- and had pre-treatment BIs ≥2.7+. For the thema nodosum leprosum (ENL), recorded subset of patients followed at the CSC by only for volunteers recruited at CSC, were re- physician-leprologists for ≥12 years, ported in 71 of 316 volunteers (23%), with 13/142 (9%) relapsed vs. 2/75 (3%) fol- 62 being graded as mild (Table 4) (27). The lowed in the field (p = 0.09, Fischer’s exact majority of RR (61%) and all ENL occurred test). For a smaller group of patients within within 5 years of MDT completion. Some the CSC subgroup followed for ≥12 yrs and volunteers developed multiple episodes of who had an initial BI ≥2.7+, the relapse rate the same reaction type, but RR and ENL was 13% (13/98) vs. 0% (0/44) in those was never reported in the same patient. All with an initial BI <2.7 (p <0.001, Fisher’s reactions resolved with oral corticosteroids, exact test). There was no correlation be- without permanent sequelae. tween relapse incubation time and BI mag- Mouse footpad studies. Lesion samples nitude at relapse (r2 = 0.26; p = 0.35). from all relapses grew in untreated mice, Using midpoint estimates, relapses were confirming viability. All relapse isolates recorded between 3 and 12 yrs, with a mean demonstrated sensitivity to all 3 tested ri- relapse incubation time of 7.9 yrs (95% CI, fampin and clofazimine dosage schedules. 6.4 to 9.3 yrs) (Table 3). Based on actual M. leprae from 12 relapsed patients were year of detection, relapses occurred be- found to be fully dapsone sensitive. Relapse 71, 4 Cellona, et al.: Relapse Rates in MB Leprosy after 2 Year WHO-MDT 313
THE FIGURE. Cumulative risk estimates (Kaplan-Meier) of relapse after 2 year MDT. Numbers in parenthe- ses in the graph represent relapses, and numbers along the x-axis represent patients in follow-up.
isolates R-499 and R-398 (Table 3) grew in for ≥12 yrs with pre-MDT BIs <2.7+ sup- mice treated with only the lowest concen- porting contentions that higher pre-treatment tration of dapsone (mild to negligible resis- BIs influence relapse risk (6). Overall, a total tance), whereas isolate R-285 grew in mice of 15 relapses yielded an absolute relapse treated with all 3 concentrations, compati- rate of 3% (0.28/100 person-years) and a ble with “high” grade resistance. Kaplan-Meier survival analysis (adjusts for differences in follow-up) estimated the cu- DISCUSSION mulative risk of relapse to be 3.9% at 15 yrs. Other notable findings included 8 of 15 Relapse was studied in a cohort of 500 relapses occurring within 7 years of MDT MB leprosy patients treated reliably with 2 completion, and in agreement with other year MDT and followed prospectively by an studies, most were in patients with high experienced study site for a mean of 10.8 pre-MDT BI (≥4+) (57). Five of 15 relapses yrs, longer than most other studies (57). For a attained a BI of 0 by MDT completion, with subset of 217 patients followed ≥12 yrs or all but one attaining a BI of 0 prior to relapse until relapse, relapse rates were 9% (13/ 142) detection, emphasizing smear negativity is among those attending the leprologist- not necessarily protective against relapse (3). staffed CSC and 3% (2/75) assessed by a All relapses, fulfilling our 2 field-expedient CSC field team, a notable difference sug- relapse criteria of new skin lesions with an gesting that relapse detection may have been increased BI, were subsequently found to affected by medical expertise. Within a contain M. leprae that multiplied in mice, smaller subset of patients followed at CSC underscoring the reliability of our relapse for ≥12 yrs and a pre-treatment BI ≥2.7, the definition. Especially encouraging was that relapse rate was 13% (13/98). In contrast, the none of the relapses were associated with relapse rate was 0% (0/44) in those followed peripheral neuropathy or disability. Post- 314 International Journal of Leprosy 2003 relapse skin Midpoint year Relapse to 1989 2000 2001 (13) 2001 (12) 1991 1997 1998 (7) 1998 (7) 2.3 1989 1992 1996 (7) 1994 (5) 0.5 1992 1997 1999 (7) 1998 (6) 0.3 1993 1997 2001 (8) 1999 (6) 2.3 1988 1994 1996 (8) 1995 (7) 0 *0 4.5* 4.0 5.0* 4.5* 4.0* 4.0* Clinical characteristics of relapses. 3. ABLE T 4.0+) ≥ pre-MDT “high” BI ( * **date (number of elapsed years) Patient New skin lesions Age at Pre-MDT Pre-MDT Post-MDT Year Prior negative smear after smear after codeR-341R-158 M F Macules, papules, localized infiltration Plaques 43 at relapse BL 3.7R-398 relapse M diagnosis Macules, localized infiltration 1.3 27 BI 1987 LL 32 BI 1987 BL enrolled 1993 (6) skin smear 1990 (3) MDT*** MDT** R-285R-366 F M Papules, localized infiltration Macules, papules, localized infiltration 16 32 LL BL 3.0 0 1990 1994 1996 (6) 1995 (5) R-408 M Nodules, localized infiltration 44 LL R-499 M Nodules, papules, localized infiltration 48 LL R-082 M Localized infiltration 20 BL R-207R-053 FR-162 MR-110 M Papules, localized infiltration Macules, nodulesR-161 M Macules, papulesR-422 M Localized infiltrationR-081 M Macules, localized infiltration M Macules, papules, localized infiltration 21 Macules, papules, localized infiltration 40 26 32 BL 25 BL 67 39 LL BL 3.6 BL 3.7 BL LL 3.3 2.7 0 2.7 3.7 1.3 3.8 1.3 0.7 1992 1988 0 1.8 1988 2.3 1987 1998 1997 1990 1987 1987 1997 1997 1999 (7) 2000 (12) 1997 2000 (12) 1994 1995 1999 (7) 1999 (11) 1997 (10) 1999 (11) 1997 (10) 2001 (11) 1999 (12) 1997 (10) 1997 (10) 1999 (9) 1996 (9) 71, 4 Cellona, et al.: Relapse Rates in MB Leprosy after 2 Year WHO-MDT 315
TABLE 4. Lepra reactions after 2 year availability of skin smears and histopathol- WHO-MDT. ogy lessens the ability to identify those pa- tients requiring the most rigorous follow- Within up. This argues for the maintenance and 5 years Range Reaction Total* Severity training of leprologists, emphasis on the of MDT (years) importance of the diagnosis of LL and its completion relapse, and the re-establishment of skin RR 62/316 57 mild 38 0Ð11 smears and histopathology. (20%) 5 moderate (61%) Recent reports suggest the relapse incu- ENL 9/316 4 mild 9 0Ð5 bation periods after rifampin-containing (3%) 5 moderate (100%) regimens like MDT likely extend beyond a * There were no enrollees with both reversal reac- previously advocated range of 3 to 7 yrs tion (RR) and erythema nodosum leprosum (ENL). (57). Indeed, Pattyn found that relapses after an intensive 6 week rifampin-containing MDT reactions responded well to oral cor- regimen began at 6 yrs, but with a doubling ticosteroids and resolved without perma- in years 8 to 9 (58). Here, 7 of 15 relapses nent sequelae. occurred at 9 or more years (midpoint esti- A notable observation was the propor- mate) after therapy, for an overall mean in- tional increase in relapse rates found in pa- cubation period of 7.9 yrs. After character- tients followed by physicians at CSC vs. izing 15 relapses through 2002, 3 others oc- those in the field. As care of leprosy pa- curred in 2003, at years 13, 14, and 15 of tients is shifted to the general health ser- follow-up, further underscoring the impor- vice, wherein even rudimentary follow-up tance of extended follow-up for defining re- may not be feasible, much less long term lapse rates. Notwithstanding, relapse incuba- follow-up, diagnosis of relapse MB leprosy tion periods will invariably depend on relapse may be delayed. This is problematic be- definitions, as would rates, in that a more rig- cause early relapses, like here, may present orous definition might require longer follow- with only new skin lesions. However, if re- up to qualify as a relapse (3, 9, 57). lapses are detected only late, the risk for Irrespective of relapse definitions, it is neurologic morbidity and deformity in- impossible to know whether relapses reflect creases. Furthermore, since failure of MDT reactivation of the “persister” organisms is generally confined to those with BL or that have survived MDT or re-infection LL and high BIs, the loss worldwide of the (35, 36, 37, 38, 39, 40, 41, 42). Indeed, although all
TABLE 5. Mouse footpad tests for viability and drug sensitivity.
Patient Control Rifampicin by gavage Clofazimine in diet Dapsone in diet code group 20 mg 10 mg 5 mg 0.01 0.001 0.0001 0.01 0.001 0.0001 R-341 16/16 0/2 0/2 0/2 0/10 0/10 0/10 0/9 0/7 0/10 R-158 7/7 0/6 0/6 NH NH 0/8 0/10 0/9 0/9 0/10 R-499 14/14 0/6 0/8 0/6 NH 0/8 0/7 0/7 0/8 8/8* R-285 10/10 0/6 NH 0/6 NH 0/7 0/6 2/8** 8/8 8/8 R-162 14/14 0/3 NH 0/6 NH 0/8 0/10 0/10 0/10 0/10 R-161 2/2 0/2 0/2 0/1 0/2 0/2 0/2 0/2 0/2 0/2 R-082 8/8 0/1 0/5 NH NH 0/7 0/6 0/7 NH NH R-110 10/10 0/2 0/5 0/3 NH 0/6 0/6 0/6 0/6 0/6 R-207 6/6 0/2 0/3 0/2 0/2 0/3 0/7 0/7 0/7 0/7 R-081 2/2 0/2 NH NH NH 0/6 0/6 0/5 0/8 0/2 R-366 6/6 0/1 0/2 0/2 NH 0/6 0/6 0/6 0/6 0/6 R-422 13/13 0/2 0/3 0/2 NH 0/9 0/7 0/8 0/7 0/9 R-053 7/7 0/4 0/7 0/5 0/6 0/4 0/6 0/4 0/6 0/3 R-408 5/5 0/5 0/5 0/5 0/5 0/6 0/6 0/6 0/6 0/6 R-398 2/2 0/6 0/5 0/5 0/6 0/6 0/6 0/5 0/5 4/6* *low dapsone resistance, **high dapsone resistance, NH (no harvest) 316 International Journal of Leprosy 2003 relapses were essentially sensitive to the Agra may have increased with additional MDT components, arguing against the de- follow-up (50). velopment of drug resistance, murine mon- In 1998, the WHO reduced the duration itoring of several rifampin-based regimens of MDT to 1 year for MB patients, with an for MB leprosy indicate that “persisters” eventual goal of 6 months for all forms of surviving rifampin therapy regularly occur leprosy and a cumulative relapse rate of in 9% of MB patients (43), coinciding with ≤5% at 5 yrs, paralleling accepted rates for the 9% relapse rate among those with ≥12 pulmonary TB (41). Previously, relapse rates yrs, CSC physician-based follow-up. In tu- greater than 5% after therapy of pulmonary berculosis (TB), RNA restriction fragment TB were rejected by the British Medical length polymorphisms show that in AIDS Research Council as inadequate (10), and re- populations, almost half the infections are cent findings of a 9% relapse rate after 6 new, whereas in populations with low en- months therapy for cavitary TB (51), like demicity, the majority are reactivation (44, 45). MB leprosy with a high bacterial burden, Genotypic assays that detect differences prompted calls to extend therapy to 9 among M. leprae strains may help to distin- months (52). Analagously, our MDT relapse guish reactivation from reinfection in lep- data, along with others, might argue that rosy, as well as whether certain M. leprae even 2 year MDT is inadequate in MB lep- strains predispose to relapse and which bio- rosy, especially for patients with high BI chemical anomalies confer treatment failure before treatment, and moreover, that 5 years (46, 47, 48). of follow-up is insufficient to accurately de- An important goal of MDT is the preven- fine relapse risks. tion of multiple drug-resistant M. leprae. Alternatives to 2 year MDT include: (i) Here, relapse M. leprae was uniformly sen- the proven modality of lifelong dapsone sitive to rifampin and clofazimine, and only therapy after 5 years of daily dapsone and ri- 3 (20%) isolates showed dapsone resistance fampin (55), (ii) extending MDT, or (iii) (2 low level, 1 fully resistant), surprising in highly bactericidal combinations that include that up to 52% of our untreated patients in rifampin or ofloxacin (or congeners), clar- the early 1990’s harbored primary dapsone ithromycin, or minocycline (54). Indeed, in resistant M. leprae (low, moderate and high) the Agra study, the 17% relapse rate found in (28). Clearly, our current data argue that 2 MB patients treated for 2 years with an initial year MDT prevented the emergence of drug- BI ≥4 was reduced to 4% when MDT was resistant M. leprae, paralleling observations extended until smear negativity, an average in effective multi-drug TB regimens (10). of 5 years on treatment (3). According to ob- In this study, even within subsets of pa- servations in mice whereby minocycline tients suggesting that the relapse rate may alone or in combination with moxifloxacin be as high as 9% or even 13%, MDT pro- added to the bactericidal activity of ri- vided a reasonably high cure rate. In con- famycin (54, 55), and in the highly bacillifer- trast, other prospective relapse studies re- ous and immunosuppressed neonatally port rates of up to 20%, with even higher thymectomized Lewis rat whereby minocy- rates among patients with a high pre-MDT cline + rifampin, but not rifampin alone, BI, usually characterized by a still positive consistently eliminated all viable M. leprae BI after MDT completion (9, 14, 19, 49, 57). In (56), minocycline and perhaps moxifloxacin particular, the Marchoux Study Group may be especially beneficial components to (Mali) reported relapses in 20% (7/35) of any future rifamycin-containing regimen. MB patients with a mean follow-up of 6 years, but a rate of 39% for those with a Acknowledgment. This study was supported by pre-treatment BI ≥4+ (5). Girdhar (India) re- grants from the World Health Organization (UNDP/ ported relapses in 7% (20/260) of MB pa- World Bank/WHO Special Programme for Research tients with a mean follow-up of 4 years, and Training in Tropical Diseases), Pacific Leprosy with a 17% (18/170) rate among those with Trust (New Zealand) and the Sasakawa Memorial ≥ Health Foundation of Japan. Rico Abella, Jay Vicada, a pre-treatment BI 4+ versus 1% (2/153) Junie Abellana, Mary Grace Bordon, Mercedita Hi- 6 with a BI <4+ ( ). Late relapses here and in naloc, and the LWM laboratory staff contributed to this Pattyn’s study, however, suggest that re- work. Guillerma Lim and Pris Reed provided adminis- lapse rates after 2 year MDT in Mali and trative support. 71, 4 Cellona, et al.: Relapse Rates in MB Leprosy after 2 Year WHO-MDT 317
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J., DESMOND, E., SIERRA, tients treated with rifampicin plus dapsone after M. F., and SCHOOLNIK, G. K. Exogenous reinfec- varying periods of dapsone monotherapy. Indian J. tion with multidrug-resistant Mycobacterium tu- Lepr. 73 (2001) 1Ð10. berculosis in patients with advanced HIV infec- 39. LOCKWOOD, D. N. Leprosy elimination—a virtual tion. N. Engl. J. Med. 328 (1993) 1137Ð1144. phenomenon or a reality? BMJ 324 (2002) 53. SOARES, D. J., NEUPANE K., and BRITTON, W. J. Re- 1516Ð1518. lapse with multibacillary leprosy caused by rifam- 71, 4 Cellona, et al.: Relapse Rates in MB Leprosy after 2 Year WHO-MDT 319
picin sensitive organisms following paucibacillary 56. THANGARAJ, R. H., and YAWALKAR S. J. Leprosy multidrug therapy. Lepr. Rev. 66 (1995) 210Ð213. for Medical Practioners and Medical Workers. 54. SUITE, M. Relapse rates following leprosy mul- 3rd ed. Basel: CIBA-GEIGY Limited, 1988. tidrug therapy. West Indian Med. J. 49 (2000) 57. WHO. Risk of Relapse in Leprosy. Geneva: 210Ð211. World Health Organization, 1994. 55. SUNEETHA, S., and REDDY, R. Histological resolu- 58. WHO STUDY GROUP. Chemotherapy of Leprosy tion and bacterial clearance with pulse ROM ther- for Control Programs. Geneva: World Health Or- apy in borderline lepromatous leprosy. Int. J. Lepr. ganization, 1982. Tech. Rep. Ser. 675. Other Mycobact. Dis. 69 (2001) 53Ð54. INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. (ISSN 0148-916X) Nail Involvement in Leprosy: A Study of 300 Patients1
Inderjeet Kaur, Aditi Chakrabarti, Sunil Dogra, Ranju Rai, and Bhushan Kumar2
ABSTRACT Three hundred leprosy patients were recruited to study the pattern and frequency of nail changes. Nail changes, like longitudinal ridging in finger nails, transverse striations involv- ing both finger and toe nails etc. which occurred with similar frequency in the PB and MB patients in comparison with the control group, were excluded from the analysis. Out of a to- tal number of 150 PB patients, 84 (56%) showed nail changes. Fifty-eight (38.6%) patients showed changes in the finger nails, with an average of 3.2 involved nails per patient. Fifty- three (35.3%) patients showed changes in the toe nails, with an average of 3.0 nails per pa- tient. The most common change observed was longitudinal melanonychia (32.4%) in the fin- ger nails and longitudinal ridging (46.3%) in the toe nails. In comparison, 131/150 (87.3%) MB patients showed nail changes. Finger nail changes were seen in 102 (68%) patients with an average of 5.5 nails affected per patient. Changes in toe nails were seen in 116 (77.3%) patients, with an average of 6.0 nails involved per pa- tient. The most common nail change observed was longitudinal melanonychia in 89/523, (17%) of the total involved finger nails and subungual hyperkeratosis in 164/702, (23.4%) of the total toe nails involvement. Out of a total of 32 colony patients, 31 (96.9%) showed nail changes both in finger and toe nails with an average of 7.9 and 8.4 affected nails per pa- tient, respectively. The most common nail change observed was rudimentary nail(s) on fin- gers (29%) and toes (21.1%). Among MB patients, a significantly higher number had finger nail involvement in LL group. The frequency of nail involvement for both fingers and toes was significantly greater in LL as compared to BL group of patients. The frequency of nail involvement was significantly more in patients having disease for more than 5 years and in those having trophic changes secondary to loss of sensations and impaired circulation. RESUMÉ Trois cent patients lépreux furent recrutés afin d’étudier la fréquence et les aspects de lé- sions des ongles. Les changements observés sur les ongles, qui apparaissent avec une fréquence similaire entre les patients PB et MB comparé au groupe témoin, comme les stries longitudinales sur les ongles de doigts de la main, les striations tranversales sur les ongles de main et de pied, etc., furent exclus de l’analyse. Parmi un total de 150 patients PB, 84 (56%) ont montré des lésions des ongles. Cinquante huit (38,6%) de ces patients ont montré des lésions des ongles des mains, avec une moyenne de 3,2 ongles atteints par malade. Cinquante trois (35,3%) de ces patients ont montré des lésions des ongles des pieds, avec une moyenne de 3,0 ongles atteints par patient. La lésion la plus fréquente était la mélanony- chie longitudinale (32,4%) pour les ongles des mains et les irrégularités longitudinales (46,3) pour les ongles des pieds. Par comparaison, 131/150 (87,3%) des patients MB ont montré des lésions unguéales. Les altérations des ongles des mains furent observées chez 102 (68%) malades, avec une moyennne de 5,5 ongles affectés par patient. Les lésions unguéales des pieds furent détec- tées chez 116 (77,3%) patients, avec une moyenne de 6,0 ongles atteints par patient. La lé- sion la plus commune observée était la mélanonychie chez 89/523 (17%) des ongles affec- tés des mains et l’hyperkératose sub-inguéale chez 164/702 (24,4%) des ongles de pied at- teints. Parmi un nombre total de 32 patients relatifs entre eux, 31 (96,9%) ont montré des lésions unguéales à la fois des mains et des pieds, avec une moyenne de 7,9 et 8,4 ongles af-
1 Received for publication 19 November 2002. Accepted for publication 26 September 2003. 2 I. Kaur, M.D., MNAMS; A. Chakrabarti, M.D.; S. Dogra, M.D., MNAMS; R. Rai, M.D.; B. Kumar, M.D., MNAMS, Department of Dermatology, Venereology & Leprology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. Reprint Requests: Inderjeet Kaur, M.D., Department of Dermatology, Venereology & Leprology, Postgraduate Institute of Medical Education and Research, Chandigarh-160 012, India. E-mail: [email protected]
320 71, 4 Kaur, et al.: Nail Involvement in Leprosy 321
fectés, respectivement. La lésion la plus communément observée était des ongle(s) rudi- mentaire(s) sur les mains (29%) et les pieds (21,1%). Parmi les patients MB, un nombre sig- nificativement supérieur de malades LL avaient une atteinte des ongles des mains. La fréquence d’atteinte des ongles à la fois des mains et des pieds était significativement plus importante chez les patients LL par rapport aux patients BL. La fréquence d’atteinte des on- gles était significativement plus élevée chez les patients malades depuis plus de cinq années et chez ceux ayant des altérations trophiques secondaires aux pertes de sensations et à une circulation sanguine altérée. RESUMEN Trescientos pacientes con lepra fueron reclutados para estudiar el patrón y la frecuencia de cambios en las uñas. Se excluyeron del análisis las alteraciones en las uñas que ocurrieron con una frecuencia similar en los pacientes PB, MB y en los controles sanos (crestas longi- tudinales en las uñas de las manos y estriaciones transversales en las uñas de pies y manos). De un total de 150 pacientes PB, 84 (56%) mostraron alteraciones en las uñas. Cincuenta y ocho (38.6%) pacientes mostraron cambios en las uñas de las manos, con un promedio de 3.2 uñas afectadas por paciente. Cincuenta y tres (35.3%) pacientes mostraron cambios en las uñas de los pies, con un promedio de 3.0 uñas por paciente. Los cambio más común- mente observados fueron melanoniquia longitudinal (32.4%) en las uñas de las manos, y crestas longitudinales (46.3%) en las uñas de los pies. En comparación, 131/150 (87.3%) pacientes MB mostraron cambios en las uñas. Los cambios en las uñas de las manos se observaron en 102 (68%) pacientes, con un promedio de 5.5 uñas afectadas por paciente. Los cambios en las uñas de los pies ocurrieron en 102 (68%) pacientes, con un promedio de 5.5. uñas afectadas por paciente. Se observaron cam- bios en las uñas de los pies en 116 (77.3%) pacientes, con un promedio de 6.0 uñas afectadas por paciente. Los cambios en las uñas más comúnmente observados fueron melanoniquia longitudinal en 89/523 (17%) del total de uñas de las manos, e hiperqueratosis subungal en 164/702, (23%) del total de uñas del pie afectadas. De un total de 32 pacientes de la colonia, 31 (96.9%) mostraron cambios en las uñas de los pies y de las manos, con un promedio de 7.9 y 8.4 uñas afectadas por paciente, respectivamente. El cambio mas comúnmente obser- vado fue el de las uñas rudimentarias en las manos (29%) y los pies (21.1%). Entre los pa- cientes MB, la frecuencia de alteración de las uñas tanto de los pies como de las manos fue significativamente mayor en el grupo LL que en el grupo BL. La frecuencia de alteración en las uñas fue significativamente mayor en los pacientes con una enfermedad de mas de cinco años y en aquellos con cambios tróficos secundarios a la pérdida de sensibilidad y circu- lación afectada.
Leprosy is a chronic infectious disease af- despite wide differences in pathology, nail fecting almost every organ of the body with changes in tuberculoid and lepromatous pa- wide ranging clinical manifestations. All or- tients were often similar and that leproma- gans and systems involved have been stud- tous patients developed bilaterally symmet- ied quite extensively in leprosy, but the in- rical nail changes late in the course of the volvement of nails has never been fully high- disease. However, there was a lack of sys- lighted. Although the dystrophic changes tematic categorization and detailed descrip- and mutilation of hands and feet are consid- tion of the pattern of nail changes observed. ered more or less synonymous with the Scanty data on the subject prompted us to symptomatology of the disease, nail changes study the frequency and pattern of nail have received only a passing reference in the changes and their correlation with the type literature (5, 9). of disease, duration of disease, deformity of Nail changes can be caused by neuropa- hands/feet, and treatment status in patients thy, repeated trauma, vascular deficit, infec- with leprosy attending our leprosy clinic tions and often more than one factor is in- and also in patients residing in a leprosy volved. There have been only few case re- colony, regardless of the duration of disease ports describing isolated nail changes in or treatment status. leprosy patients (7, 14, 19). In the only pub- lished study on large number of leprosy pa- MATERIALS AND METHODS tients, the incidence of nail changes was A total of 300 patients, 150 consecutive found to be 64% (15). It was observed that patients each with paucibacillary (PB) and 322 International Journal of Leprosy 2003
TABLE 1. Frequency of nail involvement in paucibacillary (PB) and multibacillary (MB) patients.
PB cases (N = 150) MB cases (N = 150) BT cases (N = 150) BL (N = 60) LL (N = 90) Hands (%) Feet (%) Hands (%) Feet (%) Hands (%) Feet (%) No. of patients affected 58 (38.6) 53 (35.3) 34 (56.7) 45 (75) 68 (75.5) 71 (78.9) No. of nails affected 188 (32.4) 162 (30.5) 144 (42.4) 240 (53.3) 379 (55.7) 462 (65) No. of affected nails/patient 3.2 3.0 4.2 5.7 5.6 6.3 multibacillary (MB) leprosy in the age and distribution of (fingers/toes) nails in- group 20Ð50 yrs irrespective of sex, dura- volved. Peripheral vascular status of the ex- tion of disease, treatment, and reactional sta- tremities was evaluated clinically by palpat- tus attending the leprosy clinic at Postgrad- ing the major arterial pulsations of both upper uate institute of Medical Education and Re- and lower limbs. Deformities of the hands search, Chandigarh, India were included in and feet were recorded according to the the study (Table 1). One hundred age and WHO grading (1988) (20). Fungal infections sex matched control subjects not suffering of the nails were ruled out by KOH examina- from any disease known to affect the nails tion and culture whenever indicated. X-rays and 32 treated patients of leprosy residing in of the hands and/or feet were done only in a a nearby leprosy colony were also recruited limited number of cases. The pattern of nail in the study. Patients were diagnosed ac- changes in the study and control groups cording to Ridley-Jopling classification and were compared using chi-square test. grouped into into PB (polar tuberculoid, The following nail changes, which oc- TT, and borderline tuberculoid, BT) and curred with similar frequency in the leprosy MB (borderline, BB; borderline leproma- patients (both PB and MB patients) and the tous, BL; polar lepromatous, LL) disease control group, were excluded from the for purposes of analyses. Histopathological analysis: i) longitudinal ridging and flatten- study of the skin lesions and slit skin smear ing—in finger nails; ii) longitudinal split- examination were done as a routine in all ting, transverse pigmented bands, and black- patients. In addition to the routine cuta- ish discoloration—in toe nails; iii) absence neous and neurological examination, various of lunula, leukonychia, transverse striations, nail changes were noted in a predesigned rough nails, loss of nail fold and pitting—in proforma taking into account the number both finger and toe nails. Various noted nail
TABLE 2. Pattern of nail changes in PB patients.
No. of patients Total no. of nails involved Nail changes Total Hands Feet Fingers (%)* Toes (%)* Longitudinal melanonychia 33 33 — 61 (32.4) — Subungual hyperkeratosis 26 6 20 23 (12.2) 42 (25.9) Longitudinal ridging 25 — 25 — 75 (46.3) Beau’s line 16 5 11 8 (4.2) 23 (14.2) Flattening 9 — 9 34 (18)))) Onycholysis 9 6 3 7 (3.7) 6 (3.7) Thickening of nail plate 7 7 — 9 (4.7) — Reddish brown discoloration 7 5 2 28 (14.9) 4 (2.4) Thinning of nail plate 6 — 6 — 10 (6.2)) Onchomycosis 6 6 — 6 (3.1) — Longitudinal splitting 3 3 — 7 ( 3.7) — Chronic paronychia 3 2 1 5 (2.6) 1 (0.61) Pterygium unguium 1 — 1 — 1 (0.61) * Column percentage. 71, 4 Kaur, et al.: Nail Involvement in Leprosy 323
TABLE 3. Pattern of nail changes in MB patients.
No. of patients Total no. of nails involved Nail changes Total Hands Feet Fingers (%)* Toes (%)* Subungual hyperkeratosis 71 24 47 57 (10.1) 164 (23.4) Logitudinal ridging 43 — 43 — 111 (15.8) Complete shedding 41 20 21 63 (11.2) 67 (9.5) Onychauxis 37 14 23 17 (3.0) 47 (6.7) Longitudinal melanonychia 35 35 — 89 (15.8) — Rudimentary nails 32 15 17 55 (9.8) 78 (11.1) Thickening of nail plate 29 12 17 21 (3.7) 28 (4.0) Onycholysis 25 19 6 46 (8.2) 8 (1.1) Beau’s line 19 7 12 12 (2.1) 26 (3.7) Thinning of nail plate 17 6 11 19 (3.4 ) 23 (3.3) Excessive curvature 14 11 3 38 (6.8) 14 (2.0) Pallor 12 6 6 49 (8.7) 62 (8.8) Onychomycosis 8 3 5 7 (1.2) 20 (2.8) Flattening 7 — 7 — 17 (2.4) Onychogryphosis 7 2 5 5 (0.9) 9 (1.3) Brachytelephalangia 7 4 3 6 (1.0) 13 (1.8) Longitudinal splitting 5 5 — 8 (1.4) — Reddish brown discoloration 4 4 — 24 (4.2))) — Brittle nail 3 — 3 — 8 (1.1) Pterygium unguium 2 1 1 3 (0.5) 6 (0.8) Chronic paronychia 2 2 — 2 (0.3) — Onychoheterotopia 1 — 1 — 1 (0.14) Central anonychia with polynychia 1 1 — 2 (0.3) — *Column percentage
findings were classified based on changes in grade II in 3) and 2 patients had deformities morphology and color, involving different of both hands and feet (grade 1). components of the nail unit like nail plate, Out of these 150 PB patients, 84 (56%) nail bed, nail folds viz. longitudinal melano- showed nail changes. Fifty-eight (38.6%) nychia, excessive curvature of nail plate, patients showed changes in the finger nails, subungual hyperkeratosis, rudimentary with an average of 3.2 nails per patient. nails, etc. as given in Tables 2 and 3. Fifty-three (35.3%) patients showed changes in the toe nails, with an average of RESULTS 3.0 nails per patient (Table 1). Sixty-one Paucibacillary patients. In the PB (40.7%) patients had involvement of both group, all 150 patients had BT disease. finger and toe nails. Involvement of either There were 106 (70.7%) males and 44 fingernails or toe nails alone was seen in 15 (29.3%) females. The mean age of male and (10%) and 8 (5.3%) patients, respectively. female patients was 32.8 ± 2.1 and 28.1 ± The most common nail change observed 3.2 yrs, respectively. Of these, 111 patients was longitudinal melanonychia (one to were untreated and the rest were on regular three bands) in the finger nails (32.4%) World Health Organization (WHO) Multi- (Fig. 1) and longitudinal ridging (46.3%) in drug therapy (MDT) PB regimen or had the toe nails (Table 2). Various types of nail completed the treatment. The duration of changes and their frequencies are given in the disease in these patients ranged from 1 Table 2. The presence of nail changes was month to 13 yrs (mean 13 ± 4.1 months). significantly greater in those having the dis- Thirty-four patients were in type I reaction. ease for a period of more than 5 years Duration of the reaction ranged from 20 (55/84 vs. 23/66) (p <0.05). The pattern of days to 1 yr (mean 4.7 ± 2.9 months). nail changes did not differ significantly Twenty-six patients had deformities of with an increase in the duration of the dis- hands (grade I in 10 and grade II in 16), 10 ease beyond 5 years. Nail changes were not had deformities of feet (grade I in 7 and found to be more frequent in relation to lep- 324 International Journal of Leprosy 2003
FIG. 1. Longitudinal melanonychia. rosy lesions on hands or feet, nor was any particular pattern found to correlate with the presence of these lesions. Nail changes such as nail dystrophy (Fig. 2), onycholy- sis, longitudinal ridging and brittle nails were observed to be more frequent in digits with sensorimotor deficit (p <0.01). On- chomycosis confirmed by potassium hy- droxide (KOH) and/or culture was present in 6 (4%) patients with or without nail FIG. 2. Dystrophic nail. changes of leprosy. Multibacillary patients. In the MB cally significant (p >0.1). The frequency of group (BL-60, LL-90), there were 96 (64%) nail involvement for both fingers and toes males and 54 (36%) females with a mean was significantly more in the LL as com- age of 34 ± 2.1 yrs and 31 ± 1.4 yrs, respec- pared to the BL group of patients (p <0.05) tively. Duration of the disease varied from 8 (Table 1). The most common nail change months to 19 yrs (mean 3.4 ± 1.5 yrs). observed was longitudinal melanonychia Eighteen patients had type 1 reaction with (up to five bands) in the finger nails (15.8%) duration of 1 week to 9 months (mean 4.25 and subungual hyperkeratosis (23.4%) ± 1.2 months) and 21 patients had type 2 re- (Fig. 3) in the toe nails (Table 3). The fre- action (including recurrent episodes) with quency of nail changes was significantly duration of 3 months to 2 yrs (mean 12.7 ± greater in those having neurovascular 2.1 months). Ninety-six patients had defor- deficit in the extremities (p <0.01). Certain mities of the hand (grade I in 44, grade II in patterns, like over curvature of nails (Fig. 52) and 104 patients had deformities of the 4), complete shedding, rudimentary nails feet (grade I in 56, grade II in 48). (Fig. 5) in both fingers and toes, and black- Out of a total of 150 MB patients, 131 ish discoloration of the finger nails were (87.3%) showed nail changes. Finger nail significantly more in those having the dis- changes were seen in 102 (68%) patients ease for a period of greater than 5 years and with an average of 5.5 nails per patient those having grade II deformities of the (Table 1). Toe nail changes were seen in hands and feet (p <0.05). Nail changes like 116 (77.3%) patients, with an average of nail dystrophy, onycholysis, longitudinal 6.0 nails per patient. One hundred seven ridging and brittle nails were observed to be (71.3%) patients had changes in both finger more frequent in limbs with trophic changes nails as well as toe nails. Seven (4.6%) pa- secondary to loss of sensations and impaired tients had changes only in the finger nails circulation (p <0.01). Onychomycosis con- while 17 (11.3%) showed changes in the toe firmed by KOH and or culture was present in nails alone. The number of patients having 8 (5.3%) patients with or without nail finger nail involvement was significantly changes of leprosy. more in the LL group than BL group (p Patients from leprosy colony. There <0.05). However, for toe nail involvement, were 32 patients residing in the leprosy this difference was not found to be statisti- colony. The total duration of the disease 71, 4 Kaur, et al.: Nail Involvement in Leprosy 325
FIG. 3. Subungual hyperkeratosis. was more than 10 years in all of them. All of them had received adequate MDT or dapsone monotherapy and were smear neg- ative. Thirty-one (96.9%) patients showed changes in both finger and toe nails, with involvement on an average of 7.9 finger nails per patient and an average of 8.4 toe nails per patient, respectively. The most common nail change observed was rudi- mentary nail(s) on fingers (29%) and toes (21.1%). DISCUSSION Nails are specialized keratinous cuta- neous appendages that play an important role in the everyday life of humans, and FIG. 4. Overcurvature of nails. have always provided the clinician major clues in the diagnosis of a number of dis- eases. The nail unit responds to a wide vari- changes mostly occur as a result of nerve ety of insults by a limited number of reac- involvement. Like leprosy, neuropathy is a tion patterns. In literature, there is a paucity part of diabetes mellitus in which various of comprehensive studies on the frequency nail changes like dystrophy, shortening, and pattern of nail changes in leprosy pa- fragility, and yellowish nail discoloration tients. Bryceson and Pfaltzgraf (5) and have been described (1, 12, 17). There are re- Jopling (9) first described the nail changes ports of dystrophic changes in little finger in advanced lepromatous leprosy. Patki and nail following traumatic ulnar nerve dam- Baran (15) reported nail changes in different age (13, 17). In addition, the misuse and dis- spectrum of leprosy, but these studies lack use of anesthetic and deformed hands and information about the pattern of nail feet results in repeated mechanical and ther- changes and their frequency. Nail changes mal trauma, infection, osteolysis of pha- in our patients were noted more frequently langes, and loss of nails. Trauma related in the MB (87.3%) patients than in those changes like onycholysis, Beau’s lines, lon- with PB disease (56%). This could be at- gitudinal ridging and splitting were more tributed to the extensive bilateral peripheral common in MB patients in our study, prob- neuropathy, trauma, infections, more severe ably because anaesthetic but functional degree of deformities and repeated type 2 limbs in MB cases are more frequently trau- reactions leading to immunologically medi- matized due to misuse. ated vasculopathy in the MB cases. In an- The trophic changes of leprosy have al- other published series, Patki and Baran (15) ways been attributed mostly to the nerve reported the incidence of nail changes to be damage, but it seems that the vascular com- 64% in their patients (PB/MB). Nail ponent is also important, since such dys- 326 International Journal of Leprosy 2003
FIG. 5. Rudimentary nail–index and middle finger with anonychia–ring finger. trophic nail changes are also seen in other PB patients. Though the exact explanation diseases like scleroderma, rheumatoid is not known, this has been speculated to be arthritis etc, where vascular factors play an the adverse effect of clofazimine therapy important role (10, 17, 18). The associations be- given in anti-inflammatory doses (7). Pallor tween neurovascular deficit and various of the nails noted by us in both PB and MB trophic changes including fungal infections patients, though not being specific for the have been well documented in the literature disease, can probably be attributed to ane- (1, 8). Longitudinal ridging of the toe nails mia due to the anti-leprosy drugs, notably was a common change in both PB and MB dapsone and partly due to disease itself and patients. Similar changes have been de- also vascular deficit secondary to vessels scribed with peripheral vascular deficit and involvement. Similar observations were also as an age-related change (17, 18), possi- made earlier by Patki and Baran (15). bly a result of age-related circulatory com- Complete shedding of the nails, promise. Other changes attributable to vas- brachytelephalangia, and rudimentary nails cular deficit, like onycholysis, longitudinal with loss of terminal phalanges were the splitting and brittle nails were seen more of- changes found exclusively in MB patients ten in the MB patients. Beau’s lines have and in patients residing in the leprosy been reported to be associated with type colony. This is consistent with the hypothe- 2 leprosy reactions and vasculitis was sis of Baran and Juhlin (2) which states that thought to be a probable cause. Since only development of a normal nail is dependent few of our patients were in reaction at the on the underlying bone; anonychia or hy- time of study, we did not try to correlate ponychia may result when the underlying any nail change with reactional status of the bone is either hypoplastic or completely ab- patient. sent. In leprosy it is possible that nails may Longitudinal melanonychia was a com- be affected secondary to resorption of distal mon change observed in 33/150 (22%) and phalanges. This was probably why anony- 35/150 (23.3%) of our PB and MB cases, chia and rudimentary nails were more com- respectively, and in 21.9% patients from the mon in patients from the leprosy colony leprosy colony. According to Baran (3), whose fingers and toes showed marked these bands arise due to stimulation of bony resorption. Brand (4) confirmed with melanocytes in the nail matrix following re- follow-up radiographs over a period of 5 peated trauma. Subungual hyperkeratosis years that approximately 95% of resorption was another notable change observed more resulted form open wounds developing a commonly in the MB as compared to the secondary infection. 71, 4 Kaur, et al.: Nail Involvement in Leprosy 327
Pardo-Castello and Pardo (13) noted tinea REFERENCES unguium in 32% of their leprosy patients. Ramesh and Misra (16) have postulated that 1. AKANJI, A. O., and ADETUYIDI, A. The pattern of nails could be the source of frequent tinea presentation of foot lesions in Nigerian diabetic corporis and tinea cruris infections in lep- patients. West Afr. J. Med. 9 (1990) 1Ð5. rosy patients. We noted onychomycosis in 2. BARAN, R. Frictional longitudinal melanonychia: only 4.7% of our patients. Patki and Baran a new entity. Dermatologica 174 (1987) 280Ð284. 15 3. BARAN, R., and JUHLIN, L. Bone dependent nail ( ) postulated that in contrast to frequent formation. Br. J. Dermatol. 114 (1986) 371Ð375. dermatophytic infections, candidal parony- 4. BRAND, P. W. Deformities of the hands and feet in chia is not often observed in leprosy pa- leprosy. Quaderni di Cooperazione Sanitaria 1 tients who generally have dry skin (unless (1982) 109Ð120. altered by their occupation), which is un- 5. BRYCESON, A., and PFALTZGRAF, R. E. In: Leprosy, suitable for growth of Candida albicans. 2nd ed. Ediburgh: Churchill Livingstone, 1979, We noted only 5 cases of chronic parony- p. 19. chia, none of which were of candidal etiol- 6. CHERNOSKY, M. E., and DUKE, C. D. Green nails. ogy. In our bid to find out any relationship Arch. Dermatol. 88 (1963) 548Ð553. between the nail changes and anaesthetic 7. DIXIT, V. B., CHAUDHARY, S. D., and JAIN, V. K. Clofazimine induced nail changes. Indian J. Lepr. leprosy lesions located on the correspond- 61 (1989) 476Ð478. ing hands or feet, we could not establish 8. DOGRA, S., KUMAR, B., BHANSALI, A., and any such association. Diffuse leukonychia CHAKRABARTY, A. Epidemiology of onychomyco- or pseudomacrolunula, which was de- sis in patients with diabetes mellitus in India. Int. scribed by Pardo Castello and Pardo (13) as J. Dermatol. 41 (2002) 647Ð651. an early change in leprosy, was not noted 9. JOPLING, W. H. Handbook of Leprosy. 4th ed. by us. We only observed punctate leucony- London: William Heinemann, 1988. p. 28. chia in our patients and its incidence was no 10. KAUR, S., WAHI, P. L., CHAKRAVARTI, R. N., SODHI, more than in controls. In our opinion, since J. S., VADHWA, M. B., and KHERA, A. S. Periph- loss of nails in leprosy is a consequence of eral vascular deficit in leprosy. Int. J. Lepr. Other various interacting factors like vasculopa- Mycobact. Dis. 44 (1976) 332Ð339. 11. KIKUCHI, I. Congenital onychodysplasia of the in- thy, neuropathy and their sequelae like dex finger: a case involving the thumbnails. trauma and infection, the term shedding Semin. Dermatol. 10 (1991) 7Ð11. which means “to cast off or throw off,” 12. MANN, R. J., and BURTON, J. L. Nail dystrophy due should be replaced by “acquired anony- to diabetic neuropathy. Br. Med. J. 284 (1982) 1445. chia” to describe nail dystrophy and loss. 13. PARDO-CASTELLO, V., and PARDO, O. A. Disease Central anonychia with polyonychia was of the Nails. 3rd ed. Springfield: Charles C. seen in one of our MB patients from the Thomas Publishers, Ltd., 1960. clinic and 3 patients from the leprosy 14. PATKI, A. H. Pterygium inversum unguis in a patient colony. This change has been characteristi- with leprosy. Arch. Dermatol. 126 (1990) 1110. cally described in congenital onychodyspla- 15. PATKI, A. H., and BARAN, R. Significance of nail 11 changes in leprosy: a clinical review of 357 cases. sia of the index fingers (COIF) ( ). Semin. Dermatol. 10 (1991) 77Ð81. From our study it can be concluded that 16. RAMESH, V., and MISRA, R. S. Nail changes in nail changes are common in leprosy, more leprosy and recognition of fungal nail infection. so in the MB spectrum. Attributing a partic- Indian J. Lepr. 59 (1987) 360Ð361. ular change to a single pathophysiologic 17. SAMMAN, P. D. Nails in Disease. 3rd ed. London: mechanism would be an oversimplification. William Heinemann, 1978. Peripheral neuropathy, trauma, and vascu- 18. SAMMAN, P. D., and STRICKLAND, B. Abnormalities lopathy and secondary infections all play a of the finger nails associated with impaired peripheral role in the genesis of these nail changes. blood supply. Br. J. Dermatol. 74 (1962) 165Ð173. 19. SINGH, P. K, NIGAM, P. K., and SINGH, G. Terry’s Several changes have been found in impres- nails in a case of leprosy. Indian J. Lepr. 58 (1986) sive numbers, but whether all are specific 107Ð109. for the disease will remain speculative un- 20. WHO EXPERT COMMITTEE ON LEPROSY. Sixth Re- less a clinicopathological study correlates port. Geneva: World Health Organization, 1988. our observations. Tech. Rep. Ser. 768. INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. (ISSN 0148-916X) Ultrastructural Study of Schwann Cells and Endothelial Cells in the Pathogenesis of Leprous Neuropathy1
V. Kumar and U. Sengupta 2
ABSTRACT Peripheral nerve biopsies from 4 borderline tuberculoid (BT) and 4 lepromatous (LL) pa- tients who were on multidrug therapy were investigated by light and electron microscopic studies. The variation of diameters and distribution of myelinated and unmyelinated fibers between BT and LL patients were not significant. This study has shown significant changes in peripheral nerves and endoneural blood vessels. It was revealed that besides Schwann cells (SC), the endothelial cells (EC) of endoneural blood vessels frequently harbor M. lep- rae. In BT, peripheral nerves in addition to the degenerative changes of SC and presence of perineural and perivascular cuffing by mononuclear cells, the endoneurial blood vessels showed thickening of basement membrane with hypertrophy of EC leading to narrowing or complete occlusion of lumen. On the other hand, peripheral nerves of LL patients were in- filtrated with large number of M. leprae shown to be present in the electron transparent zone (ETZ) of the SC. The EC of endoneurial blood vessels were found to be loaded with M. lep- rae, and this bacillary loaded EC was found to release M. leprae into the lumen through its ruptured membrane. RESUMÉ Les biopsies de nerfs périphériques provenant de 4 patients tuberculoïdes borderlines (BT) et de 4 patients lépromateux (LL) soumis à une polychimiothérapie (PCT) ont été étudiées par microscopies optique et électronique. Aucune différence significative fut observée en terme de diamètre et de distribution des fibres myélinisées et non myélinisées entre les patients BT et LL. L’étude a montré des différences significatives dans les nerfs périphériques en particulier au sein des vaisseaux sanguins intra-neuraux. Il a été mis en év- idence que, en plus des cellules de Schwann (CS), les cellules endothéliales (CE) des vais- seaux intra-neuraux hébergaient fréquemment des M. leprae. Au sein des nerfs pé- riphériques des patients BT, en plus des lésions dégénératives des CS et de la présence de manchons périvasculaires de cellules mononucléées, les vaisseaux sanguins intra-neuraux montraient un épaississement des membranes basales associées à une hyertrophie des CE, résultant en un rétrécissement voire une occlusion complète de la lumière. D’autre part, les nerfs périphériques des patients LL étaient infiltrés par un grand nombre de M. leprae, lo- calisées dans les CS et les ETZ ; les CE des vaisseaux sanguins intra-neuraux étaient rem- plies de M. leprae, et certaines de ces CE surchargées montraient parfois un relarguage de M. leprae dans la lumière au travers de la membrane cellulaire rompue. RESUMEN Se analizaron, por microscopía de luz y electrónica, las biopsias de nervios periféricos de 4 pacientes con lepra tuberculoide subpolar (BT) y 4 pacientes lepromatosos (LL), que estuvieron en tratamiento con poliquimioterapia. Las variaciones en el diámetro y distribu- ción de las fibras mielinizadas y no mielinizadas entre los pacientes BT y LL no fueron sig- nificativas. Sin embargo, el estudio mostró cambios importantes en los nervios periféricos y en los vasos sanguíneos endoneuriales. Se observó que además de las células de Schwann (CS), las células endoteliales (CE) de los vasos sanguíneos endoneuriales con frecuencia también contienen M. leprae. En la lepra BT, los nervios periféricos mostraron cambios de- generativos de las CS con acumulación perineural y perivascular de células mononucleares,
1 Received for publication 5 December 2002. Accepted for publication 11 August 2003. 2 V. Kumar, Ph.D., Head, Electron microscope Division, and U. Sengupta, Ph.D., Division of Immunology, Central JALMA Institute for Leprosy, Indian Council of Medical Research, Taj Ganj AgraÐ282001, India. Reprint resuests to: V. Kumar, Head, Electron Microscope Division, Central JALMA Institute for Leprosy, In- dian Council of Medical Research, Taj Ganj, Agra—282001 INDIA. E-mail: [email protected]
328 71, 4 Kumar and Sengupta: Schwann Cells and Endothelial Cells in Neuropathy 329
además de engrosamiento de la membrana basal de los vasos sanguíneos perineurales e hipertrofia de las CE, con reducción u oclusión completa del lumen. Por otro lado, los nervios periféricos de los pacientes LL estuvieron infiltrados con gran número de M. leprae, sobre todo en la zona electrotransparente (ZET) de las CS. Las CE de los vasos sanguíneos endoneuriales se encontraron repletas de bacilos y los estuvieron liberando hacia el lumen, a través de sus membranas rotas.
Leprosy is a chronic disease, caused by rae has been shown by electron microscopy Mycobacterium leprae where involvement in SC of myelinated (MY) and unmyeli- and damage of peripheral nerves is a typical nated (UMY) nerve fibers and also in and unique feature. One of the cardinal macrophages, endothelial cells (EC), and signs of clinical diagnosis of leprosy pa- perineurial cells (23, 12, 8, 9). Ultrastructurally, tients depends on the recognition of thick- M. leprae have been found in the EC of ened peripheral nerves in patients supplying blood vessels (5, 2, 3, 21, 19). It has been clear an anesthetic area in the skin, hand, legs, or therefore, that the SC are not the only target face. The histopathological demonstration cells for M. leprae growth, but organisms of M. leprae in the nerve and the presence also grow in the EC of blood vessels. Re- of inflammatory granuloma in and around a cently, in an experimental model M. leprae nerve are mandatory for confirmation of di- has been observed in the EC of epineurial agnosis. However, the bacilli have not yet and perineurial blood vessels and also in been reported in the brain and spinal cord, lymphatics (28, 27). However, it is known that possibly due to the unfavorable condition the endoneurial blood vessels supply the for survival and growth of M. leprae in these nutrients to the nerves for maintaining the areas (1). The disease is manifested in a metabolic activity of SC and is essential for spectrum based on the cell mediated immu- proper nerve fiber functioning. Therefore, nity (CMI) of the host. While a strong CMI parasitization of the EC of endoneurial against M. leprae, which limits the growth blood vessels could be also an essential fea- of the bacilli, is expressed in tuberculoid ture for initiation of nerve damage. (TT), and borderline tuberculoid (BT) lep- The present study has been carried out to rosy, in lepromatous leprosy (LL) there is a record the detailed ultrastructural changes lack CMI against the bacilli leading to un- in the SC and EC of endoneurial blood ves- limited growth of M. leprae in the host. In sels which might help in understanding the the early stage of the disease, even when morphological significance of pathogenesis only limited numbers of skin lesions are and dissemination of M. leprae. present and only small number of M. leprae are found in the skin, the organisms prefer- MATERIALS AND METHODS entially localize in the peripheral nerves. Nerve biopsies. Eight patients of leprosy Histologically, bacilli are seen within (4 BT and 4 LL) who volunteered for cells, either in myelinating or nonmyelinat- biopsy of the peripheral nerves were in- ing Schwann Cells (SC) of nerves or in cluded in this study. The patients were se- macrophages (7, 17, 18, 14). Presence of M. lep- lected from the out patient clinic of the
THE TABLE. Details of selected patients.
No. of Type of Age in Duration Status of Nerve biopsied patients disease years of the disease nerves 1BT233 months Thickened Medial cutaneous 2BT276 months Thickened Superficial peroneal 3BT388 months Thickened Infra patellar 4BT258 months Thickened Medial cutaneous 5LL471 year Thickened Superficial peroneal 6LL361 year Thickened Infra patellar 7LL481year 6 months Thickened Medial cutaneous 8LL522 years Thickened Superficial peroneal 330 International Journal of Leprosy 2003
FIGS. 1Ð2. 1, Semithin section of peripheral nerve fascicle from BT patient showing distribution of myeli- nated fibers with small endoneurial blood vessels (BV). ×600. 2, Electron micrograph of peripheral nerves from BT patient showing unmyelinated Schwann cells (USC) with axons of smallest diameters, few myelinated sheath (MS) and endoneurial blood vessels (BV). The endoneurial blood vessel showed closed lumen with enlarged nu- cleus and multilayering basement membrane (BM). ×5000.
Central JALMA Institute for Leprosy, Agra. hyde (TAAB Laboratory equipment, U.K). Peripheral nerves from all of these patients Subsequently, the tissues were washed in were biopsied by employing standard surgi- phosphate buffered saline (PBS), (Itron cal procedures (Table 1). Before conducting Laboratory Inc., Japan) post fixed in 1% os- the study an ethical clearance was obtained mium tetra oxide (OsO4) (John Matthey from the Institutional Ethical Committee. Chemical, U.K.) and again washed in PBS. One part of the biopsy was fixed in formol- The fixed tissues were then dehydrated in Zenker’s fluid and processed for histo- ascending grades of alcohol. Later, these pathology. The other part was fixed in 2.5% were immersed in propylene oxide (Fluka glutaraldehyde for electron microscopic AG, Chemische Fabrik, Switzerland) and studies. embedded in Spurr’s resin (TAAB Labora- Tissue preparation for electron micros- tories Equipment, England). Finally, the tis- copy. Small pieces of the nerve biopsies sues were polymerized overnight at 70°C were fixed overnight in 2.5% glutaralde- and blocks were made. Cross sections, 1 µ 71, 4 Kumar and Sengupta: Schwann Cells and Endothelial Cells in Neuropathy 331
FIGS 3Ð4. 3a, Ultrathin sections of peripheral nerve from BT showing Schwann cells with well preserved collagen fibers (CF) surrounded by unmyelinated fibers. The connections of mesaxon (Mx) are the extensions of the Schwann cell membrane are clearly seen. ×10,000. 3b, The Schwann cell observed on wrapping (WR) to the surrounding collagen fibers. ×15,000. 4, Ultrathin section of peripheral nerves in BT showing myelinated and unmyelinated Schwann Cells. The myelinated Schwann cells, showed attenuated Schwann cell processes arranged circumferentially forming an “Onion bulb” (OB). The cluster of unmyelinated Schwann cell can be seen with atrophied axon. The axonal cytoplasm is less electron dense than Schwann cell cytoplasm. ×7000. thick, of the entire fascicles embedded in a micrometer scale were obtained by a PM- Spurr’s resin were examined after staining 10 ADS camera, Olympus microscope and with toluidine blue (Himedia Laboratories enlarged to 1000 times. From these pho- Pvt. Ltd, Bombay). These preparations were tographs myelinated (MY) and unmyelin- used for a general survey and for counts of ated (UMY) fibers were counted and the MY and UMY fibers. Photographs of the en- measurement of their diameters were made tire cross section of each fascicle along with by the method of Espir and Harding (10). 332 International Journal of Leprosy 2003
The frequency distribution of the UMY and 5a). The diameters of UMY fibers that were external diameters of all the MY fibers in in close relation to endoneurial blood vessels each fascicle was ascertained by separating were generally very small. The external di- them into groups increasing by 1 µ. The ameter of MY fibers was measured by ex- photographs of the fascicle within the per- trapolating the population of UMY fibers in ineurium area were enlarged after finding the partial areas to the whole area of the fas- their mean radius, and the densities of the cicles. It ranged from 2 µm to 22 µm and UMY and MY fibers were calculated per their distribution varied from 9% to 22% sq. mm of the intraperineurial area. (Fig. 5b). At the ultrastructural level, it was Ultrathin sections were cut in an ultrami- observed that the UMY fibers were exten- crotome (MT2, Porter blum, U.S.A.) and sively involved with degenerative and regen- stained with uranyl acetate and lead citrate erative changes. UMY fibers manifested re- solutions (E. Merk, India). The sections generation in the form of dense groups of were observed under an electron micro- very small and intact UMY AX (Fig. 2). In scope H-300. The accuracy of the observa- the regenerative AX, mesaxon connecting tions by light microscopy was checked. the AX and the basement membrane of SC Montages were made of electron micro- was clearly seen (Fig. 3a). Proliferation of graphs taken at about 2000 times magnifi- SC and prominence of endoneurial collagen cation and enlarged to about 8000 times. fibers were also noticed. In some cases, col- Measuring the respective grids-spaces by lagen fibers increased and few SC, phago- light microscopy controlled the exact final cytic in nature, were in the process of en- enlargement and was compared with en- gulfing the adjacent collagen fibers (Fig. 3b). larged electron micrographs. One to 4 areas, Onion-like bodies were observed, which are equivelent to about 5000Ð10,000 sq. µ per probably bionecrotic parts of nerves (Fig. 4). fascicle, of 2 or 3 fascicles were studied in The MY group showed hypertrophy of each case. Quantitative studies of UMY and Schwann cells with prominent nucleus and MY fibers were made. Using the same ap- well-preserved collagen fibers. The regener- paratus, both UMY and MY fibers were ating AX were oval-shaped and some of counted. Their diameters were measured on them revealed the beginnings of MY sheath electron micrographs after being enlarged formation. However, M. leprae organism to near 8000 times, at which magnification and their debris were not observed in any of they could be separated reliably into groups these sections (Figs. 7 and 8). differing by 1 millimeter. Discrimination of Multiple layers of basal lamellae sepa- the frequency distribution of the diameter rated by ground substance accompanied of UMY axon (AX) was thus possible into the proliferation of EC of endoneurial about 8 groups increasing by 0.2 µ. Usually, blood vessels. The EC and basement over 300 UMY AX were counted in each membrane contained many granules and case. Densities of the UMY and MY were were surrounded with prominent inflam- calculated as numbers per sq. mm. No cor- matory cells. The mononuclear leukocytes rection was made for shrinkage of the tissue were often observed forming a perivascu- during preparation. lar cuff around blood vessels in the epineurium and perineurium. Such cuffing RESULTS consisted primarily of macrophages. Oc- In Borderline Tuberculoid (BT). The casionally, circulating infected monocytes semi-thin sections of the nerves revealed were also observed occasionally within uniformly distributed MY fibers with few vascular lumen. Endothelial membranes dispersed blood vessels (Fig. 1). The signifi- were seen with multiple infolding and of- cant change in the diameter of the AX and ten with finger like protrusion around the MY sheath varied between BT and LL pa- blood vessels. The EC were hypertrophied tients. Using light and electron microscopy with enlarged nuclei, often causing nar- of the entire cross sections the quantitative rowing of the vascular lumina. (Fig. 9). In measurements of UMY and MY fibers in BT some cases, this hypertrophy of EC was patients were made. The diameter of UMY up to such an extent that the lumen of the fibers ranged from 0.5 µm to 3.8 µm. Their blood vessels got completely obliterated distribution ranged from 6% to 32% (Fig. (Fig. 10). However, the degree of obstruc- 71, 4 Kumar and Sengupta: Schwann Cells and Endothelial Cells in Neuropathy 333
FIG. 5.
tion of the lumen of the blood vessels due to the hypertrophy of EC varied exten- sively. In some cases, the lumen was com- FIG. 6. pletely closed and in others the lumen had narrowed, but not closed completely. In Lepromatous Leprosy (LL). The into the lumen through endothelial cell quantitative variation of the diameter of membrane rupture (Fig. 14). UMY fibers in LL patients varied between 0.5 µm to 3.8 µm and their distribution in DISCUSSION entire cross section ranged from 2% to 20% The quantitative study showed the corre- (Fig. 6a). On the other hand, the external di- lation between axon diameter and myelin ameter of MY fibers ranged from 2 µm to sheath diameter, and indicated a linear cor- 22 µm, and their distribution varied be- relation with the thicker sheath surrounding tween 3% and 25% (Fig. 6b). The ultra- the larger AX. This feature is probably use- structural changes were characterized by ful for deciding whether an AX that has no degeneration of the SC, AX, and MY myelin sheath is truly UMY or it has be- sheath. M. leprae were present singly or in come degenerated. This might be a reflec- clusters. Clumping of cytoplasm, neural fil- tion of the changes in nerve conduction ve- aments, and neural tubules indicated degen- locities and axonal degeneration and regen- erative changes of SC. The organisms were eration or segmental demyelination in usually seen within the electron transparent neuropathies. These observations are in zone (ETZ) around the bacilli in the SC, cy- agreement with earlier findings (9). toplasm (Figs. 11 and 12). The endoneurial The ultrastructural study has shown sig- blood vessels in LL showed patent lumen nificant changes in the peripheral nerves lined by degenerative EC. The nucleus was and their endoneurial blood vessels in BT small and no hypertrophy of EC was ob- and LL patients. Various workers (23, 12, 30, 20) served. The basement membrane was thin, have reported that SC are the main target with visible endothelial cell junction (Fig. cell in leprosy. Our study using electron mi- 13). Large numbers of intact bacilli were croscopic techniques has convincingly con- also noticed in the EC with ETZ. In some firmed that beside the SC, the EC of blood EC, the bacilli were seen being released vessels also harbor M. leprae frequently. 334 International Journal of Leprosy 2003
FIGS. 7Ð8. 7, Ultrathin section of BT nerves showing two myelinated nerves surrounded by many collagen fibers. Small newly formed myelin sheath and elongated nucleus of Schwann cell is also noticed. ×8000. 8, Ultrathin sections of BT nerve showing unmyelinated Schwann cells with prominent nucleus (N) containing many small oval shaped axons (AX). In the myelinated Schwann cell the neural filaments and neural tubules (NT) are well preserved. Some of the axons seem to be in the process of small myelin sheath formation. ×7000.
Electron microscopic changes of peripheral tion of collagen fibers may be responsible nerves in BT and LL patients have indicated for the destruction of normal nerve archi- interesting findings in understanding the in- tecture and for the prevention of axonal re- terrelationship between bacilli and the host generation. Other workers (15, 29, 30) have cells. In BT nerves, the UMY SC showed proposed a similar concept. Further, they degenerative and regenerative changes with have suggested that SC in these nerves give severe destruction of the nerve elements. off multiple cytoplasmic processes, which The increases in collagen fibers suggest form close relationship with AX and also that the SC may play a very important role with the collagen formation. in the production of collagen in neuropathy, The reason for greater phagocytic activity possibly due to lack of blood supply. The of surrounding matter by SC in UMY fibers continued destruction of SC in absence of is not clearly understood. Similar phagocytic AX multiplication and excessive produc- activity of SC was also noticed by earlier 71, 4 Kumar and Sengupta: Schwann Cells and Endothelial Cells in Neuropathy 335
FIGS. 9Ð10. 9, Higher magnified ultrastructural view of endoneurial blood vessel in BT patients showing nar- rowing of lumen (L) with enlarged nucleus. The basement membranes are multilayering with finger like protrusion. ×17,000. 10, Cross section of endoneurial blood vessel in BT patients showing hypertrophy of endothelial cells con- taining many small mitochondria (MT), golgi complex with (GC) enlarged nucleus. The total obliteration of lumen and reduplication of basal lamina is clearly seen. ×10,000.
workers (24 32, 33). However in addition, the ther, it was noted that regenerating AX were phagocytic activity by the perineurial cells small in size and the mesaxons were con- was also noticed by other workers (31) who nected with the regenerating AX, and the suggested that the bacilli are ingested into surface membrane of the SC. The growing axoplasm by the phagocytic activity in the tip of the regenerating AX migrate freely in growth cones of the regenerating AX. Fur- the body fluid until they reach the final posi- 336 International Journal of Leprosy 2003
FIGS. 11, 12. 11, Ultrathin sections of peripheral nerve from LL patient showing unmyelinated Schwann cell containing numerous axons and two intact bacilli (B) in Electron transparent zone. The clumping of cytoplasm and neural fibers were also seen. ×10,000. 12, Ultrathin sections of LL nerve showing unmyelinated Schwann cell with well preserved basement membrane (BM), and single intact M. leprae (ML). ×12,000. 71, 4 Kumar and Sengupta: Schwann Cells and Endothelial Cells in Neuropathy 337
FIGS. 13, 14. 13, Ultrathin sections of endoneurial blood vessel in LL patients showing open lumen with small nucleus. Many M. leprae organisms are seen inside endothelial cell (EC) in Electron transparent zone (ETZ). The endothelial cell junctions (EJ) are also clearly noticed. ×10,000. 14, Higher magnification of en- dothelial cell of endoneurial blood vessels from LL patients containing many M. leprae organisms. The bacilli are being release from the endothelial cell into lumen of blood vessel. ×30,000. 338 International Journal of Leprosy 2003 tion in the nerve. Subsequently, SC infold caused a pronounced thickening of the the entire length of the regenerating AX be- nerve. Ultrastructurally, leaving aside the hind the growing tip. It seems that in this free SC, parasitization of other cells like EC of and naked stage of regeneration, AX engulf blood vessels and the perineurial cells was leprosy bacilli in the peripheral nerves. The also noticed. The presence of M. leprae in onion-like bodies found in the BT nerve le- SC in the ETZ confirmed the finding of other sions resembled the plasmosome of alveolar workers (11, 20, 15). The bacilli in various states cells. These onion-like bodies may be the of degenerations were seen inside phagoso- remnants of the myelin (Figs. 11 and 12). mal vacuoles of SC. However, some SC These observations are in agreement with were found loaded with intact bacilli indicat- previously published findings (12, 20, 23). In BT ing their incapability in killing M. leprae. leprosy, it was observed that almost every Some workers (25, 26) have described a molec- dermal nerve present in the localized lesion ular mechanism of M. leprae gaining entry showed a presence of inflammatory cells, into the SC of peripheral nerves. Further, they which destroyed large portion of nerves. The have demonstrated that the binding of M. lep- perivascular and perineurial cuffing by rae to the SC is mediated by surface proteins mononuclear cells is an indication of im- of the bacillus binding via α-dystroglycan to mune mediated inflammatory process. the α-2 isoform of laminin found in SC. The Ultrastructural examination of the en- present study has also indicated that there doneurial blood vessels revealed that the was no significant hypertrophy of EC and basement membranes are multilayered, sep- therefore the lumen of the blood vessels re- arated by ground substance indicating re- main patent. However, EC contained many peated episodes of injury to the vascular en- bacilli in the ETZ. These organisms ap- dothelium. The most interesting findings peared to be solid and are therefore probably were the hypertrophy of EC noticed at vari- viable. Further, the bacilli were found in ous levels as observed by others (2, 4, 6, 22). large numbers inside the cells, indicating Recently, (28) in an experimental study it their intracellular multiplication. In certain was observed that the activation of infected situations, the rupturing of EC due to exces- EC led to thickening of the cells and nar- sive bacterial growth and release of M. lep- rowing of the lumen of blood vessels. In ad- rae in the lumen of blood vessels was clearly dition, these workers suggested that, since noticed. It is therefore obvious that the re- the endoneurial blood circulation is respon- leased bacilli in the blood vessels are the di- sible for suppling the nutrients for maintain- rect evidence for hematogenous spread of ing metabolic activity of the nerves, their the disease. Therefore, the present study pro- occlusion could be a major cause of nerve vides a proof for transmission of M. leprae damage. However, the extent of swelling of infection in the nerve through blood stream EC in the experimental study did not lead to leading to active phagocytosis of M. leprae closure of the vessel. We hereby report a by SC from the circulation due to blood complete obstruction of the endoneurial nerve barrier damage (3). The present study blood vessels by hypertrophied EC which is also suggest that the small endoneurial blood frequently noticed in the nerves of BT pa- vessels may play an important role for prop- tients. The ischemia caused by this, could agation of M. leprae infection and progres- play a major role in nerve degeneration sion of the disease in the nerve in LL. leading to neuropathy and neural pain in In conclusion, we have now shown that these patients even after chemotherapy. the mechanism of nerve damage in BT and In contrast, peripheral nerves in LL cases LL forms of leprosy are different. In BT, it were infected with large numbers of M. lep- is probably the result of immune-mediated rae. The number of inflammatory cells was inflammation of the nerves damaging SC very few when compared with that of BT and further compounded by vascular occlu- cases. Further, an abundance of the organ- sion, causing ischemia. In contrast, in LL isms in SC showed foamy degeneration, there is infection of SC by M. leprae lead- followed by disintegration of AX leading to ing to their damage. There is no vascular endoneurial fibrosis. The proliferation of occlusion but the EC appear to be a source perineurial cells, increase in endoneurial col- of propagation of infection as the organisms lagen, and growth of macrophage granuloma actively multiply in them and are then re- 71, 4 Authors et al.: Short Title 339 leased into the lumen of endoneurial blood Manifestation of experimental leprosy in ar- vessels. madillo dasypus novemcinctus. Am. J. Trop. Med. Hyg. 34 (1985) 151Ð161. Acknowledgment. We thank Prof. V. I. Mathan, 14. JOB, C. K., and VERGHESE, R. Schwann cell changes Consultant, National Institute of Epidemiology (ICMR), in lepromatous leprosy. an electron microscope Chennai, and Dr. M. Mathan, Consultant, Tuberculosis study. Indian J. Med. Res. 65 (1975) 397Ð401. Research Center (ICMR), Chennai, for their comments 15. KUMAR, V. Ultrastructural demonstration of Clo- and constructive criticisms. We are grateful to Dr. fazimine crystals in Schwann cells from leproma- Ashok Mukherjee, former Director of the Institute of tous leprosy. Microscope Anal. 68 (1998) 29Ð31. Pathology (ICMR), New Delhi, for his critical sugges- 16. KUMAR, V., KATOCH, K., KATOCH, V. M., and tions. We would also like to thank to Mr. V. S. Yadav BHARADWAJ, V. P. A preliminary study of correla- for stastical analysis and Mr. H. O. Agarwal and Mr. N. tion of immuno-histological and ultrastructural Dubey for photography. characteristics of neural granuloma in leprosy pa- tients. Acta. 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RAMBUKKANA, A., YAMADA, H., ZANAZZI, G., ation of unmyelinated fibers. Brain 95 (1972) 233. MATHUS, T., SALZER, J. L., YURCHENCO, P. D., 10. ESPIR, M. L. E., and HARDING, C. T. C. Apparatus CAMPBELL, K. P., and FISCHETTI, V. A. Role of for measuring and counting myelinated nerve fibers. α-dystroglycan as a Schwann cell Receptor for M. J. Neurol. Neurosurg. Psychiat. 24 (1961) 287Ð290. leprae. Science 282 (1998) 2076Ð2079. 11. IMAEDA, T. Electron microscopic analysis of the 27. SCOLLARD, D. M. Endothelial cells and the patho- components of lepra cells. Int. J. Lepr. 28 (1960) genesis of lepromatous neuritis: insights from the 22Ð37. armadillo model. Microb. and Infect. 2 (2000) 12. JOB, C. K. Pathology of peripheral nerves lesion 1835Ð1843. in lepromatous leprosy a light and electron micro- 28. SCOLLARD, D. M., MCCORMICK, G., and ALLAN, J. scopic study. Int. J. Lepr. Other Mycobact. Dis. 39 L. Localization of M. leprae to endothelial cells (1971) 251Ð268. of epineurial and perineurial blood vessels and 13. JOB, C. K., SANCHEZ, R. M., and HASTINGS, R. C. lymphatics. Am. J. Pathol. 155 (1999) 1611Ð1620. 340 International Journal of Leprosy 2003
29. SHETTY, V. P., ANTIA, N. H., and JACOBS, J. M. 32. WEDDELL, G., PALMER, E., and REES, R. J. W. The The pathology of early leprous neuropathy. J. fate of Mycobacterium leprae in CBA mice. J. Neurol. Sci. 88 (1988) 115Ð131. Path. 104 (1971) 77. 30. SHETTY, V. P., MEHTA, L. N., IRANI, P. F., and AN- 33. WAGGENER, J. D., BUNN, S. M., and BECES, J. The TIA, N. H. Study of the evolution of nerve damage diffusion of ferritin within the peripheral nerve in leprosy. Part 1—lesions of the index branch of sheath. An electron microscopic study. J. Neu- the radial cutaneous nerve in early leprosy. Lepr. ropath. Exp. Neurol. 24 (1965) 430Ð434. India. 52 (1980) 5Ð18. 31. WEDDELL, G., and PALMER, E. The pathogenesis of leprosy. Lepr. Rev. 34 (1963) 57. INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. (ISSN 0148-916X) CASE REPORTS Leprosy and Psoriasis: An Enigmatic Relationship1
Sunil Dogra, Inderjeet Kaur, and Bhushan Kumar 2
ABSTRACT The relationship between leprosy and psoriasis has been controversial since ancient times. Based on the fundamental importance of nerve involvement in the pathogenesis of leprosy and psoriasis, it has been hypothesized that leprosy patients may be protected from developing psoriasis. There are only sporadic reports of coexistence of these two diseases as evidence of this negative association. We report a 64-year-old male patient with borderline leprosy and psoriasis. Recent advances in the elucidation of pathogenesis of both diseases have contributed to the understanding of this enigmatic relationship. Various genetic, im- munological, and structural alterations in leprosy and psoriasis as discussed could be re- sponsible for the rare co-existence of these two diseases in a given patient. RESUMÉ La relation entre la lèpre et le psoriasis est controversée depuis les temps les plus an- ciens. D’après l’importance fondamentale de la composante neurale dans la pathogenèse de la lèpre par rapport au psoriasis, il a été supposé que les patients hanséniens sont probable- ment protégés contre le développement du psoriasis. Il n’y a que quelques articles spo- radiques sur la co-existance de ces deux maladies qui suggèrent une association négative. Nous rapportons ici le cas d’un patient masculin de 64 ans souffrant de lèpre borderline et de psoriasis. Les avancées récentes sur la pathogenèse de ces deux maladies ont permis de mieux comprendre cette relation énigmatique. Des altérations variées d’origine génétique, immunologique et structurelles comme discutées ici associées à la lèpre et le psoriasis pour- raient être responsables de la co-existence rare de ces deux maladies chez un même patient. RESUMEN La relación entre lepra y soriasis ha sido controvertida desde tiempos muy antiguos. So- bre la base de la importancia de la afección a nervios en la patogénesis de la lepra y la sori- asis, se ha postulado que los pacientes con lepra podrían estar protegidos contra la soriasis. Lo esporádico de los reportes sobre la coexistencia de las dos enfermedades es más bien una evidencia de esta asociación negativa. En esta comunicación informamos el caso de un pa- ciente masculino de 64 años, con lepra BL y soriasis. Los avances recientes en la elucidación de la patogénesis de las dos enfermedades han contribuido a entender esta enigmática relación. Diversas alteraciones genéticas, inmunológicas y estructurales en la lepra y la so- riasis podrían explicar la rara coexistencia de estas dos enfermedades en un paciente dado.
Controversies about the relationship be- psoriasis (11). Undoubtedly, many psoriatic tween leprosy and psoriasis have existed patients suffered the same physical and since the time when people considered pso- mental abuses as lepers of that era. This riasis to be a form of leprosy. The biblical confusion between leprosy and psoriasis term “lepra” included what is now called lasted for almost 19 centuries when it was
1 Received for publication 19 November 2002. Accepted for publication 12 August 2003. 2 S. Dogra, M.D., MNAMS, I. Kaur, M.D., MNAMS, B. Kumar, M.D., MNAMS, Department of Dermatology, Venereology and Leprology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. Reprint requests to: Dr. Inderjeet Kaur, Additional Professor, Dept. of Dermatology, Venereology & Leprol- ogy, Postgraduate Institute of Medical Education and Research, Chandigarh-160 012, India. E-mail: [email protected]
341 342 International Journal of Leprosy 2003 realized that the two diseases are entirely different and have nothing common, not even the clinical appearance. In fact, in the latter half of the 20th century, a report on large number of leprosy patients followed up for about 40 years indicated a rarity of psoriasis among them (13). This observation stimulated the interest of many workers who have tried to explore the hypothesis that leprosy and psoriasis rarely develop in the same patient. With the better under- standing of etiopathogenesis and immuno- logical alterations in the two diseases, the basis for the rare occurrence of both leprosy and psoriasis in an individual is becoming clearer. So in a complete turn of events, the occurrence together has become a rather in- teresting rarity, and only a few such cases have been reported. We report another case of rare co-existence of the two diseases. A 64-year-old male patient, presented to us with complaints of erythematous itchy scaly lesions all over the body with a his- tory of relapses and remissions for a dura- tion of 9 yrs. The patient was a treated case of borderline lepromatous (BL) Hansen’s FIG. 1. Psoriatic plaques over lower back and ex- Disease and had received 3 yrs of multi- tremities. drug therapy MDT-MB 13 yrs back. On ex- amination, the patient had well defined ery- patients (13). To further explore this hypoth- thematous, plaque lesions with silvery esis, Kumar, et al. (5) carried out a ques- scales over the extensors of the limbs, trunk, tionnaire survey to be filled out by physi- and scalp involving almost 35% of the body cians at leprosy centers in different parts of surface area (Fig. 1). Nails were involved in the world. In this survey, out of 145,661 the form of pitting, subungual hyperkerato- cases of leprosy, only 20 individuals had sis, and onycholysis. Old lesions of psoriasis. Hansen’s disease were almost inapparent Nerve involvement is fundamental in the without any evidence of clinical activity. pathogenesis of leprosy. Mycobacterium Sensory loss was present over the area of leprae has the special characteristic of in- distribution of both lateral popliteal nerves vading nerves, resulting in neuritis and and the right ulnar nerve with grade I defor- nerve damage. However, in the pathogene- mity of both feet and the right hand. Plaque sis of psoriasis an increasing number of bio- type lesions were randomly distributed over chemical and clinical studies also provide limbs without any particular sparing of strong evidence for the functional role of anesthetic areas. The patient was diagnosed cutaneous nerves and their neuropeptides. as having extensive psoriasis. In view of the Psoriatic lesions have a significantly larger large body surface area involvement, the number of nerves with increased content of patient was started on tablet methotrexate neuropeptides. The actions of neuropep- (0.5 mg/ kg/week). Patient responded well tides like substance P (SP), vasoactive in- to the treatment in 6 weeks with reduction testinal peptide (VIP), and calcitonin-gene- in erythema, scaling, and infiltration. related peptide (CGRP) are of great signifi- There have been only sporadic reports of cance in the inflammatory and proliferative this co-existence published in the literature process and symmetrical distribution of le- (3, 8, 9, 12, 14). An early report from Israel sions in psoriasis (4, 7, 10). It has been docu- stated the rarity of psoriasis among leprosy mented that the damage to sensory nerves 71, 4 Dogra et al.: Leprosy and Psoriasis 343
results in clearance of psoriatic lesions in psoriasis the keratinocytes acquire an apop- anesthetic areas, and that neuropeptide- totic resistant phenotype attributed to the modulating drugs like capsaicin, peptide over-expression of Bcl-X, and cell survival T, somatostatin, spantide, etc. have some gene products, and other growth-regulatory beneficial role in psoriasis (2, 7). Destruction or cell cycle changes (7). of cutaneous nerve fibers and the conse- In our patient, psoriatic lesions were quent absence of neuropeptides from lep- distributed randomly over the normal as rous skin is a well-known observation. well as previously involved hypoaesthetic Hence, it could be that the neuropathy skin areas. It is known that an adequately caused by M. leprae infection results in treated patient may have some regeneration structural and functional alterations in the in the affected nerves that were not cutaneous sensory nerves, so that the completely destroyed. According to the hy- process of neurogenic inflammation, which pothesis outlined above, the occurrence of seems to be an integral part of the psoriatic lesions on the hypoaesthetic areas can be disease process, is inhibited. explained. Some workers have suggested that ge- Leprosy though is one of the oldest dis- netic factors may play a role in protecting eases known to mankind; several mysteries psoriatic patients from leprosy. Population and basic facts about M. leprae and the dis- studies have found significant association ease they produce are still unexplained. of HLA-DR 2 and HLA-DQWI with Various genetic, immunological, and struc- leprosy, whereas in psoriasis there is high tural alterations in leprosy and psoriasis as frequency of HLAÐA1,ÐBB,ÐB16,ÐB17, discussed above could be responsible for CW6 and DR7 with reference to the gen- the rare co-existence of two diseases in an eral population. Recently most investiga- individual patient. tors have focused on the MHC class I re- gion, with particular interest on the REFERENCES HLAÐCW 6 allele in psoriatic patients. Now it has been established that T lym- 1. ABULAFIA, J., and VIGNALE, R. A. Leprosy: patho- phocytes play major role in the pathogene- genesis updated. Int. J. Dermatol. 38 (1999) sis of psoriasis with Th-1 type of cytokine 321Ð324. 2. DEWING, S. B. Remission of psoriasis associated profile triggering the chain reaction of cel- with cutaneous nerve section. Arch. Dermatol. 104 lular and molecular networks that culmi- (1971) 220Ð221. nate in the formation of a psoriatic plaque. 3. FARBER, E. M., NICKOLOFF, B. J., RECHT, B., and There is increased activity of the reticu- FRAKI, J. E. Stress, symmetry, and psoriasis: pos- loendothelial system in patients with pso- sible role of neuropeptides. J. Am. Acad. Derma- riasis in the form of enhanced metabolic, tol. 14 (1986) 305Ð311. phagocytic and chemotactic functions of 4. GANAPATI, R, DESHPANDE, D. H., and CHULAWALA, the polymorphonuclear leukocytes (7). R. G. Some interesting disease combinations— This is contrary to what is observed in lep- report on two cases. Lepr. India 48 (1976) 428. rosy in which the involvement of the retic- 5. KUMAR, B., RAYCHAUDHURI, S. P., VOSSOUGH, S., and FARBER, E. M. The rare co-existence of lep- uloendothelial system and phagocytic sys- rosy and psoriasis. Int. J. Dermatol. 31 (1992) tem is characterized by reduced activity of 551Ð554. peripheral blood mononuclear leukocytes 6. NIANG, M. N., BALDE, A. T., PERRAUT, R., MANE, and macrophages and depressed T-cell I., and CARTEL, J. L. Apoptosis in leprosy pa- functional state (1). tients. Int. J. Lepr. Other Mycobact. Dis. 67 (1999) Recently, apoptosis has been implicated 473Ð474. to play an important role in the lymphocytic 7. NICKOLOFF, B. J. The immunologic and genetic alterations in leprosy because a highly sig- basis of psoriasis. Arch. Dermatol. 135 (1999) nificant increase in the level of spontaneous 1104Ð1109. apoptosis in leprosy patients as compared to 8. NIGAM, P. K., GUPTA, S. R., and NAIR, A. Leprosy 6 and psoriasis occurring together. Int. J. Dermatol. controls has been reported ( ). Apoptosis 30 (1991) 676. might represent a strategy of the immune 9. PAVITHRAN, K. Psoriasis developing in a patch of system to eliminate infected cells. If apop- leprosy as a Koebner phenomenon. Indian J. Lepr. tosis is a regular phenomenon in leprosy, in 64 (1992) 197Ð199. 344 International Journal of Leprosy 2003
10. RAYCHAUDHURI, S. P., and FARBER, E. M. Neu- rosy and psoriasis occurring together. Int. J. Der- roimmunologic aspects of psoriasis. Cutis. 66 matol. 29 (1990) 531Ð532. (2000) 357Ð362. 13. WAHBA, A., DORFMAN, M., and SHESKIN, J. Psori- 11. SHAI, A., VARDY, D., and ZVULUNOV, A. Psoriasis, asis and other common dermatoses in leprosy. Int. biblical afflictions and patients’ dignity. Harefuah J. Dermatol. 19 (1980) 93Ð95. 141 (2002) 479Ð482. 14. WAHBA-YAHAV, A. V. Disease concomitance in pso- 12. SUGATHAN, P., and RIYAZ, N. First report of lep- riasis. J. Am. Acad. Dermatol. 35 (1996) 790Ð791. INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. (ISSN 0148-916X) INTERNATIONAL JOURNAL OF LEPROSY and Other Mycobacterial Diseases
OFFICIAL ORGAN OF THE INTERNATIONAL LEPROSY ASSOCIATION REVIEW Mechanisms Involved in Peripheral Nerve Damage in Leprosy with Special Reference to Insights Obtained from In Vitro Studies and the Experimental Mouse Model1
Tannaz J. Birdi and Noshir H. Antia 2
ABSTRACT The histopathological observations of Khanolkar and Iyer, that M. leprae has a predeliction for nerves, first highlighted the central role of peripheral nerves in the pathology of leprosy. It is now well recognized that nerve damage in leprosy will still continue to be an important prob- lem in control and rehabilitation despite the presence of more efficient therapy. The multiplicity of mechanisms postulated, identified, and demonstrated in the last three decades has received little recognition from the scientific community at large. This review is therefore an attempt to collate these multiple studies on mechanisms of nerve damage into a cohesive analysis, which would facilitate the design of future studies. The objective of this review is to focus therefore only on studies which serve to illustrate mechanisms of nerve damage. RESUMÉ Les observations histopathologiques de Khanolkar et de Iyer, montrant que les M. leprae présentent une prédilection pour les nerfs, a permis de mettre en lumière le rôle central des nerfs périphériques dans la pathogénie de la lèpre. Il est maintenant bien établi que les al- térations nerveuses de la lèpre vont continuer à être un problème majeur dans le contrôle et la réhabilitation, malgré la présence de thérapies de plus en plus efficaces. Le grand nombre des mécanismes postulés, identifiés et démontrés depuis les 3 dernières décennies, n’a reçue que peu de reconnaissance de la part de la communauté scientifique en général. Cette revue est donc une tentative de rassembler ces nombreuses études sur les mécanismes de l’altéra- tion neurale au sein d’une analyse cohérente, dont le but est d’aider à l’élaboration de futures études. Cette revue a pour mission de ne se focaliser que sur les études qui permettent d’il- lustrer les mécanismes d’altération nerveuse. RESUMEN Las observaciones histopatológicas de Khanolkar e Iyer que indicaron que M. leprae tiene una predilección por nervios, subrayaron el papel central de los nervios periféricos en la patología de la lepra. Ahora está bien reconocido que el daño a nervios en la lepra contin- uará siendo un importante problema para el control y la rehabilitación de la lepra, no ob- stante la existencia de nuevas y más eficientes formas de terapia. La multiplicidad de los mecanismos postulados, identificados, y demostrados en las últimas tres décadas ha recibido, sin embargo, poco reconocimiento por parte de la comunidad científica. Esta re- visión es por lo tanto, un intento de unir, de alguna manera, estos múltiples estudios sobre los mecanismos de daño a nervios, en un análisis cohesivo que pudiera facilitar el diseño de futuros estudios. Por lo tanto, esta revisión se enfoca sólo a los estudios orientados a ilustrar los mecanismos de daño a los nervios.
1 Received for publication 12 November 2002. Accepted for publication 22 September 2003. 2 T. J. Birdi, Ph.D. and N. H. Antia, FRCS, FACS (Hon.), The Foundation For Medical Research, 84-A, R.G. Thadani Marg, Worli, Mumbai 400 0018. Reprint Requests to: Tannaz J. Birdi, The Foundation For Medical Research, 84-A, R.G. Thadani Marg, Worli, Mumbai 400 0018. E-mail: [email protected] 345 346 International Journal of Leprosy 2003
Anatomical and mechanical factors in- predominant histopathological features com- volved in leprous neuropathy have been re- mon in lepromatous and tuberculoid patients viewed extensively by Wadia (103). On the (78, 80) include: i) sub-perineural oedema con- other hand, Dastur (16, 18), Palande (61), and sisting of a proteinaceous granular matrix, Antia (1) concluded that raised intraneural interspersed with small pockets of collagen; tension was an important contributing fac- ii) axonal atrophy and secondary demyelina- tor, and that fibrous tunnels at the joints tion; iii) loss of unmyelinated fibers; iv) ac- may further excacerbate the nerve damage tivation of resident macrophages and fibrob- which results in sites of predeliction usually last within the endoneurial space. being located at the entrapment site. In lepromatous patients, M. leprae were Maintenance of the peripheral nervous detected mainly in the Schwann cells of the system is the result of a complex balance unmyelinated fibers (80, 81). Though no acid- between the two functions of the immune fast bacilli (AFB) are seen in nerves of tu- system viz. assistance in maintenance of berculoid patients, osmiophilic structures nerve physiology/ regeneration (64) and de- suggestive of bacteria are observed in the fense against infections. Leprosy serves as electron micrographs (2). In addition, viable a unique model where, due to the insidious M. leprae were recovered from homogenates nature of the infecting organism, Mycobac- of skin biopsies from tuberculoid patients in- terium leprae and its predilection for pe- noculated into the mouse footpad (86). ripheral nerve Schwann cells, the two func- Accompanying these degenerative changes tions of the immune system co-exist at least listed above, regenerative activity was also during the initial stages. observed across the spectrum (78), thus The nerve damage following M. leprae implying that there is a balance between infection of the peripheral nerve can be di- regeneration and degeneration with the out- vided into two stages. The initial phase is come depending on the predominating ac- common to both lepromatous and tubercu- tivity. loid patients and occurs despite the absence These features in the initial phase of of inflammatory cells (80, 81, 85). In the exper- nerve damage have also been studied in the imental mouse model, the onset and initial mouse model (85). The major limitation in progress of M. leprae-induced nerve dam- the mouse model has been the absence of age remained unchanged in thymectomized inflammatory cells and granulomatous reac- irradiated mice and mice treated with anti- tions mimicking the spectral disease of the Thy 1.2 or Cyclosporin A, further empha- human, as well as the inability to demon- sizing that the early events are not immuno- strate bacilli in Schwann cells of the af- logically mediated (82). A recent study by fected nerves. However, the presence of Rambukkana, et al. using Rag 1-knockout nerve abnormalities in the absence of in- mice also demonstrated that nerve damage flammatory cells reiterates the concept of a can be initiated by M. leprae infection in non-immunological mode of nerve damage the absence of an immune response (67). in the initial stages, possibly due to aberra- The studies of Brand, et al. (9) suggested tions in Schwann cell functions. On the that M. leprae survived better within the other hand, the absence of bacilli in cooler regions of the body. Therefore, the Schwann cells may merely reflect differ- metabolic alterations in Schwann cells ences in the affinity or the nature of neural and/or axon as a consequence M. leprae in- barriers in the human and mouse models. fection have been implicated as the major The Swiss White (SW) mouse is a strain in factor in this phase of nerve damage (51, 56). which the response of host cells to M. lep- In the later phase, there is an influx of rae infection parallels those observed in mononuclear cells, which is predominantly lepromatous patients as opposed to the lymphocytic in tuberculoid patients and C57BL/6 strain in which the response to M. macrophagic in the lepromatous (62). leprae is similar to that observed in tuber- Initial phase of nerve damage. The ini- culoid patients or normal individuals (3). tial phase of nerve damage observed in pa- The histopathological features of early tients across the leprosy spectrum and in a nerve damage parallel the observation in proportion of contacts is characterized by patients and are similar in experimentally an absence of inflammatory cells (80, 81). The infected SW and C57 mice (5). 71, 4 Birdi and Antia: Review: Mechanisms Involved in Nerve Damage 347
Entry of M. leprae into the nerve, medi- However, while in SW mice the damage ated by the endoneurial endothelial cells, progresses and increased demyelination is has been suggested by Mehta (46); Dastur, et observed, in C57BL/6 the damage is al. (19, 20); Boddingues, et al. (7, 8), and Scol- arrested or delayed (18). A possible ex- lard (75) who have documented the presence planation is provided by the effect of M. of M. leprae in the endothelial cells. Scol- leprae infection of Schwann cells in vitro lard (75) has proposed that no difference ex- on neural glia cell adhesion molecule ists in the mechanism of initial infection (NgCAM) expression (88), Schwann cell across the leprosy spectrum. Bacteremia proliferation (87), and production of colla- has also been reported in leprosy patients gen (90). Since none of the three parameters across the spectrum (12, 30). This is in keep- were affected by M. leprae infection of ing with the histopathological features of Schwann cells from C57BL/6 mice, the re- early nerve damage. generative capacity in this strain is not ham- M. leprae entry into Schwann cells is the pered. In contrast, M. leprae infection of most important event in the induction of SW Schwann cell cultures resulted in di- nerve damage. Based on studies utilizing minished expression of NgCAM (88), which murine dissociated Schwann cell cultures, is required for efficient Schwann cell-axon early entry of M. leprae (within 6 hours) is interaction and is normally enhanced during observed only with viable bacteria (14). Such regeneration. The aberrant myelination ob- forced entry into macrophages has also been served in infected nerves (76, 77) may be a re- reported for M. leprae as well as (49) Leish- flection of a decrease in NgCAM expression. mania donovani (11). Recent studies by Schwann cell proliferation, an important pre- Rambukkana, et al. (66) have suggested that requisite for peripheral nerve regeneration, α2 laminin and a dystroglycan are responsi- was also decreased. In addition, SW Schwann ble for the specific predeliction of M. leprae cells secreted increased levels of collagen for Schwann cells. However, since both in vitro (88), which correlated with observa- components are present in the basal lamina tions in sciatic nerves from experimentally of myelinating and non-myelinating fibers, infected SW (5, 85) mice and nerves of lep- this hypothesis fails to explain the ultra- rosy patients (78). structural observations that, at least in the In summary, the initial nerve pathology initial stages the M. leprae, are predomi- observed in the sciatic nerves of SW and nantly observed in Schwann cells of non- C57BL/6 mice infected with M. leprae in myelinating fibers (74). The studies by the footpad are similar and resemble the Choudhary, et al. (13) did not find evidence early changes in the nerves of lepromatous for a unique bacterial surface component and tuberculoid patients. Therefore, it was for bacterial entry. hypothesized that the response of Schwann An important feature of infection of host cells to the presence of M. leprae would be cells by M. leprae is the down regulation similar. In keeping with this hypothesis, cell-cell communication channels of the some common aberrations were noted in the host cell from lepromatous patients (3, 88, 89). two strains in their Schwann cell response to Once M. leprae have colonized the Schwann M. leprae infection, which included down cells, expression of nerve growth factor regulation of NGF-R expression and pro- (NGF) receptor and fibronectin secretion duction of fibronectin. Nevertheless, a num- are down regulated (89, 90). This has been ber of Schwann cell responses to M. leprae demonstrated in vitro in Schwann cells from infection were also diverse in the two SW and C57BL/6 mice. Receptor down reg- strains. SW Schwann cells showed decrease ulation would result in decreased utilization in proliferation, production of fibronectin, of NGF resulting in axonal atrophy, and the and expression of NGF-R and NgCAM, decrease in fibronectin secretion would while an increase was noted in the produc- hamper regeneration. Both of these features tion of collagens and laminin. Such differ- are characteristic of the initial phase of ing responses of Schwann cells to M. leprae nerve damage and are seen in patients across infection in the two strains in the early the spectrum (78, 80) and in the nerves of ex- stages may have important implications for perimentally infected SW and C57BL/6 the later stages of nerve damage. First, it mice (5). may result in differing degrees of Schwann 348 International Journal of Leprosy 2003 cell mediated regenerative activities in the viewed as more than just supportive cells two strains and this may explain the pro- for M. leprae multiplication, or as mere gression of early pathology to extensive antigen depots. Instead, the initial response damage only in SW strain (5). Secondly, of Schwann cells to M. leprae infection such a response in patients’ nerves would may have important and divergent influ- probably provide signals to different popu- ences on the immunological profile of lep- lations of inflammatory cells and conse- romatous and tuberculoid nerves. quently contribute to the differing composi- An alterative hypothesis is that the tion of the infiltrating cell population in lep- mononuclear cells crossing the barrier in romatous and tuberculoid nerves (44, 104). the course of normal surveillance (38) en- Concurrent with the metabolic damage counter antigen presented by antigen- induced, the bacteria continue to multiply presenting cells (APC) leading to their acti- intracellularly within the Schwann cells. It vation, which in turn signals the influx of appears that M. leprae growth is equally fresh cells. supported across the leprosy spectrum and Though the Schwann cell is capable of in Schwann cells of SW and C57BL/6 mice presentation of mycobacterial antigens on (unpublished observations). However, the its cell membrane, this expression on the immune response generated by the host is membrane is probably a result of integra- decisive in determining the later sequence tion of bacterial antigen with host mem- of events. brane components during bacterial entry Later phase of nerve damage. The hall- rather than active processing by Schwann mark of the later phase of nerve damage is cells (6). the presence of inflammatory cells. Studies Nevertheless, M. leprae-infected Schwann by Dastur, et al. (18, 19, 20); Job, et al. (31, 32); cells from both strains of mice are capable and Ridley (68), among others, have at- of sensitizing lymphoid cells in the murine tempted to elucidate the pathology of the dissociated Schwann cell culture system later phase of nerve damage across the (50). However, this ability was dependant on spectrum. The central question that arises the sensitization level of the lymphocytes is, “What is the signal for the inflammatory prior to co-culture with Schwann cells, the cell influx and how is it regulated?” antigen used, and the requirement of acces- One of the earliest theories put forward sory cells (50). In addition, an increase in the was that the inflammatory response espe- fibroblast population in culture enabled dis- cially in tuberculoid patients was the result sociated Schwann cells to induce lympho- of autoimmunity (42). Auto-antibodies in proliferation to M. leprae and 65Kd antigen leprosy have been reported against several even in the absence of adherent cells, nerve components (24, 105). In addition, hu- demonstrating that fibroblasts could under- man nerves and skin have a number of anti- take the role of accessory cells, and in con- genic determinants in common with M. lep- junction with infected Schwann cells, pre- rae (58, 65, 99). Many of these epitopes are cipitate an immunological attack (50). This heat-shock proteins (hsp) (36, 60). In animal is important since macrophages at the site models, it has been shown that M. leprae- of old lesions are paralyzed/senescent. primed macrophages attack the Schwann The role of fibroblasts, as well as cells, not only in the presence, but also in Schwann cells, in leprous neuropathy was the absence of detectable M. leprae (94), and also indicated in studies using immune M. leprae sensitized T cells also react with modulated mice (82). Progressive, late nerve Schwann cell components (92). However, damage associated with demyelination was studies by Mshana, et al. (54) and Ghas- significantly reduced in mice treated with walla, et al. (27) failed to consistently find anti-thy 1.2 antibody where, along with the antibodies against neural antigens. Lym- depletion of T-helper cells, fibroblast— phocyte proliferation in response to neural which express thy 1.2 marker—would also antigens was also absent (22, 55). Thus, it is be affected. In contrast, thymectomized and suggested that autoimmunity may only con- irradicated mice, where both T and B cell tribute to the exacerbation of the lesion. populations would be affected leaving the On the basis of the studies cited in the fibroblast population intact, showed similar previous section, Schwann cells can be intensity of nerve damage as the untreated 71, 4 Birdi and Antia: Review: Mechanisms Involved in Nerve Damage 349
animals. This indicates an important role cytes is probably curtailed by the produc- for fibroblasts in leprous neuropathy. tion of suppressor factors by the infected The presence of MHC class I products Schwann cells (47) and the invading M. and mycobacterial antigens on the surface of leprae-infected macrophages (4, 72). The data the Schwann cell (6, 94) may result in “by- of Mehta, et al. (47), indicate that M. leprae stander” damage due to direct cellular cy- infected murine Schwann cells, though ini- toxicity. Studies have demonstrated the lysis tially stimulating lymphocytes (50), release a of M. leprae-infected Schwann cells by sen- factor that results in cell death probably due sitized T cells in vitro (94). to apoptosis. These lymphoid cells are then In tuberculoid patients, the influx of in- engulfed by Schwann cells and slowly de- flammatory cells functions as a double- graded in culture (48). Apoptosis of antigen- edged sword. The influx of macrophages specific T cells in lesions of experimental enhances the regenerative process (63). In allergic encephalomyelitis (EAE) and EAN addition, the lymphocytes stimulated by M. has been identified as an effective mecha- leprae-antigens presented on Schwann cells nism in stopping neural inflammation. It are able to activate macrophages to kill in- may be the consequence of production of a tracellular M. leprae (50). Simultaneously glucocorticoid by M. leprae infected with these beneficial effects, the release of Schwann cells (108, 109). The possibility also protease results in collagen breakdown, exists that M. leprae infected Schwann cells which excacerbates granuloma formation like astrocytes (39) may be secreting a tumor (44). Reactive oxygen intermediates (ROI), necrosis factor (TNF)-like factor. The re- produced by both Schwann cells (51) and fractoriness of factor production to cyclo- macrophages as a consequence of the in- heximide treatment of M. leprae-infected flammatory process, results in further nerve Schwann cells (47) implicates the release of damage at the site of the granuloma. glucocorticoid resembling substances (97), In lepromatous patients, the invading which may explain the predominantly macrophages further down regulate the macrophage infiltration in nerve granulo- NGF-R (89), thus depriving the cell from uti- mas of lepromatous patients. lizing the available NGF, leading to slow Silent nerve damage. The studies of nerve damage. In addition, the NGF levels Shetty, et al. (29, 73, 84) and Srinivisan and have also been reported by Facer, et al. (25, 26) Gupte, et al. (93) on silent nerve damage, to be decreased in lepromatous patients. which is a major concern in patient man- Macrophages simultaneously enhance agement, have extensively documented that Schwann cell proliferation and NgCAM ex- neuropathy progresses in patients after the pression, thus aiding in the regenerative infection is cured and even in the absence process (87, 88). The transforming growth of inflammatory cells. Much less in known factor-β (TGF-β) is important in the devel- about the mechanism by which this occurs. opment and repair of the peripheral nerve Nevertheless, there is evidence that the (41). Conversely, it is also implicated in pe- demyelination observed is a consequence of ripheral neuropathies, such as experimental atrophic changes in the axonal compart- allergic neuropathy (EAN) and experimen- ment (78). It has been demonstrated that this tal Wallerian degeneration (71). TGF-β has axonal atrophy is due to hypophosphoryla- also been reported to enhance Schwann cell tion of the axonal neurofilaments (87). It is proliferation, up regulate NgCAM, and en- of interest to note that the degenerative dis- hance collagen synthesis (89). These func- orders of the central nervous system are tions of TGF-β, along with its immunosup- characterized by the presence of hyperphos- pressive action, allow us to propose that phorylation, whereas in leprous nerves TGF-β secreted by M. leprae-infected there is decreased phosphorylation of the macrophages from lepromatous patients/ neurofilaments resulting in their degrada- SW mice may be responsible for the de- tion. The observation that mycobacterial creased regenerative activity in these nerves. antigens persists in nerves long after clini- This is supported by the observation of cal cure (84) suggests that this persisting Khanolkar, et al. (35) that maximum TGF-β antigen may be important for the continued was observed within lepromatous lesions. progression of nerve damage. In addition, further invasion by lympho- Preliminary findings by Shetty, et al. (83) 350 International Journal of Leprosy 2003 demonstrated that the decreased phosphory- tous patients in the first year of multidrug lation of neurofilaments was also seen in therapy (MDT) (98). This may be due to the mice inoculated in the footpads with either cidal drugs in the MDT regimen which re- viable or heat-killed M. leprae. Treatment sult in the suppressor factors which are de- with the TGF-β and heat shock proteins has pendent on viability of M. leprae and are been shown to inhibit mammalian cyclin therefore no longer being produced by in- dependent kinase (CDK) (37). fected macrophages (4, 72) and Schwann In a culture system using macrophages cells (47), while the killed bacilli act as anti- Chan, et al. (10) have shown that lipoarabi- gen depots, which serve to stimulate an in- nomannan (LAM) from M. tuberculosis and flammatory response. The reduced levels of M. leprae inhibited the enzyme protein ki- TGF-β reported in reactional lesions further nase C (PKC). The neuron specific kinase, supports this hypothesis (35). responsible for phosphorylation of neurofil- An alternative mechanism has been pos- ament proteins, CDK5 and PKC are known tulated by Rook, et al. (70) who suggests a to share common inhibitors (43). CDK5 may neuroendocrine control of inflammation, therefore follow the same pattern of inhibi- wherein destruction of C fibers would di- tion (40, 79). Increase in phosphatase activity, minish the activity of neural feedback path- an indicator of dephosphorylation of NF, ways due to decreased secretion of active has been reported in leprous nerves and peptides. This in turn would prevent activa- skin lesions by Dastur and Dabholkar (17). tion of the hypothalamus pituitary axis, re- Thus, it is possible that the persisting anti- sulting in local deactivation of cortisol, gens may interfere with the functions of the thereby precipitating an inflammatory re- regulatory enzymes leading to hypophos- sponse. While the first hypothesis is re- phorylation. stricted to lepromatous patients where Further evidence for the role of M. leprae suppressor factors play a dominant role, de- antigens being involved directly in silent struction of C fibers, which is common to neuritis is obtained from the study of Croft lepromatous and tuberculoid patients (78, 80) who reported that in 32% of all impaired suggests that the mechanism postulated by nerves studied, nerve function did not im- Rook, et al. may explain reactions at both prove despite prednisolone therapy (15). ends of the spectrum. The involvement of Since prednisolone would only decrease in- the Cortisol-Cortisone shuttle may also ex- flammation and has no effect on the bacte- plain the clinical observations that reac- rial antigenic load, this further emphasizes tional episodes are more frequent during the importance of M. leprae antigens in me- pregnancy (23) since the enzymes of the diating silent neuritis. shuttle are affected by progesterone and Reactions in leprosy. The acute inflam- pregnancy (96). matory response in Type I reactions is of While specificity of lymphoid cells iso- special concern since it significantly exacer- lated from nerves of non-reactional tuber- bates nerve damage. Histopathologically, culoid patients is against mycobacterial the lesions show all the characteristics of a antigens and not neural antigens (22, 54), delayed-type hypersensitivity reaction (57, 68) studies on antibody responses to neural with an influx of mainly CD4 lymphocytes, antigens have resulted in conflicting results especially of the Th1 class (52, 53, 59, 102, 106, 107). [reviewed by Desikan (21)]. The observation There is an increase in IL-2 and TNF-α, that sera from all patients with erythema which confirms a shift to the Th1 subtype nodosium leprosum (ENL) contain de- during a reaction (34, 100, 101). Possibly due to myelinating antibodies is of particular inter- this shift, the humoral immunity during est (N. F. Mistry, personal communication). Type I reaction seems to be diminished (100). Nevertheless, since systemic manifestations However, a shift to Th2 may also occur are rare and there is no evidence of a spe- since there is an increase in mRNA for IL-4 cific reaction precipitating mycobacterial in some of the lesions (53, 102, 107). antigen (50), the difference may lie in local While reactional episodes can occur at factors such as differential activation and anytime during the course of the disease, tissue distribution, as well as the status of recent studies suggest that their incidence the host immune response and genetic re- has increased especially among leproma- striction. 71, 4 Birdi and Antia: Review: Mechanisms Involved in Nerve Damage 351
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A (1983) 119Ð1126. high incidence of viable Mycobacterium leprae in 73. SAMANT, G., SHETTY, V. P., UPLEKAR, M. W., and post MDT recurrent lesions in tuberculoid leprosy ANTIA, N. H. Clinical and electrophysiological patients. Lepr. Rev. 72 (2001) 337Ð344. evaluation of nerve function impairment follow- 87. SINGH, N., BIRDI, T. J., and ANTIA, N. H. Differen- ing cessation of multi drug therapy in leprosy. tial in vitro modulation of Schwann cell prolifera- Lepr. Rev. 70 (1999) 10Ð20. tion by Mycobacterium leprae and macrophages 354 International Journal of Leprosy 2003
in the murine strains, Swiss White and C57BL/6. 386 patients is West Nepal. Lepr. Rev. 65 (1994) J. Peripher. Nerv. Syst. 3(1998) 207Ð215. 190Ð203. 88. SINGH, N., BIRDI, T. J., and ANTIA, N. H. M. lep- 99. VAN DEN AKKER, T. W., NAAFS, B., KOLK, A. H. rae infection and macrophages secretory products J., et al. Similarity between mycobacteria and modulate the expression of NgCAM on Schwann human epidermal antigens. A study with human cell surface. Int. J. Lepr. Other Mycobact. Dis. 64 sera and murine anti-M. leprae monoclonal anti- (1996) 449Ð451. bodies. Brit. J. Dermatol. 127 (1992) 352Ð358. 89. SINGH, N., BIRDI, T. J., and ANTIA, N. H. Nerve 100. VERHAGEN, C. E., FABER, W. R., KLATSER, P. R., et growth factor production and expression of p75 by al. Immunohistological analyses of in situ expres- Schwann cells and neurofibroblasts in response to sion of mycobacterial antigens in the skin lesions M. leprae infection and macrophage secretory of leprosy patients across the histopathological products. Neuropathol. Appl. Neurobiol. 23 spectrum. Am. J. Path. 154 (1999) 1793Ð1804. (1997) 59Ð67. 101. VERHAGEN, C. E., VAN DER PAUW KRAAN, T. C. T. 90. SINGH, N., BIRDI, T. J., CHANDRASHEKHAR, S., AND M., BUFFING, A. A. M., et al.Type-1- and Type- ANTIA, N. H. Schwann cell ECM protein produc- 2-like lesional skin derived M. leprae-responsive tion is modulated by M. leprae and macrophage se- T-cell clones are characterized by co-expression cretory products. J. Neurol. Sci. 151 (1997) 13Ð22. of IFN-gamma/TNF-α and IL4/IL5/IL13 respec- 91. SKOFF, A. M., LISAK, R. P., BEALMEAR, B., and tively. J. Immunol. 160 (1998) 2380Ð2387. BENJAMINS, J. A. TNF-α and TGF-β act synergis- 102. VERHAGEN, C. E., WIERINGA, E. E. A., BUFFING, tically to kill Schwann Cells. J. Neurosci. Res. 53 A. A. M., et al. Reversal reaction in borderline (1998) 747Ð756. leprosy is associated with a polarized shift to 92. SPIERINGS, E., DEBOER, T., ZULIANELLO, L., and Type-1-like Mycobacterium leprae T-cell reac- OTTEHOFF, T. H. M. The role of Schwann cells, T tivity in lesional skin. A follow-up study. J. Im- cells, and M. leprae in immunopathogenesis of munol. 159 (1997) 4474Ð4483. nerve damage in leprosy. Lepr. Rev. 71 (2000) 103. WADIA, N. H. (ED.) Neurological Practice in In- 5121Ð5129. dia. Under Publication. 93. SRINIVASAN, H., and GUPTE, M. D. Experiences 104. WALLE, T. K., VARTIO, T., HELVE, T., VIRATEN, I., from studies on quiet nerve paralysis in leprosy and KURKI, P. Cellular fibronectin in rheumatoid patients. In: The Peripheral Nerve in Leprosy and synovium and synovial fluid: a possible factor Other Neuropathies. Dehli: Oxford University contributing to lymphocyte infiltration. Scand. J. Press, 1997, pp. 30Ð35. Immunol. 31 (1990) 535Ð540. 94. STANLEY, J. N. A. Pathological changes in the sci- 105. WRIGHT, D. J. M., HIRST, R. A., and WATERS, M. atic nerves of mice with leprosy neuropathy—an F. R. Neural auto-antibodies in leprosy. Lepr. electronmicroscopic study. Ph.D. thesis, Univer- Rev. 46 (1975) 157Ð169. sity of Oxford, 1988. 106. YAMAMURA, M., UYEMURA, K., DEANS, R. J., et 95. STEINHOFF, U., and KAUFMANN, S. H. E. Specific al.Defining protective responses to pathogens: lysis by CDS+T cells of Schwann cells expressing cytokine profiles in leprosy lesions. Science. 254 M. leprae antigens. Eur. J. Immunol. 18 (1998) (1991) 277Ð279. 967Ð972. 107. YAMAMURA, M., WANG, X. H., OHMEN, J. D., et 96. SUN, K., YANG, K., and CHALLIS, J. R. Regulation al. Cytokine patterns of immunologically medi- of 11β-hydroxysteroid dehydrogenase type 2 by ated tissue damage. J. Immunol. 149 (1992) progesterone, estrogen and the cyclic adenosine 1470Ð1481. 5′-monophosphate pathway in cultured human 108. ZETTL, U. K., GOLD, R., HARTUNG, H. P., and placental and chorionic trophoblasts. Biol. Re- TOYKA, K. V. Apoptotic cell death of T lympho- prod. 58 (1998) 1379Ð1384. cytes in experimental autoimmune neuritis. Neu- 97. TROGANOWSKI, J. Q., and LEE, V. Expression of rosci. Lett. 176 (1998) 75Ð79. neurofilament antigens by normal and neoplastic 109. ZETTL, U. K., GOLD, R., HARTUNG, H. P., and human adrenal chromaffin cells. N. Engl. J. Med. TOYKA, K. V. Intravenous glucocorticosteroid 313 (1985) 101Ð103. treatment augments apoptosis of inflammatory T 98. VAN BRAKEL, W., KHAWAS, I. B., and LUCAS, S. B. cells in experimental autoimmune neuritis. J. Reactions in leprosy: an epidemiological study of Neuropathol. Exp. Neurol. 54 (1995) 540Ð547. INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. (ISSN 0148-916X) INTERNATIONAL JOURNAL OF LEPROSY and Other Mycobacterial Diseases
OFFICIAL ORGAN OF THE INTERNATIONAL LEPROSY ASSOCIATION
EDITORIAL
Incomprehension1 The current resurgence of certain great endemic diseases such as tuberculosis and After reading the ILA Technical Forum trypanosomiasis, that were thought to be report, many articles and editorials in spe- under control a few decades ago, should cialized reviews, the latest Technical Bul- naturally incite us to be very cautious. letin from ILEP, the Resolution taken at the ILA Congress in Salvador (Brazil), e-mail —Dr. Pierre Bobin exchanges from an anglophone discussion General Secretary, list (leprosy mailing list), and participating Association of French Speaking Leprolo- in debates at the latest International Con- gists (ALLF) gress on Leprosy and listening to leprosy experts and personnel Ç in the field È, it is Incompréhension clear that a great difference exists in the opinion between many experts on one hand En lisant le rapport du Forum Technique and, World Health Organization (WHO) on de l’International Leprosy Association the other, about the analysis of the state of (ILA), les nombreux articles et éditoriaux leprosy in the world and the measures that de revues spécialisées, le dernier Bulletin should be taken to control it. According to Technique de l’ILEP, la récente déclaration the vast majority of experts, WHO is untir- de l’ILA, les échanges de courrier électron- ingly carrying out its leprosy Ç elimina- ique sur une liste de discussion lépro- tion È policy, apparently convinced of being logique anglophone, en participant par enlightened while, in fact, they are taking ailleurs aux débats du dernier Congrès In- risks without basing newly elaborated ternational sur la Lèpre et en écoutant des strategies on a rigorous scientific method. experts lèpre et des personnels « de ter- The split between WHO and ILEP, as rain », on constate un énorme décalage en- well as the absence of dialogue among all tre le point de vue de très nombreux experts partners involved (this could have taken et celui de l’OMS, concernant l’analyse de place, for instance, during the ILA Techni- la situation de la lèpre dans le monde et les cal Forum or during the International Lep- mesures à prendre pour la contrôler. Pour la rosy Congress), are truly regrettable. grande majorité de ces experts, l’OMS Most observers are divided between poursuit inlassablement sa politique feelings of incomprehension and indigna- Ç d’élimination » de la lèpre, apparamment tion. It therefore appears urgent to hold persuadée de détenir la vérité, mais faisant a meeting with all partners involved in en fait des Ç paris », sans une véritable dé- leprosy control to try and develop a con- marche scientifique pour argumenter les sensus policy and broad strategies based nouvelles stratégies élaborées. upon rigorous criteria. La rupture entre l’OMS et l’ILEP, ainsi que l’absence de concertation entre l’OMS 1 The following editorial was first published in the et l’ensemble des autres partenaires, alors Bulletin de l’ALLF (Review of French Speaking Lep- que cela aurait pu être possible, par exem- rologists Association) n. 12 February 2003. ple, lors du Forum Technique de l’ILA ou
355 356 International Journal of Leprosy 2003 du dernier Congrès International sur la plusieurs dizaines d’années, bien contrôlées Lèpre, sont vraiment regrettables. telles que la tuberculose ou la trypanosomi- La plupart des observateurs sont par- ase, doit, si besoin était, nous inciter à la tagés entre incompréhension et indigna- plus grande prudence. tion et il nous semble très urgent que On ne raye pas comme cela d’un trait de l’ensemble de tous les partenaires de la plume une maladie Ç millénaire », en déci- lutte contre la lèpre se retrouvent autour dant administrativement qu’elle va être très d’une table pour essayer d’obtenir un prochainement éliminée. consensus sur la politique à mener et les grandes stratégies à élaborer, en fonction —Dr Pierre Bobin de critères rigoureux. Secrétaire Général La résurgence actuelle de certaines de l’Association des Léprologues de grandes endémies qui semblaient, il y a Langue Française (ALLF)
ERRATA Due to an editing oversight in the September issue of 2003 (Vol 71.3), the title of the origi- nal article by C. K. Job, Gregory McCormick, David Scollard, and Richard Truman (p. 231) was misspelled. The correct title of the article is “Electron Microscope Appearance of Lepromatous Footpads of Nude Mice.” The editorial staff apologizes for any inconve- nience this error may have caused.
In the September 2003 issue of the Journal, an error occurred in the original article by Dr. Kaur, et. al., entitled, “‘Relatively spared zones’ in leprosy: a clinicopathological study of 500 patients.” The footnote to Table 2 should have read: “*TT, tuberculoid leprosy; BT, borderline tuberculoid; BB, borderline; BL, borderline lepromatous; LL, lepromatous lep- rosy.” We apologize for the error. ERRATA Due to an editing oversight in the September issue of 2003 (Vol 71.3), the title of the origi- nal article by C. K. Job, Gregory McCormick, David Scollard, and Richard Truman (p. 231) was misspelled. The correct title of the article is “Electron Microscope Appearance of Lepromatous Footpads of Nude Mice.” The editorial staff apologizes for any inconve- nience this error may have caused.
In the September 2003 issue of the Journal, an error occurred in the original article by Dr. Kaur, et. al., entitled, “‘Relatively spared zones’ in leprosy: a clinicopathological study of 500 patients.” The footnote to Table 2 should have read: “*TT, tuberculoid leprosy; BT, borderline tuberculoid; BB, borderline; BL, borderline lepromatous; LL, lepromatous lep- rosy.” We apologize for the error. INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. (ISSN 0148-916X) CORRESPONDENCE
This department is for the publication of informal communications that are of interest because they are informative and stimulating, and for the discussion of controversial matters. The mandate of this JOURNAL is to disseminate information relating to leprosy in particular and also other mycobacterial diseases. Dissident comment or interpretation on published research is of course valid, but personality attacks on individuals would seem unnecessary. Political comments, valid or not, also are unwelcome. They might result in interference with the distribution of the JOURNAL and thus interfere with its prime purpose.
Leprosy in Greece at the End of the 20th Century (1988–2000)1
TO THE EDITOR: aminations, presented with new signs and symptoms of the disease verified by histo- Historical data indicate that Greece be- pathologic and smear examinations (bacte- longs to the group of countries with very rial index, BI). Drug sensitivities were not low leprosy endemicity. Sporadic newly de- assessed in the relapsed cases. tected Greek cases of active disease are Case classification across the disease spec- mainly referred by dermatologists to our trum was based on clinical picture, histo- unit with a prevalence rate much lower than pathology, bacterial index from skin lesions 1:100,000 dermatologic outpatients. The (BI), lepromin test, and epidemiologic his- disease has therefore been eliminated as a tory. Therapeutic decisions are always based public health problem, but not completely on WHO treatment recommendations (7). eradicated (4). For leprosy patients and their Disease type distribution, yearly relative contacts, consultation, laboratory investiga- relapse rates (relapsed leprosy cases were tion, World Health Organization (WHO) rec- the numerator and yearly followed up ex- ommended multi-drug treatment (MDT), hos- leprosy patients were the denominator), and pitalization, and follow-up are free of charge. prevailing symptom at diagnosis (progres- As has occurred in other European coun- sive skin lesions or a leprosy reaction) were tries, since 1990 an influx of approximately analyzed either alone or as related to dis- one million migrants from Eastern Europe ease duration, age, gender, and residence (mainly Albania), the Middle East, Africa, (rural or urban). the Indian Subcontinent, and South-East There was no significant difference be- Asia have entered Greece (total population tween Greek incident and relapsed cases with ten million). regard to disease type distribution (Table 1). A retrospective study (1988Ð2000) re- After 1992, no more paucibacillary (PB) garding 25 Greek and five foreign newly cases were detected. Between incident and detected (incident) leprosy patients, as well relapsed cases, there was no difference by as 40 relapsed Greek cases was carried out. gender (Table 2) nor prevailing symptom Relapses were old cases who, years after that led to diagnosis. Skin lesions were a being discharged from the hospital and with more common presenting symptom (inci- repeatedly negative clinical and smear ex- dent cases 70%, 21/30; relapsed cases 55%, 22/40) than leprosy reactions (incident cases 30%, 9/30; relapsed cases 45%, 18/40). 1 Received for publication 19 November 2002. Ac- cepted for publication 15 October 2003. When estimating disease progression in Mailing Address: Kyriakos P. Kyriakis, M.D., the relapsed cases, it was observed that four Koumanoudi Str. 52, 11474 Athens, Greece. remained PB (10%), seven progressed from
357 358 International Journal of Leprosy 2003
TABLE 1. Leprosy in Greece by classification 1988–2000.
Leprosy classification Cases ITTBTBBBLLLTotal Greek Incident 1.5 1.5 3.5 4.5 88.5 25 PB vs. MB 5 (20%, 6.8Ð40.7) 20 (80%, 59.3Ð93.2)
Relapsed — 1.5 3.5 5.5 9. 22.5 40 PB vs. MB 4 (10%, 2.8Ð33.6) 36 (90%, 76.3Ð97.2)
Total 1.5 2.5 6.5 9. 17.5 30.5 65 Percent 1.5 3.1 9.2 13.8 26.2 46.2 100 95% CI 0.04Ð8.3 0.4Ð10.7 3.5Ð19 6.5Ð24.8 16Ð38 33.7Ð58.9 — PB vs. MB 9 (13.8%, 6.5Ð24.6) 56 (86.2%, 75.3Ð93.5) 65
Other Nationalities Filipino 2 Egyptian 1 S. Leonian 1 Albanian 1 Total 1 2 2 5 95% CI: 95% “exact” confidence interval.
PB to multibacillary (MB) (17.5%), and the incident cases (19 of 25, 76%, “exact” CI remaining 29 cases were MB from the mo- 54.9 to 90.6) when compared to relapsed ment of diagnosis (72.5%). Despite disease cases, who are mainly living in cities, mostly duration of the relapsed cases, which from in the Athens greater area and throughout the the moment of first diagnosis was 29.4 ± country (25 of 40, 62.5%, “exact” CI 45.8 to 10.6 yrs, range 8 to 48 yrs, there was no dif- 77.3, χ2, Yates corrected, p = 0.005, odds ra- ference in disease type distribution within tio (OR) 5.3, “exact” CI for OR 1.5 to incident cases at first diagnosis. Relapse 19.4). In contrast, there was no association rate and disease duration did not differ be- of patient residence with gender (incident, tween men and women. The number of ex- relapsed, and overall). leprosy patients followed up annually series Foreign patients represented a small pro- presented a declining trend and a negative portion of total active leprosy cases (N = 5, compound growth rate (exponential trend, Y total N = 70, 7.1%, “exact” CI 2.4 to 15.9), = 395.7 (0.92)t, t-statistic Ð5.96, p <0.001, coming from endemic countries (Table 1). compound growth rate Ð7.6%) (Table 2). After 1992, only one imported case (LL) Regarding the age of Greek active lep- was detected. rosy cases, there was a significant differ- ence between incident PB (N = 5, mean DISCUSSION 26.6, range 6 to 56 yrs) compared with inci- In Greece during the second half of the dent MB (N = 20, 55.9, range 33 to 79 yrs, 20th century, rapid socioeconomic develop- p <0.001), but not between incident MB ment, rapid urbanization, substantial im- and the relapsed cases (N = 40, 64.05, range provement of living standards, nuclear 42 to 90 yrs, Bonferroni p-values, one way family pattern predominance, and a lack of ANOVA). Only 2 children younger than 14 differentials in the access to health care yrs were detected in the whole study period services were gradually achieved. These (diagnosed in 1989). factors are closely associated with a disease Comparisons regarding possible associa- decline, reduced exposure and transmission tions of active case detection with residence even in endemic areas, irrespective of lep- (rural or urban) revealed that rural residence rosy control interventions (2). Thereafter, (villages) was five times more frequent in secondary prevention, a good dapsone 71, 4 Correspondence 359
TABLE 2. Yearly active leprosy cases (numbers, incident and relapsed) and relative re- lapse rates (RRR, per thousand rate) in Greece, 1988Ð2000.
Incident Examined Relapsed (RRR ‰) Total Year active MF MF MFcases 1988 1 1 187 181 2 (10.7) 4 (22.1) 8 1989 5 5 177 223 4 (22.6) 3 (13.5) 17 1990 — 1 167 206 5 (29.9) 2 (9.7) 8 1991 1 1 129 145 2 (15.5) 1 (6.9) 5 1992 — 1 143 159 1 (7.0) 1 (6.3) 3 1993 — — 81 84 (— (—0 1994 — 1 103 112 (—1 (8.9) 2 1995 2 — 76 96 1 (13.2) 2 (20.8) 5 1996 — 2 102 100 4 (39.2) (—6 1997 — 1 85 87 1 (11.8) (—2 1998 — — 80 81 (—1 (12.3) 1 1999 — 1 80 81 1 (12.5) 1 (12.3) 3 2000 2 — 100 81 2 (20.0) 1 (12.3) 5 Total 11 14 1510 1636 23 (15.2) 17 (10.4) 65 Mean/y 0.85 1.1 116.2 125.8 1.8 1.3 5.0 Foreign patients are not taken into account in this table M = males, F = females; Mean/y: mean yearly rate.
monotherapy program in conjunction with tions resulted in concealment which, in as- yearly clinical and BI examinations aiming sociation with socioeconomic develop- a permanent smear negativity of registered ments, might explain why the majority of cases and the significant BCG coverage of relapsed cases lives in urban areas. the general population have further con- The number of yearly examined, promi- tributed to the epidemiologic pattern of a nently elderly, ex-leprosy patients is not dying out disease where the few cases that constant and gradually declines as a func- occur have a predominance of lepromatous tion of mortality, life conditions influencing leprosy (1, 5). compliance of elderly people in general, A constant policy of our center is that all and stigma-related fatigue. According to current and former household contacts of new previous regimens, all smear negative MB patients should be invited for examination. As cases first treated with dapsone monother- a rule, nuclear family members do present at apy had to remain under lifelong treatment least once for clinical, BI and histopatho- with dapsone. Re-treatment with MDT of logic examinations, as the only cost effec- all these old cases requires intense health tive method of active case finding (1). education and is gradually obtained. The majority of new cases and especially those seen after 1992 seem to represent a —Kyriakos Kyriakis, M.D. “hidden prevalence” which was not per- Dermatologist in Charge ceived before (6). A clear evidence for this West Attica General Hospital and is documented by more frequent rural resi- Leprosy Center dence of incident cases, the lack of differ- Athens, Greece ence in age between incident MB and re- REFERENCES lapsed cases, as well as by the homoge- neous disease type distribution in both 1. FEENSTRA, P. Sustainability of leprosy control ser- vices in low-endemic situations. Int. J. Lepr. Other patient categories at first diagnosis. Mycobact. Dis. 62 (1994) 599Ð608. In our elderly ex-leprosy patients, stigma 2. GILBODY, J. S. Aspects of rehabilitation in leprosy. Int. is based on memories of compulsory segre- J. Lepr. Other Mycobact. Dis. 60 (1992) 608Ð640. gation, isolation in leprosaria, and discrimi- 3. JOPLING, W. H. Leprosy stigma. Lepr. Rev. 62 natory lows in the past (2, 3). These condi- (1991) 1Ð12. 360 International Journal of Leprosy 2003
4. KYRIAKIS, K. P., KONTOCHRISTOPOULOS, G. J., and 6. SMITH, W. C. S. We need to know what is happen- PANTELEOS, D. N. Current profile of active leprosy ing to the incidence of leprosy. Lepr. Rev. 68 (1997) in Greece; a five-year retrospective study (1988– 195Ð200. 1992). Int. J. Lepr. Other Mycobcat. Dis. 62 (1994) 7. WHO EXPERT COMMITTEE ON LEPROSY. Epidemiol- 547Ð551. ogy. Geneva: World Health Organization, 1998. 5. NOORDEEN, S. K. The epidemiology of leprosy. In: Tech. Rep. Ser. 874, pp. 5Ð11. Leprosy. Edinburgh: Churchill-Livingstone, 1985, pp. 15Ð30. INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. (ISSN 0148-916X) OBITUARIES PAUL WILSON BRAND 1914–2003
His graphic descriptions of disease presen- tations were brilliant and were long remem- bered by many of his students; I was fortu- nate to be one of them. His deep concern and care for patients touched and moved each one of his students to whom he was a role model. It seems to me that it was God’s plan that brought together the world famous leprologist, Dr. Robert Cochrane and Dr. Brand, the young enthusiastic, ingenious and compassionate surgeon. In 1947 when, at the leprosy hospital at Chinglepet, Dr. Cochrane showed him the useless, de- formed and disabled hands of leprosy pa- tients that no surgeon had ever cared to touch and to repair, Dr. Brand saw the chal- lenge before him. He started his careful research into the pathology and pathogene- Dr. Paul W. Brand, one of the greatest sis of deformities in leprosy and into physicians of all time, well known to all methods to reconstruct their paralyzed leprologists of his generation, passed away hands and feet. His pioneering work in the on 8th of July, 2003, at the age of 89. He correction of deformities caused by leprosy was a pioneering surgeon, an excellent changed the lives of thousands of grateful teacher, a compassionate leprologist, and a patients. He also trained many surgeons dedicated missionary. from different parts of the world using the He was born in South India in July, 1914 facilities at Schieffelin Leprosy Research of British parents, studied medicine in the Center, Karigiri, and at Christian Medical University College Hospital, London, and College, Vellore. later qualified for the FRCS diploma. His Another area of research he was engaged father Jessi Brand died of malaria while in was the pathogenesis of plantar ulcers. serving as a missionary to the tribal people He established that the main course of these in Kolli Hills, situated in the mountain ulcers was loss of sensation due to nerve ranges of South India. His mother took over damage and not just leprosy. This finding the work of her husband and served the led to the adoption of measures to heal, to tribal folk until her death at the ripe old age protect, and to prevent the damage to insen- of 96. Service to the suffering, underprivi- sitive hands and feet. Specialized sandals, leged, and the poor was his inheritance. made out of microcellular rubber, were The call to serve as a medical missionary found suitable to prevent the formation of in India came to him in 1946 from Dr. plantar ulcers and to stop their recurrence. Robert G. Cochrane, the then principal of A rubber mill to manufacture microcellular the Christian Medical College, Vellore, who rubber was established at Schieffelin Lep- was desperately searching for a surgeon to rosy Research and Training Center, Karigiri satisfy the university requirements for because it was not profitable for any com- recognition of the MBBS degree offered by mercial rubber company to manufacture the college. Dr. Brand was a born teacher. this product.
361 362 International Journal of Leprosy 2003
Dr. Brand was the first one to demon- Laskar award in 1960. Queen Elizabeth strate that nerve damage was localized to honored him with a title of the Commander subcutaneously placed nerves and to sug- of the Order of the British Empire in 1961. gest that M. leprae multiplied at the cooler He served as President of the Leprosy Mis- regions of the body. This finding led to the sion International based in London and was path breaking experimental studies by on the Panel of Experts on leprosy of the Charles Shepard who succeeded in growing World Health Organization. He was one of M. leprae in the footpads of mice. the main architects of the All Africa Lep- At various periods during his tenure in rosy Rehabilitation and Training Center in Christian Medical College, Vellore, Dr. Brand Addis Ababa, Ethiopia, and the Schieffelin held the posts of Professor of Orthopedic Leprosy Research and Training Center at surgery, Associate Director and Principal. He Karigiri, India. A biography was written on was also consultant surgeon at Schieffelin him entitled, “Ten fingers for God,” by Leprosy Research Center at Karigiri. He es- Dorothy Clarke Wilson. He authored sev- tablished the New Life Center, Vellore, as a eral books that were best sellers among model rehabilitation center for leprosy pa- Christian literature. They are “Fearfully and tients. This Center simulated a village envi- Wonderfully made,” 1981, “In His Image,” ronment and was located at the residential 1984, and “Gift of Pain,” 1993. He also area of the Christian Medical College cam- wrote a standard reference book for hand pus, in an effort to dispel the stigma that was surgeons entitled “Clinical Mechanics of so prevalent even among medical profes- the Hand.” sionals. Correcting deformities to restore the Dr. Paul W. Brand, with all the honors he self-respect of patients and to integrate them received and with all his greatness, re- into society was his cherished goal. mained a simple and humble Christian lep- In 1966, after 19 years of service in India rosy worker. He was a man of integrity and he moved to the U.S.A. on invitation to take witnessed for his convictions forcefully and up the position of Chief of Rehabilitation effectively with gentleness and respect. He Branch at the National Hansen’s Disease exemplified in his life that excellence in Center at Carville. He worked there for 20 medicine was not just knowledge and skills years and established a well-equipped and but the application of them to serve and to well-staffed research unit to study the com- give one’s best to the cause of the poor, plications of insensitive hands and feet, needy and the neglected. His many contri- their prevention and management. His butions to the care of leprosy patients will methods for prevention and management of be long remembered. plantar ulcers are now extensively used for He leaves behind his loving wife, treatment of patients with diabetes mellitus Dr./Mrs. Margaret Brand, six children and who have similar problems. His contribu- twelve grandchildren. Mrs. Brand is also a tion to the understanding of pain is monu- leprologist who has received international mental. He emphasized the role of pain, acclaim in the study and management of which protects and preserves and is a bless- eye complications in leprosy. We, his stu- ing. When he retired in 1986 from the U.S. dents, friends, colleagues and patients, share Public Health Service, he moved to Seattle with the family the sadness and the loss. and continued his teaching work as emeri- Nonetheless, we celebrate his great contri- tus professor of Orthopedics in the Univer- butions and the privilege of having known sity of Washington. him, having had him as a teacher, friend, During his career, Dr. Brand received colleague, and caregiver. We thank and many awards and honors. He was awarded praise God for his wonderful and blessed the Hunterian professorship of the Royal life that has been a blessing to many. College of Surgeons in 1952, and the Albert —Dr. C. K. Job 71, 4 Obituaries 363 DR. PAUL WILSON BRAND 1914–2003
My first meeting with Paul Brand was at mities, where I was familiarized with the the Proctor House Mission in Bombay care of the ulcerated foot in leprosy. At this when I had gone to enquire of him the dan- meeting, Ernest Price also demonstrated his ger of contracting leprosy during surgery. interesting observations on the footprints of He assured me that this was a mere myth as leprosy patients. This meeting provided due he and his staff had kept a record of needle recognition to the pioneering work of Brand pricks during surgery for over a decade in this field. It also led to support from the with no untoward consequences. U.S. Department of Health, Education, and We next met when I advertized for a Welfare for several of our leprosy and burn physiotherapist for our hospital resulting in activities including surgery, research, and an interview visit by Furness, Brand’s se- rehabilitation. nior physiotherapist and the husband of This was followed by a series of ex- Brand’s secretary (also an old leprosy pa- changes between our institutions and my tient). This resulted in obtaining his personal interaction, not only with Paul and brother-in-law, Walter Jennings, as our Margaret, but aslo with the Karats, Selva- physiotherapist, who served our hospital for pandian and Anthony Samy at Katpadi—a almost two decades, a former leprosy pa- fruitful exchange. tient who had both his hands operated on by During a six week visit to our department Brand. The excellence of Brand’s surgery at the J. J. Hospital in Bombay, Dr. Robert not only permitted him to undertake excel- Cochrane provided interesting information lent physiotherapy, but also to keep typed as to how he had inveigled a new orthope- records of patients and their surgeries. As a dic surgeon of Vellore into undertaking the result of our discussions, he eventually per- correction of deformities of patients sent to formed “many tail” operations on paralyzed him from the Victoria Hospital in Chen- hands with excellent results, for he could gleput. Thanks to Paul, Vellore became a judge the tensions of the graft better than mecca of surgery for leprosy. Many of the most surgeons. surgeons from various countries of the Another outcome of Furness’ visit was world while visiting Vellore also visited us the visit of Ernest Fritschi to Pune in order in Bombay and Kondhwa. to observe our plastic surgery approach to The contributions of Cochrane and Brand deformities of the face in this disease, un- will always remain as landmarks in leprosy dertaken under primitive conditions with with Vellore as their “home.” patients as the only assistants. It is heartening for me to see that leprosy This led to several decades of interaction is one of the few diseases of poverty that between Dr. Brand, myself, and his col- has shown a definite decrease. This is also leagues in Vellore and Karigiri. Paul visited reflected in the decrease of its deformities, Kondhwa and observed Sir Harold Gillies even though eradication will remain a dis- undertaking surgery during his visit in 1958. tant dream till necessary poverty is ban- My first visit to Karigiri was in 1959, to ished by concerted political action at all attend the First International WHO confer- levels and in all countries. ence on Rehabilitation on Leprosy Defor- —Dr. N. H. Antia 364 International Journal of Leprosy 2003 DR. GOPAL RAMU 1924–2003
Dr. Ramu was an outstanding expert on all aspects of clinical leprosy and blended his keen observational instincts with good sci- ence. Together with the veteran leprologist Dr. K. Ramanujam and with the support of Dr. C. G. S. Iyer and Dr. K. V. Desikan, he studied several aspects of the clinical mani- festations of leprosy and its complications. Thus, the 1960’s saw some important contri- butions in leprosy from him through this group. He was also instrumental in develop- ing a rational method for scoring of clinical lesions in leprosy which greetly facilitated the follow-up of patients during clinical trials. When Dr. Ramu moved to CJIL at Agra in 1986, he saw an opportunity not only to build good clinical services but also to ex- Dr. Gopal Ramu, the well known leprolo- ploit important research opportunities that gist from India, passed away on 25 July had become available by then through de- 2003, at the age of 79. velopments in the serology of leprosy. This Dr. Ramu was born in Kerala and had his work very much complimented his earlier early medical education at Indore. His ini- interest in lepromin reaction among patients tial career started in the state of Holkar. and contacts. Even while working in general medicine, Of his several accomplishments, one of he showed a keen interest in leprosy. He the most important was his ability to moti- pursued his leprosy interest both in the field vate young workers who came in contact and in the clinic when he started working at with him. The environment and encourage- Rewa (M.P.). It was at this time that he was ment provided by him resulted in several involved in research on lathyrism under the individuals joining in leprosy research. Indian Council of Medical Research. His Wherever he was, he set a good example of association with Dr. C. G. S. Iyer during the diligence and perseverance. lathyrism research enabled him to look for a As an individual, Dr. Ramu was greatly better place to pursue his interst in leprosy, liked by everyone who interacted with him. and to join the Central Leprosy Teaching He was most helpful and generous, both to and Research Institute, Chengalpattu. his associates and patients. Deeply inter- Dr. Ramu was greatly instrumental in ested in music and religion, he spent the last building up the clinical research at two ma- part of his days with his daughter at Coim- jor leprosy institutions in India, the Central batore (Tamilnadu) continuing to provide Leprosy Teaching and Research Institute advice on leprosy research. (CLTRI), Chengalpattu (Tamilnadu) from The 1960’s saw a great deal of resurgence 1962Ð1976, and the Central JALMA Insti- in leprosy research in India and Dr. Ramu tute for Leprosy Research (CJIL), Agra from came onto the scene at the right time and 1976Ð1986. Besides these two major institu- made a great impact in better understanding tions, Dr. Ramu made significant contribu- the disease and its management. His mem- tions to leprosy research in his earlier days at ory will be cherished for a long time both by Rewa and in his post-retirement days at his fellow workers and patients. Kumbakonam and Coimbatore (Tamilnadu). —Dr. S. K. Nordeen INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. (ISSN 0148-916X) NEWS and NOTES This department furnishes information concerning institutions, organizations, and individuals engaged in work on leprosy and other mycobacterial diseases, and makes note of scientific meetings and other matters of interest.
Notice. Several extra copies of the old is- study of the treatment details given to pa- sues of The International Journal of Lep- tients either referred or institute cases, help- rosy are available from the business office. ful in deciding a rationale management. Due to a shortage of storage space, some of Clinically interesting cases (staying in these must be discarded soon. If you wish Kalyan area) with recurrent type II reaction to obtain any of these back issues of the put on Thalidomide were also demon- JOURNAL, please contact Dr. Paul Saunder- strated and discussed. The seminar was son by e-mail: [email protected]. sponsored by M/s Jansen’s Cilag Pharma- ceuticals Ltd.
Notice. The International Journal of Lep- rosy is now available on-line by visiting our The Editorial office received the follow- website at http://www.leprosy-ila.org./ This ing letter from the Leonard Wood Memorial. provides the most convenient access to the JOURNAL on-line. You can also renew your TO: Friends of the Leonard Wood membership, or join if you are not already a Memorial member of the ILA. The JOURNAL will ac- Subject: New Scientific Director cept submissions electronically, as well. I am most happy to inform you that effec- tive September 1, 2003, Dr. Robert Gelber will officially join the Leonard Wood Academic Meeting at Kalyan. The In- Memorial as its new Scientific Director. dian Association of Leprologists— Maha- As you may know, Dr. Gelber has been rashtra Branch in collaboration with Bom- working in leprosy, both as a clinician and bay Leprosy Project organized a seminar on researcher, particularly in the field of “Leprosy—from a Practicing Dermatolo- chemotherapy, for almost four decades. gists Point of View” on Sunday 22.06.2003 During this time, he has published well over at Kalyan. 100 articles and written major chapters in This seminar was organized for the mem- prestigious textbooks. He comes to us from bers of the “Kalyan-Dombivli Dermatolo- his position of Clinical Professor at the Uni- gists Club,” a newly formed local academic versity of California, San Francisco and also association. Senior attending physician of the TB control Issues such as the Role of standard Pred- program at San Francisco General Hospital. nisolone in management of reactions, Role We are delighted to have hired someone of newer drugs like Cyclosporin, Pentoxy- who is highly qualified, both in the field of phylline and other useful drugs like leprosy and tuberculosis. He is excited and Thalidomide availability to needy patients enthusiastic about this position and we look in managing chronic/recurrent reactions, as forward to a rich and rewarding association an alternative line of treatment were dis- with Dr. Gelber. cussed at length. Dr. R. Ganapati and Dr. V. Please join us in welcoming him. V. Pai were the Resource Persons. Discussion on methods of recording the Sincerely, past treatment details of patients in a “Treat- August Zinsser III ment Graph” experimented and prepared by President BLP was also demonstrated. The objective LWM Board of Trustees of such innovative exercise was a scientific
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ILA GLOBAL PROJECT ON THE and music. The idea is to develop a network HISTORY OF LEPROSY that is dedicated to making oral history, as ACADEMIC NETWORK MINUTES there are not enough resources for Anwei OF INAUGURAL MEETING, Law and her team to carry out all the SORIA MORIA CONFERENCE recording themselves. She pointed out that CENTER, OSLO anyone over the age of seventy is a “fragile Friday, 5 September 2003 resource,” so their identification and oral history will be a priority. Present: Jo Robertson (chair), Jaime Ben- 2. INDIVIDUAL RESEARCH AREAS chimol, Harriet Deacon, Deborah Emmitt, This item on the agenda was postponed, Mark Harrison, George Joseph, Sanjiv Kakar, but Jo Robertson pointed out that many Simonne Horwitz, Anwei Law, Laurinda Ma- members’ research interests are described ciel, John Manton, Renisa Mawani, Yara on the Academic Network page of the Proj- Monteiro, Chandi Nanda, Diana Obregón, ect website. Shubha Pandya, Biswamoy Pati, David Scol- lard, Magali Romero Sá 3. RESEARCH TOPICS OF INTEREST FROM THE POINT OF VIEW OF Apologies: Bernardino Fantini PEOPLE IN THE FIELD 1. THE PROJECT AND ITS INTERESTS David Scollard, as Editor of the Interna- tional Journal of Leprosy and Other My- Jo Robertson summarized the Project’s cobacterial Diseases, talked about the activities to date. The last funding period of submission of articles by members of the twenty months ended in May 2003, and the network. The journal is a bio-science pub- Project has now entered a bridging stage of lication, but has always accepted the occa- funding, provided by the Sasakawa Foun- sional article of historical interest. There is dation, until future funding of a further a commitment to support the history of three years is assured. leprosy, and submitted papers from social The main aims of the Project are, firstly, science disciplines will be reviewed by to build an online database of archives on appropriate social scientists. Readers of the leprosy, held in numerous locations journal, mainly leprosy doctors and re- throughout the world, and secondly, to es- searchers around the world, must see the ar- tablish and maintain a network of re- ticles as useful. David Scollard invited searchers who are working on different as- members of the leprosy history network to pects of the history of leprosy. This network submit articles of historic interest and to ask is expected to be self-perpetuating, that is, themselves how their submission is useful the members will maintain contact amongst in the field, how it could help medical themselves once the Project has made them workers, scientists, and others. aware of each other’s existence through its website and activities. 4. RESEARCH OPPORTUNITIES Mark Harrison explained how he intends Oral History to make the history of leprosy one of the Anwei Law explained the oral history main areas of research at the Wellcome component, which will begin once further Unit, Oxford, and will invite applications funding is assured. Oral history “makes his- for this. He will be putting in research pro- tory more rounded,” as it is related by the posals to the Grants Committee—at least people actually involved. When making one large one, or maybe two smaller ones. oral histories, it is important to include In addition, individual research applications families, and both younger and older gener- can also be made to the Wellcome Trust, ations as this establishes continuity. Guide- from academics from the European Eco- lines on making oral histories will be devel- nomic area. Wellcome collaborative grants oped, and other expressions produced by make available a relatively small amount of people who have been affected by leprosy money to develop specific research proj- will be identified, such as poetry, artwork ects, in order to facilitate travel between the 71, 4 News and Notes 367 two units for meetings and conferences, as an African University. It would be difficult well as the employment of researchers. to get funding but it is important to do so. Mark Harrison invited suggestions for col- Jo Robertson—If anyone knows of any laborative projects. Biswamoy Pati asked other academics in the leprosy history field, whether Ph.D. students could apply, Mark let her know and she will establish commu- replied that there was nothing in the rubric nication with them. against it, and that all the basic details con- Henk Menke—It may be useful to define cerning this can be found on the Wellcome one main historical problem as a group, and Unit website. see how the research of each country relates to it. 5. STRATEGIES FOR THE FUTURE Jo Robertson—This approach is impor- OF THE NETWORK tant, and could be fulfilled by the research Jo Robertson outlined three main strate- projects outlined by Mark Harrison. Also, gies. Firstly, an electronic discussion forum on the academic network web page, there is will be set up, the importance of which was information on members’ publications and made clear by the recent pre-conference ex- research interests. Academics need the free- change of emails, mainly concerning use of dom to go in the direction that their work terminology in historical articles. Secondly, leads them, and would prefer not to be pinned suggestions on papers to be submitted to his- down to one particular research area. tory of medicine conferences are welcome. George Joseph—Maybe three or four ar- Thirdly, possible collaborations among mem- eas could be established to begin with but bers of the network, which Jo Robertson left one would be too constraining. open for discussion. Jo Robertson—We could look at the cur- rent areas being researched by the network Discussion and compile a list of core issues. Diana Obregon—The electronic forum Jaime Benchimol—Agreed that it is too could be used to share bibliographic infor- early to identify specific research areas. mation as each academic is not necessarily What would be valuable is an appraisal of aware of other publications in the field. what has been done in the field, e.g., lep- Sanjiv Kakar—It will also be useful for rosy and public health. sharing information on conferences world- Sanjiv Kakar—Oral history is one domi- wide. nant theme that we already have. George Joseph—The submission of pa- Jo Robertson—The politics of oral his- pers to conferences is normally more pro- tory are being handled carefully in the pro- ductive when in panels, rather than sending posal for further funding, as there were individual papers, which are often difficult problems with this area previously. We are for organizers to place in the program. He is trying to incorporate it once more. in the early stages of planning a conference Harriet Deacon—Oral history is a for late April or early May in the United methodology, not an analytical approach. By States. The American Association for the pointing out that it is part of every history History of Medicine (AAHM) will meet in may be a convincing argument in its favor. Birmingham, Alabama, during the first Anwei Law—The problem arises from week of May and in conjunction with the not seeing oral history as a good source of leprosy conference most likely to be held in history due to a lack of understanding of its New Orleans, Louisiana, it may be possible use in different contexts. to arrange a visit to Carville at the same Harriet Deacon—Asked whether the Proj- time. George Joseph suggested the circula- ect has to clear all methodologies with WHO. tion of the papers prior to the conference to Jo Robertson—Now that the Project is allow a more advanced level of discussion. strong, we have received an email from John Manton—There is a problem with WHO that is virtually contractual, stating studying leprosy history in “tropical” Africa that the website content must be cleared by (i.e., Africa except South Africa). It would WHO. This issue will go to the Steering therefore be useful to underpin networks of Group to be debated. Jo Robertson listed Africa scholars and/or have a symposium in the members of the Steering Group, as not 368 International Journal of Leprosy 2003 all the network members were aware of INDEPENDENT EVALUATION OF whom it comprised. There is now a page on THE GLOBAL ALLIANCE FOR THE the website with this information. ELIMINATION OF LEPROSY (GAEL) Chandi Nanda—Asked whether it is pos- June 13, 2003 sible to identify some people who have been cured of leprosy, who can be brought in to the network. Jo Robertson—This is an academic net- Released by WHO 4 July 2003, the eval- work. Anwei Law will develop a network uation of the Global Alliance for the Elimi- of people to gather oral histories. nation of Leprosy, GAEL, was drafted by Henk Menke—The main group working an independent panel of six, led by Dr. for leprosy patients to date has been doctors Richard Skolnik. With the exception of Pro- and nurses. The historical method is rela- fessor Michel Lechat, the evaluators are not tively new. We need to make our work clear part of the leprosy community. They based to those in the field, not only through publi- the evaluation on a review of literature, cations but also meetings. If it is limited to communications, documents and approxi- historians, we may miss the important goal. mately 100 widely representational inter- David Scollard—The last ILA Congress views. in Salvador, Brazil, is an example of how historians and present day medical workers Richard Skolnik (Team Leader), Florent have already come together. The history Agueh, Judith Justice, Michel Lechat. symposia during this Congress were very The George Washington University, The successful. There are people from all fields University of Louvain, and The University at this regular, international conference, so of California at San Francisco. to have history also represented is very good. Hopefully we can find future ways in congresses to put forward particular histor- ical problems and issues. Jo Robertson—It was a very international ABSTRACT gathering, and people who had had the dis- This is an independent evaluation of the ease were also responding. Global Alliance for the Elimination of Lep- Harriet Deacon—One of the dangers of rosy. It assesses the extent to which the Al- classing us as a leprosy network limits the liance has contributed to the goal of elimi- focus to that disease. However, it is useful nating leprosy as a public health problem. to make comparisons with issues surround- This evaluation was based on a review of ing syphilis, AIDS and other diseases, to literature, documents, communications, and see how policies that develop around these almost 100 informant interviews. matters relate to leprosy. The evaluation team believes that the Al- Jaime Benchimol—Emphasis also needs liance has added important value to the goal to be given to national studies; tuberculosis of eliminating leprosy as a public health is an important comparison. problem. It has mobilized political commit- Anwei Law—This kind of study should ment, financial resources, and free drugs. It not be limited to diseases, but human rights has helped to improve the management and issues too. reach of multi-drug therapy. It has ener- Jo Robertson—Bernardino Fantini is de- gized a number of leprosy programs. Dur- veloping a human rights program and wants ing the course of the Alliance, 16 of 22 en- to include leprosy. demic countries have been deemed to have David Scollard—Expressed a desire to met the goal of elimination. publish the abstracts of the current confer- In addition, at the country level, the Al- ence in the International Journal of Lep- liance appears to be functioning well. Most rosy. Members were asked to make any fi- countries are actively leading and coordi- nal adjustments to their abstracts, and send nating their leprosy programs. Collabora- them to Jo Robertson as soon as possible. tion is good, with the World Health Organi- zation (WHO) playing an advisory role and non-government organizations (NGOs) in- 71, 4 News and Notes 369 volved in a range of leprosy efforts in con- organize their leadership around a country- junction with WHO and government. level leprosy task force. WHO should play Despite these important successes, the the advisory role to country programs, with Alliance is not adding the value that it could effective use of input from other collabora- add and this poses threats to country lep- tors. The WHO should also convene a rosy programs and to the reputations of col- group of technical advisors, selected with laborators on leprosy work. Relations the advice of others involved in leprosy, to among some collaborators at the global carry out independent monitoring and eval- level are very bad. Concerned NGOs, uation of leprosy activities. The Technical physicians, and scientists have raised im- Advisory Group (TAG) of WHO would portant questions to WHO about technical, have its membership strengthened, again operational, and strategic matters but they with the advice of others. have not been resolved. In addition, some It is also hoped that the Novartis Corpo- collaborators do not have a clear under- ration, working with the Novartis Founda- standing of the aims of the Alliance, or a tion for Sustainable Development, would clear agreement on how the Alliance should continue to provide drugs and that the be governed. There are also strong views Sasakawa Memorial Health Foundation and among some collaborators that the Alliance the Nippon Foundation would continue to is too embedded in WHO and that WHO support technical cooperation and research, has not been sufficiently consultative in its including through its important financial as- management of the Alliance. sistance. This is already mid-2003, and the target The above approach would carry on from date for elimination that was set by the the work done effectively to date and would World Health Assembly and extended by build on the comparative advantages of dif- the Alliance is very close. There will con- ferent actors engaged in leprosy efforts. It tinue to be significant numbers of leprosy would also build on the unique role and patients after the goal of elimination has commitment in leprosy work of NGOs. It been achieved. In addition, there will also would have clear and accountable roles for be needs at the global level for advocacy ef- all actors and would be inclusive. It would forts, funds for leprosy activities, and ex- also have to be based on open, transparent, changes of information and best practices and collegial relations, the lack of which among those working on leprosy. At the lo- would preclude any alliance from effec- cal level, all countries will need to lead tively supporting the important work on their leprosy programs in sound ways. If leprosy that will remain, even after 2005. these measures are not addressed effec- Finally, these arrangements would help pro- tively, some of the important gains on lep- vide a sound transition to further leprosy rosy will be lost. control and rehabilitation efforts. For these reasons, the panel believes that Obtained directly from the WHO web-site, the Alliance must be rebuilt and refined im- http://www.who.int/lep/GlobalAlliance/ mediately. Much of the global work of the evaluation.doc, at which the full report may Alliance would be convened and lead by be examined. the NGO and foundation movement. These activities would focus on ensuring effective advocacy, as needed, and promoting learn- ing and input into country programs on Notice. On 13Ð15 October 2003 a Work- technical, operational, and strategic issues. shop was held in Amsterdam on Leprosy They would build on earlier work by the In- Transmission and Diagnosis. During this ternational Association of Anti-Leprosy As- workshop it was decided to co-ordinate re- sociations (ILEP), the International Leprosy search activities in this field. Association (ILA), and the Sasakawa A consortium supported by the WHO/TDR Memorial Health Foundation. They would Special Program therefore now issues a include all who work with leprosy, includ- call for interest for partners to engage in ing the private sector and groups of people this comprehensive research program to affected by the disease. apply modern developments in the mo- If not already doing so, countries should lecular typing of M. leprae and specific 370 International Journal of Leprosy 2003 antigen/epitope definition to field studies If you are interested in participating, towards better understanding of the epi- please submit a letter of interest to the In- demiology and transmission of leprosy, terim Steering Committee of the consortium and the improved diagnosis of leprosy in- briefly stating the extent of your interest in fection. The purpose of this call is to re- these areas, your experience and your asso- cruit partners to participate in working ciation with leprosy field studies. A standard groups on: form and more information is available at: http://www.kit.nl/biomedical_research/htm/ Assays for molecular epidemiology leprosy_research_consortium.asp Immunology-based diagnostic assays Field studies related to transmission Dr. Linda Oskam Ph.D. (secretary of the and diagnosis Interim Steering Committee) Research Co-ordinator Mycobacteriology The purpose of the working groups is to KIT (Royal Tropical Institute) Biomedical (i) raise funds to advance the necessary ba- Research sic and operational research; and (ii) set Meibergdreef 39, 1105 AZ Amsterdam, policies, proposals, protocols, under the The Netherlands umbrella of the consortium. http://www.kit.nl/biomedical_research/
Calendar of Meetings and Events Day mm/yy Location Details Contact e-mail 9Ð13 12/3/2003 Valencia ILEP Working Session and ILEP Secretariat [email protected],uk General Assembly 19Ð22 12/3/2003 Chattisgarh National Conference on Dr. S.K. Noordeen vinodkoomar@ Elimination of Leprosy rediffmail.com
AMERICAN SOCIETY FOR MICROBIOLOGY DIVISION U SYMPOSIUM Washington Covention Center Washington D.C. 18–22 May 2003 The 103rd General Meeting of the Ameri- ber of Division U whose outstanding can Society for Microbiology was held at achievements have contributed to advances the Washington Convention Center in Wash- in mycobacteriology. ington D.C., U.S.A., on May 18Ð22, 2003. The conveners of the symposium were At this meeting, Division U (Mycobacteriol- Drs. James L. Krahenbuhl and Diana L. ogy) sponsored a symposium entitled, “Ad- Williams, both of the National Hansen’s vances in Leprosy Research 2003 and Be- Disease Programs Laboratory, Baton yond: Following in Shepard’s FootPads.” Rouge, LA, U.S.A. After a brief introduc- This symposium featured a distinguished tion by Dr. Krahenbuhl, the symposium list of speakers covering a range of topics commenced with this year’s Division U lec- which emphasized the rapid gains in knowl- turer, Dr. Warwick J. Britton from the Uni- edge since the sequencing of the Mycobac- versity of Sydney, Sydney, Australia, who terium leprae genome. This symposium presented an overview of his research on also featured the prestigious Division U lec- leprosy and tuberculosis vaccines. His lec- ture, which is awarded each year to a mem- ture was followed by a presentation by Dr. 71, 4 News and Notes 371
Williams on her work in defining a partial that past BCG immunization, as indicated M. leprae transcriptome. Dr. Patrick J. Bren- by the presence of BCG a scar, was associ- nan, Colorado State University, Fort Collins, ated with approximately 50% reduction in Colorado, U.S.A., presented his work on the clinical leprosy (range 20 to 81%). The im- genes encoding M. leprae cell wall compo- pact of widespread BCG implementation on nents. Last, Dr. Thomas P. Gillis, National leprosy control is difficult to quantitate, Hansen’s Disease Programs Laboratory, dis- however, it is probable that BCG is one fac- cussed his research on the use of bioinfor- tor which has contributed to the decline in matics to search for potential vaccine and leprosy in some countries. The recent South skin test antigen candidates. India vaccine trial provided further evi- This informative and timely symposium dence of the effectiveness of vaccines was attended by over 350 meeting partici- against clinical leprosy (2). In this study the pants and each presentation stimulated addition of heat-killed M. leprae to BCG thoughtful discussion. In an effort to share resulted in improvement in the vaccine effi- the proceedings of this symposium with the cacy from 34 to 61%. This is contrast to readership of the International Journal of earlier studies in Malawi and Venezuela, Leprosy who were unable to attend the which showed no benefit from the addition meeting, the four lecturers have here pro- of heat-killed M. leprae to BCG (1). An ad- vided a overview of their presentations. ditional finding was that the cultivatable —Linda Adams bacillus ICRC, probably a member of the Chair, Division U (Mycobacteria) M. avium-intracellulare family, also con- American Society for Microbiology ferred significant protection (VE, 65%) when given as a dead mycobacterium. This provides further evidence that immuniza- ABSTRACTS tion with heterologous mycobacteria pro- Britton, W. J. On the vaccine trail: from tects against M. leprae. Leprosy to Tuberculosis. Subunit vaccines against leprosy. The potential of subunit vaccines against lep- The control of leprosy has improved rosy was raised by the early studies of Gel- markedly since the introduction of multi- ber and Brennan, which showed that crude drug therapy with dramatic falls in the preva- M. leprae cell wall fractions and native cell lence of leprosy patients receiving anti- wall-derived proteins protected against M. microbial therapy during the 1990’s. Despite leprae footpad infection in mice. More re- this widespread implementation of MDT, cently, Ngamying and colleagues showed the incidence of leprosy as measured by case that M. leprae cytosol and membrane frac- detection rate has not yet fallen in major en- tions protected against mouse footpad in- demic countries. This justifies continuing re- fection, but the cell wall skeleton of search to understand the transmission, host mycolyl-AG peptidoglycan from M. leprae response in protective immunity against My- was not protective (3). Therefore, protein cobacterium leprae. Immunization to im- components are essential for effective sub- prove host response against M. leprae infec- unit vaccines. The trail to determine which tion will also be an important component in M. leprae protein antigens induce effective the long term control of leprosy. immunity dates back to studies with mono- Current leprosy vaccines. The anti- clonal antibody-defined proteins in the tuberculosis vaccine, Mycobacterium bovis 1980’s. These included the M. leprae bacille Calmette-guerin (BCG) has partial GroEs, GroEl and 70 kDa heat shock pro- efficacy against clinical leprosy. In four teins, widely shared with other mycobacte- randomized clinical trials, BCG stimulated ria, and the M. leprae 18 kD protein, which a degree of vaccine efficacy (VE) ranging contains M. leprae-specific T cell epitopes from 34% in India to 80% in Uganda. The but has a homologue in M. avium. Subse- prospective vaccine study in Malawi quently, native proteins were purified from demonstrated a 52% VE for BCG, with a M. leprae, including the cytoplasmic 10 further 50% reduction in clinical leprosy kDa GroEs protein and the membrane- following repeat BCG immunization (1). In associated proteins MMPI and MMPII. addition, 9 case control studies have shown Fractions of M. leprae containing GroEs 372 International Journal of Leprosy 2003 and MMPI stimulated some protective ef- mouse model, single protein or DNA vac- fect in the mouse footpad model. More re- cines against tuberculosis infection have cently, a bioinformatics approach has been generally been less effective than BCG in employed to define M. leprae homologues mice (9). Therefore, we and others have of M. tuberculosis antigens known to in- been examining ways of increasing the pro- duce protective immunity against tubercu- tective efficacy of subunit vaccines against losis, such as the secreted proteins, Antigen mycobacterial infections. A number of cy- 85B and ESAT-6. tokines were compared as adjuvants for Our own group has focused on the M. DNA immunization and plasmid IL-12 was leprae 35 kDa (MMPI) as a subunit vaccine the most effective. Co-immunization with against leprosy. This protein was first rec- DNA-85B and plasmid IL-12 resulted in a ognized by M. leprae-specific monoclonal rise in IFN-γ and T cell responses, a fall in antibodies to conformational determinants specific antibody responses with increased on the protein, which are also the dominant protection against M. tuberculosis infection epitopes for human leprosy sera. We cloned (10). Co-immunization with DNA-35 and the gene for the M. leprae 35 kDa protein plasmid IL-12 produced significantly (4) and found it had no homologues in M. greater protection against virulent M. avium tuberculosis or BCG, but one was present in infection than BCG (11). IL-12 was more ef- M. avium with 94% amino acid identity (5). fective than IL-18, another Th1-promoting Recombinant 35 kDa protein expressed in cytokine, at improving DNA vaccine effi- the rapidly growing M. smegmatis forms cacy against mycobacterial infections (12). highly immunogenic multimers of >900 We have also examined the interactions kDa. This protein is recognized across the of subunit vaccines and BCG against M. tu- leprosy spectrum, so that paucibacillary berculosis infection. Priming with a DNA leprosy patients and contacts of leprosy pa- vaccine expressing the M. tuberculosis anti- tients develop a strong T cell response with gen 85B, followed by boosting with BCG, low levels of antibody and multi-bacillary significantly improved the effective efficacy patients demonstrate a strong antibody re- against M. tuberculosis to a level greater sponse and weak T cell response (4). Fur- than that achieved with BCG alone (10). This ther, the protein elicits delayed type hyper- may be due to focusing of the immune re- sensitivity (DTH) in M. leprae sensitized sponse against a dominant secreted antigen guinea pigs. of M. tuberculosis. These findings demon- DNA vaccines expressing the M. leprae strate that protection against mycobacterial or M. avium 35 kDa proteins induced pro- infections in experimental models is not tective immunity against M. leprae and M. limited to that achieved with BCG alone. avium infection in mice, which was equiva- Challenges for new anti-leprosy vac- lent to BCG in both cases (6, 7). This was ac- cines. Although considerable progress has companied by strong specific Interferon been made, there remain uncertainties in (IFN)-γ T cell responses, as well as high the understanding the complex host im- titre antibody responses to the conforma- munological response to mycobacteria and tional determinant on the protein. This es- this influences the development of more ef- tablishes that immunization with a single fective anti-leprosy and anti-tuberculosis antigen can be effective against experimen- vaccines. First, the factors which determine tal leprosy infection. Major antigens shared the immunological dominance of antigens with M. tuberculosis and BCG may also are still not resolved. Factors which may af- induce protection when used as a subunit fect this include the quantity of mycobacte- vaccines. For example, immunization with rial protein and the timing of exposure. For the M. tuberculosis antigen 85B as a DNA example, the GroES protein is the major cy- vaccine induced heterologous protection toplasmic protein in armadillo-derived M. against M. leprae footpad infection in leprae, and is a dominant antigen in host re- Swiss albino mice. (8). sponse to M. leprae (13). Secreted proteins, Improving subunit vaccines against including antigen A85 complex, may be the mycobacterial infections. Although sub- first antigens encountered and appear to unit vaccines against leprosy infection stimulate protective immunity against a show an equivalent protection to BCG in a number of mycobacteria (8, 10). There may 71, 4 News and Notes 373 be intrinsic properties to certain proteins, progress. In addition, it is important to con- such as the multimeric form of the 35 kDa firm that the vaccine antigens induce protec- M. leprae protein, which contribute to their tive immunity in mice with differing genetic persistence in the phago-lysosome and so backgrounds to confirm their applicability in their antigenicity. human populations. Second, the importance of species speci- The third and most important challenge ficity in determining the protective efficacy for introduction of new anti-leprosy vac- of individual proteins is unclear. In fact, cines is the capacity to test these in endemic species-specific proteins may not be the op- regions. Will it be possible to conduct an- timal vaccine candidates, although they other major human leprosy vaccine trial on have obvious importance as a diagnostic the scale of the recent Indian vaccine study? reagents. Recent studies by Black and col- This would require sufficient number of leagues in Malawi (14) have demonstrated new cases and the extensive infrastructure apparent cross-reactivity between the major required for such a study. Another approach antigens of M. leprae, including the 35kDa would be to include leprosy in future tuber- and 18kDa proteins, and environmental culosis vaccine trials. A number of candi- mycobacterial species isolated in Malawi. date TB vaccines are moving into Phase I These antigens may stimulate pre-existing and Phase II clinical trials. If these are to be T cell responses in infected subjects in of used in leprosy endemic countries, it is be endemic regions and this may blunt the ap- important that they have an effective anti- parent effectiveness of mycobacterial vac- leprosy component, either because the anti- cines in that environment. gens are shared between M. tuberculosis and Third, the relative contribution of different M. leprae, or M. leprae-related components T cell subsets to protective immunity may are added to the vaccine. This will require vary between different species of mycobac- the design and conduct of the vaccine trials teria. Both CD4 and CD8 T cells appear to to measure the effects on leprosy as well as contribute to effective immunity against M. tuberculosis in leprosy-endemic regions. tuberculosis, although the exact role of CD8 Acknowledgement. Laboratory research has been T cells inducing protection against M. leprae funded by the National Health and Medical Research infection has not been established. Council of Australia, the World Health Organization, Fourth, understanding the factors control- the Co-operative Research Center for Vaccine Tech- ling of T cell memory, particularly in CD4 T nology and the New South Wales Department of cells is incomplete and this has major impli- Health. I thank E. Martin, J. Triccas, U. Palendira and cations for subunit vaccines against myco- A. Kamath in Sydney and Dr. P. Roche, Anandaban bacterial infections. Although protein and Leprosy Hospital, Kathmandu, Nepal, for their collab- DNA subunit vaccines can stimulate short oration and discussions. I am grateful to Dr. J. Vele- term protective immunity against tubercu- mer, Leprosy Mission International for his analysis of losis and leprosy in animal models, their the effectiveness of BCG vaccines. ability to stimulate long term protection is REFERENCES yet to be determined. These subunit vac- 1. BLACK, G. F., WEIR, R. E., and CHAGULUKA, S. D., cines may prove most useful in boosting et al. Gamma interferon responses induced by a immunity established by BCG or other vi- panel of recombinant and purified mycobacterial able vaccines. antigens in healthy, non-Mycobacterium bovis The second major challenge for new anti- BCG-vaccinated Malawian young adults. Clin. leprosy vaccines are limitations of the mod- Diagn. Lab. Immunol. 10 (2003) 602Ð611. els for testing protective efficacy. The dy- 2. BRITTON, W. J., and PALENDIRA, U. Improving namic range of the mouse footpad infection vaccines against tuberculosis. Immunol. Cell Biol. model is low, and it is difficult to measure in- 81 (2003) 34Ð45. creased effective efficacy above that achieved 3. GUPTE, M. D., VALLISHAYEE, R. S., and ANANTHARA- with BCG. The lack of immunological MAN, D. S., et al. Comparative leprosy vaccine trial 70 reagents and complexibility of the armadillo in south India. Indian J. Lepr. (1998) 369Ð388. 4. KARONGA PREVENTION TRIAL GROUP.Random- model mean that it is not currently applica- ized controlled trial of single BCG, repeated BCG, ble to testing vaccines. Further, the length or combined BCG and killed Mycobacterium lep- of time for testing individual vaccines, cur- rae vaccine for prevention of leprosy and tubercu- rently 9 to 12 months, restricts the rate of losis in Malawi. Lancet 348 (1996) 17Ð24. 374 International Journal of Leprosy 2003
5. MARTIN, E., KAMATH, A. T., BRISCOE, H., and The genome of Mycobacterium leprae BRITTON, W. J. The combination of plasmid has been completely sequenced and anno- interleukin-12 with a single DNA vaccine is more tated. Approximately, 1604 open reading effective than Mycobacterium bovis (BCG) in pro- frames, encoding potentially functional pro- tecting against systemic Mycobacterim avium in- fection. Immunology 109 (2003) 308Ð314. teins, and 1104 inactivated genes (pseudo- 6. MARTIN, E., KAMATH, A. T., TRICCAS, J. A., and genes) have been identified. However, the BRITTON, W. J. Protection against virulent Myco- minimum gene set required for intracellular bacterium avium infection following DNA vacci- growth and survival (transcriptome) has not nation with the 35 kDa antigen is accompanied by yet been defined. To address this, we have induction of IFN-γ secreting CD4+ T cells. Infect. initiated studies to determine the potential Immun. 68 (2000) 3090Ð3096. transcriptome using RT-PCR and cross- 7. MARTIN, E., ROCHE, P. W., TRICCAS, J. A., and species DNA microarray analysis using a BRITTON, W. J. DNA encoding a single mycobac- comprehensive M. tuberculosis array using terial antigen protects against leprosy infection. a commercially available oligonucleotide Vaccine 19 (2001) 1391Ð1396. set (Operon Technologies, Alameda, CA) as 8. MEHRA, V., BLOOM, B. R., and BAJARDI, A. C., et al.Amajor T cell antigen of Mycobacterium lep- a prelude to evaluating global gene ex- rae is a 10-kD heat-shock cognate protein. J. Exp. pression using an M. leprae cDNA array, Med. 175 (1992) 275Ð284. which is not currently available. For RT- 9. NGAMYING, M., SAWANPANYALERT, P., BUTRAPORN, PCR, RNA was obtained from two R., NIKASRI, J., CHO, S. N., LEVY, L., and BREN- geographically distinct strains of M. leprae NAN, P. J. Effect of vaccination with refined com- and cDNA was produced by reverse- ponents of the organism on infection of mice with transcription using random priming. Gene Mycobacterium leprae. Infect. Immun. 71 (2003) transcripts of interest were amplified from 1596Ð1598. cDNA using PCR with primer sets flanking 10. PALENDIRA, U., KAMATH, A. T., FENG, C. G., and gene fragments of several potentially func- BRITTON, W. J. Co-expression of Interleukin-12 chains by a self splicing vector increases the pro- tional families of M. leprae. PCRs were tective cellular immune response of DNA and initially characterized using DNA from M. BCG vaccines against Mycobacterium tuberculo- leprae T-53 resultant PCR fragments were sis. Infect. Immun. 70 (2002) 1949Ð1956. analyzed by gel electrophoresis. Cross- 11. ROCHE, P. W., NEUPANE, K. D., FAILBUS, S. S., KA- species DNA microarray analysis was ac- MATH, A. T., and BRITTON, W. J. Vaccination with complished using 5 ug total RNA from DNA of the Mycobacterium tuberculosis 85B anti- T-53 and 4089 and labeled with either Cy3 gen protects the mouse footpad against infection or Cy5 fluorochromes using RT. The la- with Mycobacterium leprae. Int. J. Lepr. Other beled cDNAs will be hybridized to the Mycobact. Dis. 69 (2001) 93Ð98. slides, the slides will be washed and 12. TRICCAS, J. A., ROCHE, P. W., WINTER, N., FENG, C. G., BUTLIN, C. R., and BRITTON, W. J. A 35 scanned using an Axon Scanner. The inten- kilodalton protein is a major target of the human sities of the two dyes at each spot will be immune response to Mycobacterium leprae. In- quantified using the GenePix software fect. Immun. 64 (1996) 5171Ð5177. package. Results of RT-PCR and cross- 13. TRICCAS, J. A., SUN, L., PALENDIRA, U., and BRIT- species microarray experiments demon- TON, W. J. Comparative affects of plasmid- strated that genes encoding a variety of en- encoded interleukin 12 and interleukin 18 on the zymes were transcribed in both strains. protective efficacy of DNA vaccination against These include enzymes involved with folic Mycobacterium tuberculosis. Immunol. Cell Biol. acid synthesis, iron utilization, cofactor 80 (2002) 346Ð350. biosynthesis, gluconeogenesis, glycolysis, 14. TRICCAS, J. A., WINTER, N., ROCHE, P. W., GILPIN, A., KENDRICK, K. E., and BRITTON, W. J. Molec- glyoxylate bypass, those associated with ular and immunological analyses of the Mycobac- beta oxidation of fatty acids, degradation terium avium homolog of the immunodominant of phosphorous compounds, degradation of Mycobacterium leprae 35-kilodalton protein. In- DNA, detoxification and virulence associ- fect. Immun. 66 (1998) 2684Ð2690. ated proteins, synthesis of mycolic acids, modification and maturation of ribosomes, synthesis of RNA, stress proteins, proteins Williams, D. L. Establishing the Transcrip- of the SecA-dependent secretion pathway tome of Mycobacterium leprae: A Mini- and 25 proteins containing secretion motifs mal Gene Set for Survival. or, and several proteins with unknown 71, 4 News and Notes 375 functions. These data have provided us murD, murX, murF, murE) and other related with the first insight into the transcriptome genes (e.g., murA, murB, ponA1, ponA2). of M. leprae and further demonstrated the Polyprenyl-phosphates are the membrane homogeneity of this species. It is antici- carrier lipids for many aspects of cell wall pated that this analysis will help to identify synthesis, such as arabinose, arabino- a larger set of functional genes in M. leprae galactan, peptidoglycan, and, as expected, which will potentially help us to under- M. leprae contains the majority of genes stand the minimal requirements for growth encoding enzymes of the non-mevalonate and replication of this pathogen. This infor- pathway for polyprenyl-phosphate synthesis mation may lead to the identification of (e.g., dxsI and ispCÐG). It is devoid of the new drug targets, skin test antigens, and to dxsII of M. tuberculosis, which helped in identify factors that allow this pathogen to deciding that dxsI is the functional gene for evade the immune system and destroy pe- deoxylulose-5-phosphate synthase. The en- ripheral nerves. tire array of genes required for rhamnose synthesis (rmlB, rmlC, rmlD, rmlA) are present in the M. leprae genome, as Patrick J. Brennan and Varalakshmi D. expected, since rhamnose is a component of Vissa. Maintenance of Genes for Myco- the key diglycosyl-phosphoryl unit joining bacterium leprae Cell Wall Synthesis. the mycolyl-arabinogalactan complex to peptidoglycan. Most of the known genes Sequencing of the Mycobacterium leprae responsible for the synthesis of mannose, the genome by S.T. Cole, et al. (http://www. PIMs (phosphatidylinositol mannosides), nature.com/nature/v409/n6823/fig_tab/ LM (lipomannan), and LAM (lipoarabino- 4091007aO_F1.html) was a momentous mannan), are present in M. leprae, such as event, comparable to the introduction of pmmA, the Rv3256c homolog, pmi, manB, MDT (multiple drug therapy) in the 1980’s. the Rv2609c homolog, pimA, Rv2611c Initial analysis indicated a genome size of homolog, and pgsA. However pimB is about 3.3 megabases, a G/C content of apparently missing, which requires new 57.8%, only 1604 protein genes, 1116 thinking on the mechanism of synthesis of pseudogenes, and hence a protein coding PIMs/LM/LAM; we do know from chem- capacity of 49.5% (these latter figures are ical analysis that all three types of products to be compared to a size of about 4.4 are present in M. leprae. Likewise, most of megabases for the Mycobacterium tuberc- the genes for mycolic acid synthesis, ulosis H37Rv genome, a G/C content of modification, and deposition are present in 65.6%, 3959 protein genes, only about 6 the M. leprae genome, such as fasI, fabD, pseudogenes, and thus a protein coding acpM, kasA, kasB, accD6, mabA, inhA, capacity of 90.8%). (These data are being umaA2, mmaA4, mmaA1, fbpA, fbpB, fbpC, constantly revised in light of more recent and fbpC2. However, as noted initially by and ongoing analysis of bacterial genomes.) Cole, et al., umaA1, mmaA3, and mmaA2 Thus, the M. leprae genome has undergone are missing as whole genes, and this absence reductive evolution, becoming trapped and is exactly in accord with the absence of crippled. In light of such a paucity of methoxymycolates in M. leprae as reported protein coding genes, it is worthwhile to by several workers in the 1980’s. examine the cell wall of M. leprae from The pks (polyketide synthase)-like genes both the perspectives of known biochemical of Mycobacterium tuberculosis responsible information and in silico analysis. This was for the synthesis of the phthiocerol, the purpose of this review. phenolphthiocerol, and methyl branched For instance, we have known from the fatty acids of DIM (dimycocerosyl phthio- early chemical analysis of the cell wall cerol) and the phenolic glycolipids (PGL) peptidoglycan of M. leprae by P. Draper, et are receiving considerable current atten- al., that this essential component of all tion, since these products have been eubacteria is intact and comparable to that implicated in disease processes. In the case of M. tuberculosis and other bacteria. of M. tuberculosis, it has been dem- Indeed, M. leprae apparently retains the full onstrated that disruption of the pks10 and mur operon (ftsZ, ftsQ, murC, murG, ftsW, pks7 genes which are clustered with pks8, 376 International Journal of Leprosy 2003 pks17, pks9 and pks11 resulted in mutants missing from M. leprae. Likewise, a cluster deficient in the synthesis of DIM. Simi- of at least 8 glycosyltransferases in the larly, the pks12 gene has also been impli- Rv1500 to Rv1526 region of the M. cated in synthesis of DIM. However, a tuberculosis genome is missing from M. careful analysis of the M. leprae genome leprae. This evidence supports the chemical indicates that pks10, pks7 and pks12 are evidence for “stunted” or “truncated” pseudogenes; and pks 8, 17, 9, and 11 are versions of some polysaccharides, such as absent. LAM, in M. leprae. An analysis of the intact pks genes of M. Thus, an analysis of the genome of M. leprae from a different perspective, i.e., leprae versus those of M. tuberculosis, M. from the perspective of identification of the bovis, and other Mycobacterium spp. genes responsible for the synthesis of PGL-I, supports the chemical analytical evidence yields interesting information. The entire of an intact but minimal cell wall in M. array of genes attributed to the synthesis of leprae. phthiocerol (ppsAÐE) is located between ML2357 and ML2353. At a different Some relevant publications: location (ML0139) is the mas gene responsible for mycocerosic acid synthesis. Vissa, V.D., and P.J. Brennan. Impact of the The pks1 and pks15 of M. tuberculosis Mycobacterium leprae genome sequence on genes are fused as one gene in the M. bovis leprosy research. In: Genomics of GC Rich Gram- genome, which has been demonstrated to Positive Bacteria, Wymondham, U.K.: Caister be involved in the synthesis of phenol- Academic Press, 2002. pp. 85-Ð118. Crick, D.C., P.J. Brennan, and M.R. McNeil. The cell phthiocerol, most likely a precursor of the wall of Mycobacterium tuberculosis. In: Tuberc- M. bovis specific PGL. The M. leprae ulosis. Second Edition Philadelphia: Lippincott genome also contains the fused gene in Williams & Wilkins, 2004. pp. 115Ð134. concordance with the elaboration of its phenolic glycolipids. Elongation occurs with the ppsAÐE cluster. Associated with Gillis, T. The Use of Bioinformatics in Lep- ppsAÐE are the genes fadD26 and drrAÐC rosy Research. implicated in the attachment of myco- cerosic acid and phthiocerol followed by Bioinformatics encompasses all aspects transport through the membrane. Putative of biological information acquisition and glycosyltransferases and methyltransferases analysis, and combines the tools of com- possibly involved in the synthesis of the puter science and biology with the aim of trisaccharide segment of PGL-I can be understanding biological significance. The located in one cluster (ML0125-ML0130). combination of enhanced sequencing and We have previously commented on the computing power has allowed for unprece- “cell wall gene cluster” of M. leprae dented advances in assimilating huge characterized by the presence of embAÐC amounts of raw data and the initiation of implicated in arabinan synthesis, the fbp meaningful molecular modeling. Bioinfor- genes responsible for mycolic acid matic approaches are particularly attractive deposition, and glf and glfT involved in for aiding studies of M. leprae since many D-galactofuranose and D-galactan synthe- conventional biological tools are unavail- sis. A detailed comparison of gene arrange- able to investigators working with noncul- ments in this cluster in the M. leprae and M. turable agents. tuberculosis genomes demonstrates the Three major areas of bioinformatics (ge- absence of several genes (Rv3784-3788) from nomics, proteomics, and transcriptional the M. leprae genome, one of which is a profiling) have had and should continue to putative glycosyltransferase (Rv3786c). have an impact on our understanding of M. Additionally, a dedicated analysis of puta- leprae and the disease it causes. Genomics tive glycosyltransferases over the entire has provided a working genetic blueprint genome of M. leprae versus M. tuberculosis for M. leprae allowing for comparisons shows that several orthologs (e.g., Rv1212c, with other mycobacterial genomes to assess Rv0539, and Rv2957) representative of the M. leprae’s basic physiological capabilities, glycosyltransferase families 1 and 2 are potential virulence factors and establish 71, 4 News and Notes 377
molecular markers for drug resistance and sponses during infection. In addition, newly strain variation. Newly developed strain discovered proteins can be tested for their identification markers using simple repeated ability to induce protective immunity and DNA sequences should provide tools to in- may constitute a new group of proteins with vestigate transmission patterns of leprosy vaccine or diagnostic potential. Transcrip- and may help define risk factors involved in tional profiling may allow investigators to reinfection versus relapse of disease. Algo- study M. leprae’s gene expression at differ- rithms for predicting open reading frames ent stages of growth as well. For example, and for categorizing location and function of gene expression in growth-permissive cells, M. leprae proteins have initiated basic stud- such as the macrophage and Schwann cell ies on secreted proteins as well as proteins could differ and, therefore, may reveal as- involved in nerve invasion and M. leprae- pects of M. leprae’s unique tissue tropism. specific proteins potentially useful for de- Functional genomics is the integration of tecting exposure to the leprosy bacillus predictive bioinformatics with validation through skin testing or serological testing. through experimental biological analysis. Proteomic studies of the leprosy bacillus This part of the equation remains a major have been hampered by low protein yields challenge to workers in the leprosy field. from purified bacilli derived from infected Surrogate genetics to study M. leprae genes animals, but have established a baseline in cultivable mycobacteria as well as new profile of highly expressed proteins from approaches for “knocking in” genes to M. M. leprae. Combining transcription profil- leprae are areas in need of research and de- ing of the leprosy bacillus with proteomic velopment. Both approaches will benefit studies may provide new insights into pro- from bioinformatics and should continue to teins previously lost during purification further our understanding of the leprosy from infected host tissues and provide new bacillus in particular, and the host-parasite antigens useful for studying immune re- relationship in general. INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. (ISSN 0148-916X) NEWS and NOTES This department furnishes information concerning institutions, organizations, and individuals engaged in work on leprosy and other mycobacterial diseases, and makes note of scientific meetings and other matters of interest.
Notice. Several extra copies of the old is- study of the treatment details given to pa- sues of The International Journal of Lep- tients either referred or institute cases, help- rosy are available from the business office. ful in deciding a rationale management. Due to a shortage of storage space, some of Clinically interesting cases (staying in these must be discarded soon. If you wish Kalyan area) with recurrent type II reaction to obtain any of these back issues of the put on Thalidomide were also demon- JOURNAL, please contact Dr. Paul Saunder- strated and discussed. The seminar was son by e-mail: [email protected]. sponsored by M/s Jansen’s Cilag Pharma- ceuticals Ltd.
Notice. The International Journal of Lep- rosy is now available on-line by visiting our The Editorial office received the follow- website at http://www.leprosy-ila.org./ This ing letter from the Leonard Wood Memorial. provides the most convenient access to the JOURNAL on-line. You can also renew your TO: Friends of the Leonard Wood membership, or join if you are not already a Memorial member of the ILA. The JOURNAL will ac- Subject: New Scientific Director cept submissions electronically, as well. I am most happy to inform you that effec- tive September 1, 2003, Dr. Robert Gelber will officially join the Leonard Wood Academic Meeting at Kalyan. The In- Memorial as its new Scientific Director. dian Association of Leprologists— Maha- As you may know, Dr. Gelber has been rashtra Branch in collaboration with Bom- working in leprosy, both as a clinician and bay Leprosy Project organized a seminar on researcher, particularly in the field of “Leprosy—from a Practicing Dermatolo- chemotherapy, for almost four decades. gists Point of View” on Sunday 22.06.2003 During this time, he has published well over at Kalyan. 100 articles and written major chapters in This seminar was organized for the mem- prestigious textbooks. He comes to us from bers of the “Kalyan-Dombivli Dermatolo- his position of Clinical Professor at the Uni- gists Club,” a newly formed local academic versity of California, San Francisco and also association. Senior attending physician of the TB control Issues such as the Role of standard Pred- program at San Francisco General Hospital. nisolone in management of reactions, Role We are delighted to have hired someone of newer drugs like Cyclosporin, Pentoxy- who is highly qualified, both in the field of phylline and other useful drugs like leprosy and tuberculosis. He is excited and Thalidomide availability to needy patients enthusiastic about this position and we look in managing chronic/recurrent reactions, as forward to a rich and rewarding association an alternative line of treatment were dis- with Dr. Gelber. cussed at length. Dr. R. Ganapati and Dr. V. Please join us in welcoming him. V. Pai were the Resource Persons. Discussion on methods of recording the Sincerely, past treatment details of patients in a “Treat- August Zinsser III ment Graph” experimented and prepared by President BLP was also demonstrated. The objective LWM Board of Trustees of such innovative exercise was a scientific
365 366 International Journal of Leprosy 2003
ILA GLOBAL PROJECT ON THE and music. The idea is to develop a network HISTORY OF LEPROSY that is dedicated to making oral history, as ACADEMIC NETWORK MINUTES there are not enough resources for Anwei OF INAUGURAL MEETING, Law and her team to carry out all the SORIA MORIA CONFERENCE recording themselves. She pointed out that CENTER, OSLO anyone over the age of seventy is a “fragile Friday, 5 September 2003 resource,” so their identification and oral history will be a priority. Present: Jo Robertson (chair), Jaime Ben- 2. INDIVIDUAL RESEARCH AREAS chimol, Harriet Deacon, Deborah Emmitt, This item on the agenda was postponed, Mark Harrison, George Joseph, Sanjiv Kakar, but Jo Robertson pointed out that many Simonne Horwitz, Anwei Law, Laurinda Ma- members’ research interests are described ciel, John Manton, Renisa Mawani, Yara on the Academic Network page of the Proj- Monteiro, Chandi Nanda, Diana Obregón, ect website. Shubha Pandya, Biswamoy Pati, David Scol- lard, Magali Romero Sá 3. RESEARCH TOPICS OF INTEREST FROM THE POINT OF VIEW OF Apologies: Bernardino Fantini PEOPLE IN THE FIELD 1. THE PROJECT AND ITS INTERESTS David Scollard, as Editor of the Interna- tional Journal of Leprosy and Other My- Jo Robertson summarized the Project’s cobacterial Diseases, talked about the activities to date. The last funding period of submission of articles by members of the twenty months ended in May 2003, and the network. The journal is a bio-science pub- Project has now entered a bridging stage of lication, but has always accepted the occa- funding, provided by the Sasakawa Foun- sional article of historical interest. There is dation, until future funding of a further a commitment to support the history of three years is assured. leprosy, and submitted papers from social The main aims of the Project are, firstly, science disciplines will be reviewed by to build an online database of archives on appropriate social scientists. Readers of the leprosy, held in numerous locations journal, mainly leprosy doctors and re- throughout the world, and secondly, to es- searchers around the world, must see the ar- tablish and maintain a network of re- ticles as useful. David Scollard invited searchers who are working on different as- members of the leprosy history network to pects of the history of leprosy. This network submit articles of historic interest and to ask is expected to be self-perpetuating, that is, themselves how their submission is useful the members will maintain contact amongst in the field, how it could help medical themselves once the Project has made them workers, scientists, and others. aware of each other’s existence through its website and activities. 4. RESEARCH OPPORTUNITIES Mark Harrison explained how he intends Oral History to make the history of leprosy one of the Anwei Law explained the oral history main areas of research at the Wellcome component, which will begin once further Unit, Oxford, and will invite applications funding is assured. Oral history “makes his- for this. He will be putting in research pro- tory more rounded,” as it is related by the posals to the Grants Committee—at least people actually involved. When making one large one, or maybe two smaller ones. oral histories, it is important to include In addition, individual research applications families, and both younger and older gener- can also be made to the Wellcome Trust, ations as this establishes continuity. Guide- from academics from the European Eco- lines on making oral histories will be devel- nomic area. Wellcome collaborative grants oped, and other expressions produced by make available a relatively small amount of people who have been affected by leprosy money to develop specific research proj- will be identified, such as poetry, artwork ects, in order to facilitate travel between the 71, 4 News and Notes 367 two units for meetings and conferences, as an African University. It would be difficult well as the employment of researchers. to get funding but it is important to do so. Mark Harrison invited suggestions for col- Jo Robertson—If anyone knows of any laborative projects. Biswamoy Pati asked other academics in the leprosy history field, whether Ph.D. students could apply, Mark let her know and she will establish commu- replied that there was nothing in the rubric nication with them. against it, and that all the basic details con- Henk Menke—It may be useful to define cerning this can be found on the Wellcome one main historical problem as a group, and Unit website. see how the research of each country relates to it. 5. STRATEGIES FOR THE FUTURE Jo Robertson—This approach is impor- OF THE NETWORK tant, and could be fulfilled by the research Jo Robertson outlined three main strate- projects outlined by Mark Harrison. Also, gies. Firstly, an electronic discussion forum on the academic network web page, there is will be set up, the importance of which was information on members’ publications and made clear by the recent pre-conference ex- research interests. Academics need the free- change of emails, mainly concerning use of dom to go in the direction that their work terminology in historical articles. Secondly, leads them, and would prefer not to be pinned suggestions on papers to be submitted to his- down to one particular research area. tory of medicine conferences are welcome. George Joseph—Maybe three or four ar- Thirdly, possible collaborations among mem- eas could be established to begin with but bers of the network, which Jo Robertson left one would be too constraining. open for discussion. Jo Robertson—We could look at the cur- rent areas being researched by the network Discussion and compile a list of core issues. Diana Obregon—The electronic forum Jaime Benchimol—Agreed that it is too could be used to share bibliographic infor- early to identify specific research areas. mation as each academic is not necessarily What would be valuable is an appraisal of aware of other publications in the field. what has been done in the field, e.g., lep- Sanjiv Kakar—It will also be useful for rosy and public health. sharing information on conferences world- Sanjiv Kakar—Oral history is one domi- wide. nant theme that we already have. George Joseph—The submission of pa- Jo Robertson—The politics of oral his- pers to conferences is normally more pro- tory are being handled carefully in the pro- ductive when in panels, rather than sending posal for further funding, as there were individual papers, which are often difficult problems with this area previously. We are for organizers to place in the program. He is trying to incorporate it once more. in the early stages of planning a conference Harriet Deacon—Oral history is a for late April or early May in the United methodology, not an analytical approach. By States. The American Association for the pointing out that it is part of every history History of Medicine (AAHM) will meet in may be a convincing argument in its favor. Birmingham, Alabama, during the first Anwei Law—The problem arises from week of May and in conjunction with the not seeing oral history as a good source of leprosy conference most likely to be held in history due to a lack of understanding of its New Orleans, Louisiana, it may be possible use in different contexts. to arrange a visit to Carville at the same Harriet Deacon—Asked whether the Proj- time. George Joseph suggested the circula- ect has to clear all methodologies with WHO. tion of the papers prior to the conference to Jo Robertson—Now that the Project is allow a more advanced level of discussion. strong, we have received an email from John Manton—There is a problem with WHO that is virtually contractual, stating studying leprosy history in “tropical” Africa that the website content must be cleared by (i.e., Africa except South Africa). It would WHO. This issue will go to the Steering therefore be useful to underpin networks of Group to be debated. Jo Robertson listed Africa scholars and/or have a symposium in the members of the Steering Group, as not 368 International Journal of Leprosy 2003 all the network members were aware of INDEPENDENT EVALUATION OF whom it comprised. There is now a page on THE GLOBAL ALLIANCE FOR THE the website with this information. ELIMINATION OF LEPROSY (GAEL) Chandi Nanda—Asked whether it is pos- June 13, 2003 sible to identify some people who have been cured of leprosy, who can be brought in to the network. Jo Robertson—This is an academic net- Released by WHO 4 July 2003, the eval- work. Anwei Law will develop a network uation of the Global Alliance for the Elimi- of people to gather oral histories. nation of Leprosy, GAEL, was drafted by Henk Menke—The main group working an independent panel of six, led by Dr. for leprosy patients to date has been doctors Richard Skolnik. With the exception of Pro- and nurses. The historical method is rela- fessor Michel Lechat, the evaluators are not tively new. We need to make our work clear part of the leprosy community. They based to those in the field, not only through publi- the evaluation on a review of literature, cations but also meetings. If it is limited to communications, documents and approxi- historians, we may miss the important goal. mately 100 widely representational inter- David Scollard—The last ILA Congress views. in Salvador, Brazil, is an example of how historians and present day medical workers Richard Skolnik (Team Leader), Florent have already come together. The history Agueh, Judith Justice, Michel Lechat. symposia during this Congress were very The George Washington University, The successful. There are people from all fields University of Louvain, and The University at this regular, international conference, so of California at San Francisco. to have history also represented is very good. Hopefully we can find future ways in congresses to put forward particular histor- ical problems and issues. Jo Robertson—It was a very international ABSTRACT gathering, and people who had had the dis- This is an independent evaluation of the ease were also responding. Global Alliance for the Elimination of Lep- Harriet Deacon—One of the dangers of rosy. It assesses the extent to which the Al- classing us as a leprosy network limits the liance has contributed to the goal of elimi- focus to that disease. However, it is useful nating leprosy as a public health problem. to make comparisons with issues surround- This evaluation was based on a review of ing syphilis, AIDS and other diseases, to literature, documents, communications, and see how policies that develop around these almost 100 informant interviews. matters relate to leprosy. The evaluation team believes that the Al- Jaime Benchimol—Emphasis also needs liance has added important value to the goal to be given to national studies; tuberculosis of eliminating leprosy as a public health is an important comparison. problem. It has mobilized political commit- Anwei Law—This kind of study should ment, financial resources, and free drugs. It not be limited to diseases, but human rights has helped to improve the management and issues too. reach of multi-drug therapy. It has ener- Jo Robertson—Bernardino Fantini is de- gized a number of leprosy programs. Dur- veloping a human rights program and wants ing the course of the Alliance, 16 of 22 en- to include leprosy. demic countries have been deemed to have David Scollard—Expressed a desire to met the goal of elimination. publish the abstracts of the current confer- In addition, at the country level, the Al- ence in the International Journal of Lep- liance appears to be functioning well. Most rosy. Members were asked to make any fi- countries are actively leading and coordi- nal adjustments to their abstracts, and send nating their leprosy programs. Collabora- them to Jo Robertson as soon as possible. tion is good, with the World Health Organi- zation (WHO) playing an advisory role and non-government organizations (NGOs) in- 71, 4 News and Notes 369 volved in a range of leprosy efforts in con- organize their leadership around a country- junction with WHO and government. level leprosy task force. WHO should play Despite these important successes, the the advisory role to country programs, with Alliance is not adding the value that it could effective use of input from other collabora- add and this poses threats to country lep- tors. The WHO should also convene a rosy programs and to the reputations of col- group of technical advisors, selected with laborators on leprosy work. Relations the advice of others involved in leprosy, to among some collaborators at the global carry out independent monitoring and eval- level are very bad. Concerned NGOs, uation of leprosy activities. The Technical physicians, and scientists have raised im- Advisory Group (TAG) of WHO would portant questions to WHO about technical, have its membership strengthened, again operational, and strategic matters but they with the advice of others. have not been resolved. In addition, some It is also hoped that the Novartis Corpo- collaborators do not have a clear under- ration, working with the Novartis Founda- standing of the aims of the Alliance, or a tion for Sustainable Development, would clear agreement on how the Alliance should continue to provide drugs and that the be governed. There are also strong views Sasakawa Memorial Health Foundation and among some collaborators that the Alliance the Nippon Foundation would continue to is too embedded in WHO and that WHO support technical cooperation and research, has not been sufficiently consultative in its including through its important financial as- management of the Alliance. sistance. This is already mid-2003, and the target The above approach would carry on from date for elimination that was set by the the work done effectively to date and would World Health Assembly and extended by build on the comparative advantages of dif- the Alliance is very close. There will con- ferent actors engaged in leprosy efforts. It tinue to be significant numbers of leprosy would also build on the unique role and patients after the goal of elimination has commitment in leprosy work of NGOs. It been achieved. In addition, there will also would have clear and accountable roles for be needs at the global level for advocacy ef- all actors and would be inclusive. It would forts, funds for leprosy activities, and ex- also have to be based on open, transparent, changes of information and best practices and collegial relations, the lack of which among those working on leprosy. At the lo- would preclude any alliance from effec- cal level, all countries will need to lead tively supporting the important work on their leprosy programs in sound ways. If leprosy that will remain, even after 2005. these measures are not addressed effec- Finally, these arrangements would help pro- tively, some of the important gains on lep- vide a sound transition to further leprosy rosy will be lost. control and rehabilitation efforts. For these reasons, the panel believes that Obtained directly from the WHO web-site, the Alliance must be rebuilt and refined im- http://www.who.int/lep/GlobalAlliance/ mediately. Much of the global work of the evaluation.doc, at which the full report may Alliance would be convened and lead by be examined. the NGO and foundation movement. These activities would focus on ensuring effective advocacy, as needed, and promoting learn- ing and input into country programs on Notice. On 13Ð15 October 2003 a Work- technical, operational, and strategic issues. shop was held in Amsterdam on Leprosy They would build on earlier work by the In- Transmission and Diagnosis. During this ternational Association of Anti-Leprosy As- workshop it was decided to co-ordinate re- sociations (ILEP), the International Leprosy search activities in this field. Association (ILA), and the Sasakawa A consortium supported by the WHO/TDR Memorial Health Foundation. They would Special Program therefore now issues a include all who work with leprosy, includ- call for interest for partners to engage in ing the private sector and groups of people this comprehensive research program to affected by the disease. apply modern developments in the mo- If not already doing so, countries should lecular typing of M. leprae and specific 370 International Journal of Leprosy 2003 antigen/epitope definition to field studies If you are interested in participating, towards better understanding of the epi- please submit a letter of interest to the In- demiology and transmission of leprosy, terim Steering Committee of the consortium and the improved diagnosis of leprosy in- briefly stating the extent of your interest in fection. The purpose of this call is to re- these areas, your experience and your asso- cruit partners to participate in working ciation with leprosy field studies. A standard groups on: form and more information is available at: http://www.kit.nl/biomedical_research/htm/ Assays for molecular epidemiology leprosy_research_consortium.asp Immunology-based diagnostic assays Field studies related to transmission Dr. Linda Oskam Ph.D. (secretary of the and diagnosis Interim Steering Committee) Research Co-ordinator Mycobacteriology The purpose of the working groups is to KIT (Royal Tropical Institute) Biomedical (i) raise funds to advance the necessary ba- Research sic and operational research; and (ii) set Meibergdreef 39, 1105 AZ Amsterdam, policies, proposals, protocols, under the The Netherlands umbrella of the consortium. http://www.kit.nl/biomedical_research/
Calendar of Meetings and Events Day mm/yy Location Details Contact e-mail 9Ð13 12/3/2003 Valencia ILEP Working Session and ILEP Secretariat [email protected],uk General Assembly 19Ð22 12/3/2003 Chattisgarh National Conference on Dr. S.K. Noordeen vinodkoomar@ Elimination of Leprosy rediffmail.com
AMERICAN SOCIETY FOR MICROBIOLOGY DIVISION U SYMPOSIUM Washington Covention Center Washington D.C. 18–22 May 2003 The 103rd General Meeting of the Ameri- ber of Division U whose outstanding can Society for Microbiology was held at achievements have contributed to advances the Washington Convention Center in Wash- in mycobacteriology. ington D.C., U.S.A., on May 18Ð22, 2003. The conveners of the symposium were At this meeting, Division U (Mycobacteriol- Drs. James L. Krahenbuhl and Diana L. ogy) sponsored a symposium entitled, “Ad- Williams, both of the National Hansen’s vances in Leprosy Research 2003 and Be- Disease Programs Laboratory, Baton yond: Following in Shepard’s FootPads.” Rouge, LA, U.S.A. After a brief introduc- This symposium featured a distinguished tion by Dr. Krahenbuhl, the symposium list of speakers covering a range of topics commenced with this year’s Division U lec- which emphasized the rapid gains in knowl- turer, Dr. Warwick J. Britton from the Uni- edge since the sequencing of the Mycobac- versity of Sydney, Sydney, Australia, who terium leprae genome. This symposium presented an overview of his research on also featured the prestigious Division U lec- leprosy and tuberculosis vaccines. His lec- ture, which is awarded each year to a mem- ture was followed by a presentation by Dr. 71, 4 News and Notes 371
Williams on her work in defining a partial that past BCG immunization, as indicated M. leprae transcriptome. Dr. Patrick J. Bren- by the presence of BCG a scar, was associ- nan, Colorado State University, Fort Collins, ated with approximately 50% reduction in Colorado, U.S.A., presented his work on the clinical leprosy (range 20 to 81%). The im- genes encoding M. leprae cell wall compo- pact of widespread BCG implementation on nents. Last, Dr. Thomas P. Gillis, National leprosy control is difficult to quantitate, Hansen’s Disease Programs Laboratory, dis- however, it is probable that BCG is one fac- cussed his research on the use of bioinfor- tor which has contributed to the decline in matics to search for potential vaccine and leprosy in some countries. The recent South skin test antigen candidates. India vaccine trial provided further evi- This informative and timely symposium dence of the effectiveness of vaccines was attended by over 350 meeting partici- against clinical leprosy (2). In this study the pants and each presentation stimulated addition of heat-killed M. leprae to BCG thoughtful discussion. In an effort to share resulted in improvement in the vaccine effi- the proceedings of this symposium with the cacy from 34 to 61%. This is contrast to readership of the International Journal of earlier studies in Malawi and Venezuela, Leprosy who were unable to attend the which showed no benefit from the addition meeting, the four lecturers have here pro- of heat-killed M. leprae to BCG (1). An ad- vided a overview of their presentations. ditional finding was that the cultivatable —Linda Adams bacillus ICRC, probably a member of the Chair, Division U (Mycobacteria) M. avium-intracellulare family, also con- American Society for Microbiology ferred significant protection (VE, 65%) when given as a dead mycobacterium. This provides further evidence that immuniza- ABSTRACTS tion with heterologous mycobacteria pro- Britton, W. J. On the vaccine trail: from tects against M. leprae. Leprosy to Tuberculosis. Subunit vaccines against leprosy. The potential of subunit vaccines against lep- The control of leprosy has improved rosy was raised by the early studies of Gel- markedly since the introduction of multi- ber and Brennan, which showed that crude drug therapy with dramatic falls in the preva- M. leprae cell wall fractions and native cell lence of leprosy patients receiving anti- wall-derived proteins protected against M. microbial therapy during the 1990’s. Despite leprae footpad infection in mice. More re- this widespread implementation of MDT, cently, Ngamying and colleagues showed the incidence of leprosy as measured by case that M. leprae cytosol and membrane frac- detection rate has not yet fallen in major en- tions protected against mouse footpad in- demic countries. This justifies continuing re- fection, but the cell wall skeleton of search to understand the transmission, host mycolyl-AG peptidoglycan from M. leprae response in protective immunity against My- was not protective (3). Therefore, protein cobacterium leprae. Immunization to im- components are essential for effective sub- prove host response against M. leprae infec- unit vaccines. The trail to determine which tion will also be an important component in M. leprae protein antigens induce effective the long term control of leprosy. immunity dates back to studies with mono- Current leprosy vaccines. The anti- clonal antibody-defined proteins in the tuberculosis vaccine, Mycobacterium bovis 1980’s. These included the M. leprae bacille Calmette-guerin (BCG) has partial GroEs, GroEl and 70 kDa heat shock pro- efficacy against clinical leprosy. In four teins, widely shared with other mycobacte- randomized clinical trials, BCG stimulated ria, and the M. leprae 18 kD protein, which a degree of vaccine efficacy (VE) ranging contains M. leprae-specific T cell epitopes from 34% in India to 80% in Uganda. The but has a homologue in M. avium. Subse- prospective vaccine study in Malawi quently, native proteins were purified from demonstrated a 52% VE for BCG, with a M. leprae, including the cytoplasmic 10 further 50% reduction in clinical leprosy kDa GroEs protein and the membrane- following repeat BCG immunization (1). In associated proteins MMPI and MMPII. addition, 9 case control studies have shown Fractions of M. leprae containing GroEs 372 International Journal of Leprosy 2003 and MMPI stimulated some protective ef- mouse model, single protein or DNA vac- fect in the mouse footpad model. More re- cines against tuberculosis infection have cently, a bioinformatics approach has been generally been less effective than BCG in employed to define M. leprae homologues mice (9). Therefore, we and others have of M. tuberculosis antigens known to in- been examining ways of increasing the pro- duce protective immunity against tubercu- tective efficacy of subunit vaccines against losis, such as the secreted proteins, Antigen mycobacterial infections. A number of cy- 85B and ESAT-6. tokines were compared as adjuvants for Our own group has focused on the M. DNA immunization and plasmid IL-12 was leprae 35 kDa (MMPI) as a subunit vaccine the most effective. Co-immunization with against leprosy. This protein was first rec- DNA-85B and plasmid IL-12 resulted in a ognized by M. leprae-specific monoclonal rise in IFN-γ and T cell responses, a fall in antibodies to conformational determinants specific antibody responses with increased on the protein, which are also the dominant protection against M. tuberculosis infection epitopes for human leprosy sera. We cloned (10). Co-immunization with DNA-35 and the gene for the M. leprae 35 kDa protein plasmid IL-12 produced significantly (4) and found it had no homologues in M. greater protection against virulent M. avium tuberculosis or BCG, but one was present in infection than BCG (11). IL-12 was more ef- M. avium with 94% amino acid identity (5). fective than IL-18, another Th1-promoting Recombinant 35 kDa protein expressed in cytokine, at improving DNA vaccine effi- the rapidly growing M. smegmatis forms cacy against mycobacterial infections (12). highly immunogenic multimers of >900 We have also examined the interactions kDa. This protein is recognized across the of subunit vaccines and BCG against M. tu- leprosy spectrum, so that paucibacillary berculosis infection. Priming with a DNA leprosy patients and contacts of leprosy pa- vaccine expressing the M. tuberculosis anti- tients develop a strong T cell response with gen 85B, followed by boosting with BCG, low levels of antibody and multi-bacillary significantly improved the effective efficacy patients demonstrate a strong antibody re- against M. tuberculosis to a level greater sponse and weak T cell response (4). Fur- than that achieved with BCG alone (10). This ther, the protein elicits delayed type hyper- may be due to focusing of the immune re- sensitivity (DTH) in M. leprae sensitized sponse against a dominant secreted antigen guinea pigs. of M. tuberculosis. These findings demon- DNA vaccines expressing the M. leprae strate that protection against mycobacterial or M. avium 35 kDa proteins induced pro- infections in experimental models is not tective immunity against M. leprae and M. limited to that achieved with BCG alone. avium infection in mice, which was equiva- Challenges for new anti-leprosy vac- lent to BCG in both cases (6, 7). This was ac- cines. Although considerable progress has companied by strong specific Interferon been made, there remain uncertainties in (IFN)-γ T cell responses, as well as high the understanding the complex host im- titre antibody responses to the conforma- munological response to mycobacteria and tional determinant on the protein. This es- this influences the development of more ef- tablishes that immunization with a single fective anti-leprosy and anti-tuberculosis antigen can be effective against experimen- vaccines. First, the factors which determine tal leprosy infection. Major antigens shared the immunological dominance of antigens with M. tuberculosis and BCG may also are still not resolved. Factors which may af- induce protection when used as a subunit fect this include the quantity of mycobacte- vaccines. For example, immunization with rial protein and the timing of exposure. For the M. tuberculosis antigen 85B as a DNA example, the GroES protein is the major cy- vaccine induced heterologous protection toplasmic protein in armadillo-derived M. against M. leprae footpad infection in leprae, and is a dominant antigen in host re- Swiss albino mice. (8). sponse to M. leprae (13). Secreted proteins, Improving subunit vaccines against including antigen A85 complex, may be the mycobacterial infections. Although sub- first antigens encountered and appear to unit vaccines against leprosy infection stimulate protective immunity against a show an equivalent protection to BCG in a number of mycobacteria (8, 10). There may 71, 4 News and Notes 373 be intrinsic properties to certain proteins, progress. In addition, it is important to con- such as the multimeric form of the 35 kDa firm that the vaccine antigens induce protec- M. leprae protein, which contribute to their tive immunity in mice with differing genetic persistence in the phago-lysosome and so backgrounds to confirm their applicability in their antigenicity. human populations. Second, the importance of species speci- The third and most important challenge ficity in determining the protective efficacy for introduction of new anti-leprosy vac- of individual proteins is unclear. In fact, cines is the capacity to test these in endemic species-specific proteins may not be the op- regions. Will it be possible to conduct an- timal vaccine candidates, although they other major human leprosy vaccine trial on have obvious importance as a diagnostic the scale of the recent Indian vaccine study? reagents. Recent studies by Black and col- This would require sufficient number of leagues in Malawi (14) have demonstrated new cases and the extensive infrastructure apparent cross-reactivity between the major required for such a study. Another approach antigens of M. leprae, including the 35kDa would be to include leprosy in future tuber- and 18kDa proteins, and environmental culosis vaccine trials. A number of candi- mycobacterial species isolated in Malawi. date TB vaccines are moving into Phase I These antigens may stimulate pre-existing and Phase II clinical trials. If these are to be T cell responses in infected subjects in of used in leprosy endemic countries, it is be endemic regions and this may blunt the ap- important that they have an effective anti- parent effectiveness of mycobacterial vac- leprosy component, either because the anti- cines in that environment. gens are shared between M. tuberculosis and Third, the relative contribution of different M. leprae, or M. leprae-related components T cell subsets to protective immunity may are added to the vaccine. This will require vary between different species of mycobac- the design and conduct of the vaccine trials teria. Both CD4 and CD8 T cells appear to to measure the effects on leprosy as well as contribute to effective immunity against M. tuberculosis in leprosy-endemic regions. tuberculosis, although the exact role of CD8 Acknowledgement. Laboratory research has been T cells inducing protection against M. leprae funded by the National Health and Medical Research infection has not been established. Council of Australia, the World Health Organization, Fourth, understanding the factors control- the Co-operative Research Center for Vaccine Tech- ling of T cell memory, particularly in CD4 T nology and the New South Wales Department of cells is incomplete and this has major impli- Health. I thank E. Martin, J. Triccas, U. Palendira and cations for subunit vaccines against myco- A. Kamath in Sydney and Dr. P. Roche, Anandaban bacterial infections. Although protein and Leprosy Hospital, Kathmandu, Nepal, for their collab- DNA subunit vaccines can stimulate short oration and discussions. I am grateful to Dr. J. Vele- term protective immunity against tubercu- mer, Leprosy Mission International for his analysis of losis and leprosy in animal models, their the effectiveness of BCG vaccines. ability to stimulate long term protection is REFERENCES yet to be determined. These subunit vac- 1. BLACK, G. F., WEIR, R. E., and CHAGULUKA, S. D., cines may prove most useful in boosting et al. Gamma interferon responses induced by a immunity established by BCG or other vi- panel of recombinant and purified mycobacterial able vaccines. antigens in healthy, non-Mycobacterium bovis The second major challenge for new anti- BCG-vaccinated Malawian young adults. Clin. leprosy vaccines are limitations of the mod- Diagn. Lab. Immunol. 10 (2003) 602Ð611. els for testing protective efficacy. The dy- 2. BRITTON, W. J., and PALENDIRA, U. Improving namic range of the mouse footpad infection vaccines against tuberculosis. Immunol. Cell Biol. model is low, and it is difficult to measure in- 81 (2003) 34Ð45. creased effective efficacy above that achieved 3. GUPTE, M. D., VALLISHAYEE, R. S., and ANANTHARA- with BCG. The lack of immunological MAN, D. S., et al. Comparative leprosy vaccine trial 70 reagents and complexibility of the armadillo in south India. Indian J. Lepr. (1998) 369Ð388. 4. KARONGA PREVENTION TRIAL GROUP.Random- model mean that it is not currently applica- ized controlled trial of single BCG, repeated BCG, ble to testing vaccines. Further, the length or combined BCG and killed Mycobacterium lep- of time for testing individual vaccines, cur- rae vaccine for prevention of leprosy and tubercu- rently 9 to 12 months, restricts the rate of losis in Malawi. Lancet 348 (1996) 17Ð24. 374 International Journal of Leprosy 2003
5. MARTIN, E., KAMATH, A. T., BRISCOE, H., and The genome of Mycobacterium leprae BRITTON, W. J. The combination of plasmid has been completely sequenced and anno- interleukin-12 with a single DNA vaccine is more tated. Approximately, 1604 open reading effective than Mycobacterium bovis (BCG) in pro- frames, encoding potentially functional pro- tecting against systemic Mycobacterim avium in- fection. Immunology 109 (2003) 308Ð314. teins, and 1104 inactivated genes (pseudo- 6. MARTIN, E., KAMATH, A. T., TRICCAS, J. A., and genes) have been identified. However, the BRITTON, W. J. Protection against virulent Myco- minimum gene set required for intracellular bacterium avium infection following DNA vacci- growth and survival (transcriptome) has not nation with the 35 kDa antigen is accompanied by yet been defined. To address this, we have induction of IFN-γ secreting CD4+ T cells. Infect. initiated studies to determine the potential Immun. 68 (2000) 3090Ð3096. transcriptome using RT-PCR and cross- 7. MARTIN, E., ROCHE, P. W., TRICCAS, J. A., and species DNA microarray analysis using a BRITTON, W. J. DNA encoding a single mycobac- comprehensive M. tuberculosis array using terial antigen protects against leprosy infection. a commercially available oligonucleotide Vaccine 19 (2001) 1391Ð1396. set (Operon Technologies, Alameda, CA) as 8. MEHRA, V., BLOOM, B. R., and BAJARDI, A. C., et al.Amajor T cell antigen of Mycobacterium lep- a prelude to evaluating global gene ex- rae is a 10-kD heat-shock cognate protein. J. Exp. pression using an M. leprae cDNA array, Med. 175 (1992) 275Ð284. which is not currently available. For RT- 9. NGAMYING, M., SAWANPANYALERT, P., BUTRAPORN, PCR, RNA was obtained from two R., NIKASRI, J., CHO, S. N., LEVY, L., and BREN- geographically distinct strains of M. leprae NAN, P. J. Effect of vaccination with refined com- and cDNA was produced by reverse- ponents of the organism on infection of mice with transcription using random priming. Gene Mycobacterium leprae. Infect. Immun. 71 (2003) transcripts of interest were amplified from 1596Ð1598. cDNA using PCR with primer sets flanking 10. PALENDIRA, U., KAMATH, A. T., FENG, C. G., and gene fragments of several potentially func- BRITTON, W. J. Co-expression of Interleukin-12 chains by a self splicing vector increases the pro- tional families of M. leprae. PCRs were tective cellular immune response of DNA and initially characterized using DNA from M. BCG vaccines against Mycobacterium tuberculo- leprae T-53 resultant PCR fragments were sis. Infect. Immun. 70 (2002) 1949Ð1956. analyzed by gel electrophoresis. Cross- 11. ROCHE, P. W., NEUPANE, K. D., FAILBUS, S. S., KA- species DNA microarray analysis was ac- MATH, A. T., and BRITTON, W. J. Vaccination with complished using 5 ug total RNA from DNA of the Mycobacterium tuberculosis 85B anti- T-53 and 4089 and labeled with either Cy3 gen protects the mouse footpad against infection or Cy5 fluorochromes using RT. The la- with Mycobacterium leprae. Int. J. Lepr. Other beled cDNAs will be hybridized to the Mycobact. Dis. 69 (2001) 93Ð98. slides, the slides will be washed and 12. TRICCAS, J. A., ROCHE, P. W., WINTER, N., FENG, C. G., BUTLIN, C. R., and BRITTON, W. J. A 35 scanned using an Axon Scanner. The inten- kilodalton protein is a major target of the human sities of the two dyes at each spot will be immune response to Mycobacterium leprae. In- quantified using the GenePix software fect. Immun. 64 (1996) 5171Ð5177. package. Results of RT-PCR and cross- 13. TRICCAS, J. A., SUN, L., PALENDIRA, U., and BRIT- species microarray experiments demon- TON, W. J. Comparative affects of plasmid- strated that genes encoding a variety of en- encoded interleukin 12 and interleukin 18 on the zymes were transcribed in both strains. protective efficacy of DNA vaccination against These include enzymes involved with folic Mycobacterium tuberculosis. Immunol. Cell Biol. acid synthesis, iron utilization, cofactor 80 (2002) 346Ð350. biosynthesis, gluconeogenesis, glycolysis, 14. TRICCAS, J. A., WINTER, N., ROCHE, P. W., GILPIN, A., KENDRICK, K. E., and BRITTON, W. J. Molec- glyoxylate bypass, those associated with ular and immunological analyses of the Mycobac- beta oxidation of fatty acids, degradation terium avium homolog of the immunodominant of phosphorous compounds, degradation of Mycobacterium leprae 35-kilodalton protein. In- DNA, detoxification and virulence associ- fect. Immun. 66 (1998) 2684Ð2690. ated proteins, synthesis of mycolic acids, modification and maturation of ribosomes, synthesis of RNA, stress proteins, proteins Williams, D. L. Establishing the Transcrip- of the SecA-dependent secretion pathway tome of Mycobacterium leprae: A Mini- and 25 proteins containing secretion motifs mal Gene Set for Survival. or, and several proteins with unknown 71, 4 News and Notes 375 functions. These data have provided us murD, murX, murF, murE) and other related with the first insight into the transcriptome genes (e.g., murA, murB, ponA1, ponA2). of M. leprae and further demonstrated the Polyprenyl-phosphates are the membrane homogeneity of this species. It is antici- carrier lipids for many aspects of cell wall pated that this analysis will help to identify synthesis, such as arabinose, arabino- a larger set of functional genes in M. leprae galactan, peptidoglycan, and, as expected, which will potentially help us to under- M. leprae contains the majority of genes stand the minimal requirements for growth encoding enzymes of the non-mevalonate and replication of this pathogen. This infor- pathway for polyprenyl-phosphate synthesis mation may lead to the identification of (e.g., dxsI and ispCÐG). It is devoid of the new drug targets, skin test antigens, and to dxsII of M. tuberculosis, which helped in identify factors that allow this pathogen to deciding that dxsI is the functional gene for evade the immune system and destroy pe- deoxylulose-5-phosphate synthase. The en- ripheral nerves. tire array of genes required for rhamnose synthesis (rmlB, rmlC, rmlD, rmlA) are present in the M. leprae genome, as Patrick J. Brennan and Varalakshmi D. expected, since rhamnose is a component of Vissa. Maintenance of Genes for Myco- the key diglycosyl-phosphoryl unit joining bacterium leprae Cell Wall Synthesis. the mycolyl-arabinogalactan complex to peptidoglycan. Most of the known genes Sequencing of the Mycobacterium leprae responsible for the synthesis of mannose, the genome by S.T. Cole, et al. (http://www. PIMs (phosphatidylinositol mannosides), nature.com/nature/v409/n6823/fig_tab/ LM (lipomannan), and LAM (lipoarabino- 4091007aO_F1.html) was a momentous mannan), are present in M. leprae, such as event, comparable to the introduction of pmmA, the Rv3256c homolog, pmi, manB, MDT (multiple drug therapy) in the 1980’s. the Rv2609c homolog, pimA, Rv2611c Initial analysis indicated a genome size of homolog, and pgsA. However pimB is about 3.3 megabases, a G/C content of apparently missing, which requires new 57.8%, only 1604 protein genes, 1116 thinking on the mechanism of synthesis of pseudogenes, and hence a protein coding PIMs/LM/LAM; we do know from chem- capacity of 49.5% (these latter figures are ical analysis that all three types of products to be compared to a size of about 4.4 are present in M. leprae. Likewise, most of megabases for the Mycobacterium tuberc- the genes for mycolic acid synthesis, ulosis H37Rv genome, a G/C content of modification, and deposition are present in 65.6%, 3959 protein genes, only about 6 the M. leprae genome, such as fasI, fabD, pseudogenes, and thus a protein coding acpM, kasA, kasB, accD6, mabA, inhA, capacity of 90.8%). (These data are being umaA2, mmaA4, mmaA1, fbpA, fbpB, fbpC, constantly revised in light of more recent and fbpC2. However, as noted initially by and ongoing analysis of bacterial genomes.) Cole, et al., umaA1, mmaA3, and mmaA2 Thus, the M. leprae genome has undergone are missing as whole genes, and this absence reductive evolution, becoming trapped and is exactly in accord with the absence of crippled. In light of such a paucity of methoxymycolates in M. leprae as reported protein coding genes, it is worthwhile to by several workers in the 1980’s. examine the cell wall of M. leprae from The pks (polyketide synthase)-like genes both the perspectives of known biochemical of Mycobacterium tuberculosis responsible information and in silico analysis. This was for the synthesis of the phthiocerol, the purpose of this review. phenolphthiocerol, and methyl branched For instance, we have known from the fatty acids of DIM (dimycocerosyl phthio- early chemical analysis of the cell wall cerol) and the phenolic glycolipids (PGL) peptidoglycan of M. leprae by P. Draper, et are receiving considerable current atten- al., that this essential component of all tion, since these products have been eubacteria is intact and comparable to that implicated in disease processes. In the case of M. tuberculosis and other bacteria. of M. tuberculosis, it has been dem- Indeed, M. leprae apparently retains the full onstrated that disruption of the pks10 and mur operon (ftsZ, ftsQ, murC, murG, ftsW, pks7 genes which are clustered with pks8, 376 International Journal of Leprosy 2003 pks17, pks9 and pks11 resulted in mutants missing from M. leprae. Likewise, a cluster deficient in the synthesis of DIM. Simi- of at least 8 glycosyltransferases in the larly, the pks12 gene has also been impli- Rv1500 to Rv1526 region of the M. cated in synthesis of DIM. However, a tuberculosis genome is missing from M. careful analysis of the M. leprae genome leprae. This evidence supports the chemical indicates that pks10, pks7 and pks12 are evidence for “stunted” or “truncated” pseudogenes; and pks 8, 17, 9, and 11 are versions of some polysaccharides, such as absent. LAM, in M. leprae. An analysis of the intact pks genes of M. Thus, an analysis of the genome of M. leprae from a different perspective, i.e., leprae versus those of M. tuberculosis, M. from the perspective of identification of the bovis, and other Mycobacterium spp. genes responsible for the synthesis of PGL-I, supports the chemical analytical evidence yields interesting information. The entire of an intact but minimal cell wall in M. array of genes attributed to the synthesis of leprae. phthiocerol (ppsAÐE) is located between ML2357 and ML2353. At a different Some relevant publications: location (ML0139) is the mas gene responsible for mycocerosic acid synthesis. Vissa, V.D., and P.J. Brennan. Impact of the The pks1 and pks15 of M. tuberculosis Mycobacterium leprae genome sequence on genes are fused as one gene in the M. bovis leprosy research. In: Genomics of GC Rich Gram- genome, which has been demonstrated to Positive Bacteria, Wymondham, U.K.: Caister be involved in the synthesis of phenol- Academic Press, 2002. pp. 85-Ð118. Crick, D.C., P.J. Brennan, and M.R. McNeil. The cell phthiocerol, most likely a precursor of the wall of Mycobacterium tuberculosis. In: Tuberc- M. bovis specific PGL. The M. leprae ulosis. Second Edition Philadelphia: Lippincott genome also contains the fused gene in Williams & Wilkins, 2004. pp. 115Ð134. concordance with the elaboration of its phenolic glycolipids. Elongation occurs with the ppsAÐE cluster. Associated with Gillis, T. The Use of Bioinformatics in Lep- ppsAÐE are the genes fadD26 and drrAÐC rosy Research. implicated in the attachment of myco- cerosic acid and phthiocerol followed by Bioinformatics encompasses all aspects transport through the membrane. Putative of biological information acquisition and glycosyltransferases and methyltransferases analysis, and combines the tools of com- possibly involved in the synthesis of the puter science and biology with the aim of trisaccharide segment of PGL-I can be understanding biological significance. The located in one cluster (ML0125-ML0130). combination of enhanced sequencing and We have previously commented on the computing power has allowed for unprece- “cell wall gene cluster” of M. leprae dented advances in assimilating huge characterized by the presence of embAÐC amounts of raw data and the initiation of implicated in arabinan synthesis, the fbp meaningful molecular modeling. Bioinfor- genes responsible for mycolic acid matic approaches are particularly attractive deposition, and glf and glfT involved in for aiding studies of M. leprae since many D-galactofuranose and D-galactan synthe- conventional biological tools are unavail- sis. A detailed comparison of gene arrange- able to investigators working with noncul- ments in this cluster in the M. leprae and M. turable agents. tuberculosis genomes demonstrates the Three major areas of bioinformatics (ge- absence of several genes (Rv3784-3788) from nomics, proteomics, and transcriptional the M. leprae genome, one of which is a profiling) have had and should continue to putative glycosyltransferase (Rv3786c). have an impact on our understanding of M. Additionally, a dedicated analysis of puta- leprae and the disease it causes. Genomics tive glycosyltransferases over the entire has provided a working genetic blueprint genome of M. leprae versus M. tuberculosis for M. leprae allowing for comparisons shows that several orthologs (e.g., Rv1212c, with other mycobacterial genomes to assess Rv0539, and Rv2957) representative of the M. leprae’s basic physiological capabilities, glycosyltransferase families 1 and 2 are potential virulence factors and establish 71, 4 News and Notes 377
molecular markers for drug resistance and sponses during infection. In addition, newly strain variation. Newly developed strain discovered proteins can be tested for their identification markers using simple repeated ability to induce protective immunity and DNA sequences should provide tools to in- may constitute a new group of proteins with vestigate transmission patterns of leprosy vaccine or diagnostic potential. Transcrip- and may help define risk factors involved in tional profiling may allow investigators to reinfection versus relapse of disease. Algo- study M. leprae’s gene expression at differ- rithms for predicting open reading frames ent stages of growth as well. For example, and for categorizing location and function of gene expression in growth-permissive cells, M. leprae proteins have initiated basic stud- such as the macrophage and Schwann cell ies on secreted proteins as well as proteins could differ and, therefore, may reveal as- involved in nerve invasion and M. leprae- pects of M. leprae’s unique tissue tropism. specific proteins potentially useful for de- Functional genomics is the integration of tecting exposure to the leprosy bacillus predictive bioinformatics with validation through skin testing or serological testing. through experimental biological analysis. Proteomic studies of the leprosy bacillus This part of the equation remains a major have been hampered by low protein yields challenge to workers in the leprosy field. from purified bacilli derived from infected Surrogate genetics to study M. leprae genes animals, but have established a baseline in cultivable mycobacteria as well as new profile of highly expressed proteins from approaches for “knocking in” genes to M. M. leprae. Combining transcription profil- leprae are areas in need of research and de- ing of the leprosy bacillus with proteomic velopment. Both approaches will benefit studies may provide new insights into pro- from bioinformatics and should continue to teins previously lost during purification further our understanding of the leprosy from infected host tissues and provide new bacillus in particular, and the host-parasite antigens useful for studying immune re- relationship in general. INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. (ISSN 0148-916X) THIRTY-EIGHTH U.S.-JAPAN TUBERCULOSIS-LEPROSY RESEARCH CONFERENCE Public Health Research Institute Newark, New Jersey 21–22 July 2003 Sponsored by the U.S.-Japan Cooperative Medical Sciences Program National Institute of Allergy and Infectious Diseases National Institutes of Health
US-Japan Co-operative Tuberculosis and Leprosy Research Panel, 2003. Front Row: Hatsumi Taniguchi, Masamichi Goto, Masao Mitsuyama, Patrick Brennan and Toru Mori. Back Row: Kiyoshi Takatsu, Gail Jacobs, Tom Gillis, Gilla Kaplan, Christine Sizemore, Philip Hopewell and David McMurray.
378 71, 4 News and Notes 379
ABSTRACTS Spencer, J. S., Kim, H. J., Gonzalez- Western blot showed no detectable levels Juarerro, M., Marques, M. A. M., Wil- of any of these proteins in the native sub- liams, D. L., Sang-Nae Cho, and Bren- cellular fractions. Nevertheless, by RT- nan, P. J. Lessons learned from cloning, PCR analysis of M. leprae cDNA isolated expression, and immunologic analysis of from two geographically different strains unique Mycobacterium leprae proteins. of M. leprae (Thai-53, originally isolated from a patient from Thailand, and strain With the recent completion and annota- 4089, originally isolated from a patient tion of the genomes of M. tuberculosis and from Mexico), it appears that all of these M. leprae, and additional sequence data genes have mRNA transcripts, and, thus, from environmental mycobacteria (M. are potentially transcribed. However, fur- avium, M. marinum, and M. smegmatis), ther testing of these five recombinant pro- our ability to identify and test leprosy- teins in M. leprae-sensitized guinea pigs specific antigens is much improved. Com- did not reveal any detectable delayed type parative genomic analysis of the M. leprae hypersensitivity (DTH) response. In addi- genome has identified 1604 open reading tion, the proteins were examined by ELISA frames, as well as 1116 inactivated genes assay using leprosy patient sera (including (pseudogenes), and up to 165 genes with sera from multibacillary lepromatous and no orthologue in M. tuberculosis. Through paucibacillary tuberculoid patients). Re- comparative genomic analysis of all myco- sponses to the unique antigens showed bacterial databases, we have targeted 28 of much lower responses overall, with the these novel M. leprae genes for cloning majority of the O.D. readings falling in the and expression as recombinant proteins. low to background level response range, This report characterizes our initial find- consistent with the level of these antigens ings of five of these selected unique pro- produced, while responses to well ex- teins, with a comparison of several other pressed antigens were much higher. It is M. leprae proteins that have homologues possible that some of these novel proteins in M. tuberculosis. The unique proteins are just not that immunogenic. Alterna- tested were ML0008, ML0394, ML0126, tively, despite the lack of a significant anti- ML1057, and ML2567, while proteins with body response, T cells from PBMC of lep- homologues include the well-known 10 rosy patients may react better in IFN-γ kDa GroES (ML0380), Ag85B (ML2028), assays to some of these unique proteins or ESAT-6 (ML0049) and CFP-10 (ML0050) their peptides, a possibility we are cur- proteins. After cloning the genes from M. rently exploring. In light of our current leprae genomic DNA by PCR, all were findings, we should seriously question the produced as single recombinant proteins in strategy of choosing antigens for diagnos- E. coli. Our strategy was to produce poly- tic purposes based on comparative genom- clonal antisera against all of these single ics alone. It would appear from these re- proteins, and then to use each antiserum to sults that some genes unique to M. leprae, examine the relative amounts of the indi- although capable of generating a transcript, vidual proteins in the native subcellular are not expressed in sufficient quantities in fractions of M. leprae (cytosol, membrane, the bacillus itself to induce a meaningful and soluble cell wall antigens). In this way, immune response, and hence should not be we had previously shown the existence of pursued for diagnostic purposes. Alterna- native M. leprae ESAT-6 in the cell wall tive strategies for the selection of promising fraction in amounts significant enough to antigens will be discussed. suggest that it could stimulate antibody or T cell responses in a natural infection. In- Groathouse, N. A., Rivoire, B., Sang-Nae. deed, several investigators have recently Cho, Brennan, P. J., and Vissa, V. D. shown that leprosy patient PBMC respond Polymorphisms in Short Tandem Repeat well to both M. leprae ESAT-6 and CFP-10 Sequences of Mycobacterium leprae al- proteins and peptides. Our initial analysis low for the Epidemiological Characteri- of the five unique M. leprae proteins by zation of Strains. 380 International Journal of Leprosy 2003
Up until now, the characterization of found to have the same length as the TN strain and lineage of Mycobacterium leprae strain, in all isolates tested. isolates has been a great obstacle to study- Our findings clearly indicate that poly- ing the epidemiology of leprosy. In this morphisms are present at various sites in post-genome era, the availability of a com- the genomes of different strains of M. lep- plete DNA sequence for M. leprae has pro- rae, and consequently could be developed vided an opportunity to target and analyze to track their lineage. Further emphasis will specific sites within the genome. Our ap- be placed on the use of fluorescent 5′ la- proach for molecular typing of M. leprae beled primers for amplification, detection includes the detection of variable number of and fragment length analysis of the multi- short tandem repeat (STR) sequences, in plexed products from the six DNA isolates different isolates. The specific goal of this on sequencing gels to improve throughput. research is to identify several polymorphic The next phase will be expansion of the sites and combine them into a multiplex analysis to some of the remaining selected PCR-based molecular typing tool, which loci, followed by inclusion of a larger col- could be used on clinical biopsy samples lection of DNA isolates. The final goal is to collected in endemic parts of the world. We produce a fingerprinting system for M. lep- hope to develop a product (technique and rae obtained from clinical isolates and to reagents) that is portable, easy to apply and provide a tool for studying other aspects of analyze, and inexpensive. leprosy such as transmission, virulence, The first step, localization of regions of drug resistance and relapse. the genome within the sequenced Tamil Nadu (TN) strain containing a succession of single, di- and tri-nucleotide repeats, Williams, D. L. Gene Expression and Sta- was accomplished by utilizing the search bility of mRNA in Mycobacterium leprae. pattern program of the Leproma database (http://genolist.pasteur.fr/leproma/). A panel Evaluation of gene expression in Myco- of 31 distinct sites in non-coding regions bacterium leprae under different experimen- that contained 5 or more repeat units was tal conditions should provide important in- identified. From these, several primary sites sight into the physiology and therefore, the were selected. Primer sets were designed to life-style of this noncultivable mycobacte- flank the individual STR sites and elicit rium. Recently, we have identified mRNA PCR products of incremental length to fa- transcripts for 194/200 genes analyzed by cilitate multiplexing in the future. A total of RT-PCR and the presence of polyadenylated six M. leprae DNA sources (armadillo de- mRNAs in 15/15 transcripts analyzed by rived human clinical isolates) were used to oligodT priming of total RNA. Since search for polymorphisms by comparing polyadenylation in bacteria leads to mRNA the STR lengths. The PCR products were stabilization under some conditions and cloned to enable sequence confirmation of degradation under other conditions, poly- the entire amplified fragment, including the adenylation of mRNA in M. leprae may not repeat region. be a useful indicator of mRNA stability. So far, we have confirmed repeat length Nothing is known about the stability of polymorphisms in four of the DNA sources, mRNA in this slow growing pathogen. The and in all but one STR sequence. One of the purpose of the current study was to determine AT repeat regions, showed as few as 25 bp the relative stability of selected polyadeny- and as many as 37 bp within the STR se- lated mRNAs in M. leprae after termination quence of the various DNA sources when of transcription. To accomplish this, a sus- compared to the 37 bp sequence reported in pension of mouse footpad-derived M. leprae the TN strain. Likewise, a different AT re- in 7H12 medium was killed by subjecting peat sequence varied from 20 to 30 bp in bacteria to 5 × 106 rads of γ-radiation for 4 hr the different DNA isolates while it is at 34 followed by rifampin (8 µg/ml final con- bp in the TN strain. A multi-C repeat se- centration) treatment to terminate mRNA quence that is 20 bp in the TN strain ranged transcription. RNA was purified from un- from 8 to 14 bp in the M. leprae isolates. treated bacteria (live) and the from dead Alternatively, a CG repeat sequence was bacteria stored at 33°C for 24, 48, 72 hr and 71, 4 News and Notes 381
1 week post treatment. The expression of limited value because M. leprae appeared five M. leprae genes from various gene to show limited heterogeneity at the pheno- families, previously found to be transcribed typic and genomic levels. Insertion se- in the mouse foot pad (rpoB, gyrA, folP1, quences are not abundant in M. leprae and, hsp18, and sodA), was determined using therefore, are not expected to provide suffi- semiquantitative RT-PCR. Appropriate con- cient intrusion into the genome to create trols for DNA contamination were included variation useful for developing a strain typ- for each template analyzed. The 16S rRNA ing system. Similarly, neither restriction PCR product from each template was used fragment length polymorphism analysis to normalize these data for template varia- (RFLP) nor 16S rDNA sequencing has iden- tions. Results demonstrated that transcripts tified diversity among M. leprae isolates for all genes studied were detected in the from patients or natural animal hosts. More live bacteria for the entire experiment. In recently, various repeating elements have addition, transcripts for rpoB, folP1, and been shown to be effective in genotyping hsp18 were observed up to one week post other highly conserved bacterial species. death, although transcript levels were lower The completed sequence of the M. leprae than that of the untreated controls and pro- genome reveals a number of such elements gressively decreased with time. Transcripts and some limited diversity already has been for gyrA were observed up to 48 hr post reported among a few clinical specimen treatment, although transcript levels were within these repetitive sequences. lower than those found in the live bacteria. In Japan, limited variation in a 6 bp re- Transcripts for sodA were not observed in peat in the rpoT gene was found among iso- the 24 hr treatment cDNA indicating that lates from one region but it was absent this mRNA is less stable than the rest of the among isolates in other locales. Somewhat mRNAs studied. These results demon- greater diversity has been observed among strated that the relative stability of the se- M. leprae derived from a number of patient lected polyadenylated mRNAs varied sug- samples in the Philippines, where polymor- gesting that polyadenylation in M. leprae phism was observed ranging from 10 to 37 can not be used as an indicator of mRNA copy numbers of a TTC (GAA) triplet repeat stability. Therefore, it is recommended that occurring in an intergenic segment down- mRNA stability analysis be conducted on stream of pseudogene. Along with a few genes of interest prior to designing gene ex- other repeating elements of unknown diver- pression experiments for M. leprae because sity, the significance of these variable num- down regulation of the expression of a gene ber tandem repeat (VNTR) markers in M. may not be detected in the background of a leprae and their utility for epidemiological stable transcript encoded by that gene. In or clinical study has not yet been described. addition, the relatively short stability of the We examined the diversity of GAA sodA transcripts suggests that further stud- (same as TTC repeat described by Shin, et ies should be conducted to determine if al.) repeats along with 3 other VNTR mark- mRNA is a suitable target for the develop- ers using a battery of clinical samples and ment of rapid real time RT-PCR assay for M. leprae reference strains derived from the viability of M. leprae. patients and wild animals in different geo- graphic regions. To help assess the suitabil- ity of these VNTR markers for laboratory Truman, R., Fontes, A., Suffys, P., Gillis, and community based studies, we also ex- T. Characterization of VNTR Polymor- amined the stability of the reference strain phisms in Mycobacterium leprae. VNTR genotype with passage in different animals under varied conditions and in dif- Differentiating mycobacteria at the sub- ferent tissues. species (strain) level based on genomic di- versity can be useful in understanding their evolution and for discriminating relation- Lahiri, R., Randhawa, B., and Krahen- ships between different clinical cases or buhl, J. Further definition of viability of laboratory strains. Early attempts to define Mycobacterium leprae as a research re- strain variants of M. leprae have been of source. 382 International Journal of Leprosy 2003
A rapid and reliable method to compare showed variety of TTC repeat numbers. M. viability between two suspensions of Myco- leprae genotype of four leprosy cases in two bacterium leprae is needed. We have shown houses was examined. M. leprae isolates previously that a radiorespirometry method, from a couple of the father and a son which assesses the metabolic activity of M. showed the same TTC genotype but the fa- leprae, compares well with the mouse foot- ther and the son in another house were in- pad assay. In this study, we tested a two- fected by different M. leprae distinguished color (Syto9 and Propidium iodide) fluores- from TTC repeat. The findings suggested cence assay to determine if a rapid direct the transmission of the bacilli might be from count viability staining can be applied to M. various infectious sources, though it is gen- leprae. A variety of experimental conditions erally believed the main infectious source is were employed to assess this “viability multi-bacillary patients and household con- stain.” We also applied both radiorespirom- tact with such patient has the highest risk of etry and viability staining on extracellular being infected. Infectious sources other than and intracellular M. leprae treated with dif- multi-bacillary patients in the household ferent known antimycobacterial drugs to as- would be important for leprosy control espe- sess the reliability of these two methods cially in endemic countries. The results indi- taken together, in screening anti-leprosy cated the existence of the infectious source drugs. We used Rifampin, Clofazamine, other than patients in the household. Dapsone, Ofloxacin, and Minocycline, on axenic and intracellular cultures of M. lep- rae. We found that the intracellular model Ohyama, H., Takeuchi, K., Yamada, H., of drug testing was more efficient in detect- Uemura, Y., and Matsushita, S. SNPs ing anti bacterial effect of drugs in M. lep- on IL-12 receptor gene associated with rae than axenic cultures. the susceptibility to leprosy.
Activated T cells from lepromatous lep- Matsuoka, M., Liangfen, Z., and Budi- rosy patients are likely to produce low awan, T. Analysis of leprosy transmis- amounts of IFN-gamma even in the pres- sion based on genotyping. ence of IL-12. The objective of this study is to reveal the low productivity of IFN- Mycobacterium leprae isolates were di- gamma in leprosy patients from an im- vided into two groups by the polymorphism munogenetical viewpoint. The polymor- in the rpoT gene. Geographic distribution phism of 5′ flanking regions of both IFNG of the rpoT genotype of Mycobacterium and IL-12RB2 were determined to compare leprae in Latin American countries was in- the allele frequencies between patients and vestigated in connection with human pre- healthy donors, using direct sequencing historic migration. All M. leprae isolates technique. The results of the study are as from Peru and Paraguay showed three tan- follows. i) No polymorphism was detected dem repeats of 6 bp. On the contrary, 25 out in the promoter region of IFNG. ii) Several of 27 isolates from Mexico revealed four SNPs were detected in the 5′ flanking re- tandem repeats. It was assumed that the lep- gion of IL-12RB2, and SNPs located at the rosy was carried into these countries by dif- positions, Ð1035, Ð1023, Ð650 and Ð464 ferent groups of ancient Mongoloids mi- were more frequently detected in LL pa- grated to Latin America at different periods. tients than in TT patients. Genotyping by TTC repeat polymor- These results suggest that the polymor- phism was applied for epidemiological phism in the 5′ flanking region of IL12RB2 analysis of leprosy transmission. M. leprae may be implicated to determine disease on the nasal mucous membrane of villagers form of leprosy. 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 INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. REVIEWERS 2003
The Editor, on behalf on the INTERNATIONAL JOURNAL OF LEPROSY and the membership of the International Leprosy Association, expresses his deepest appreciation to the following reviewers who have provided invaluable expertise, criticism, and advice in preparing Vol- ume 71.
Linda Adams Kathleen McDonough Warwick Britton Sam Moschella Anne Burdick Bernard Naafs Paul Converse Sharon Nations Richard Croft Peter Nicholls Mannam Ebenezer S. K. Noordeen Richard Frankel T.H.M. Ottenhoff Thomas Gillis Francois Portaels Shigeo Hashimoto Rajni Rani Robert Hastings Thomas H. Rea Carlotta Hill Jo Robertson Robert Jacobson Larry Schlesinger Baohong Ji Edward J. Shannon Charles K. Job Robert Shebert Judith Justice Cairns Smith Vishwa Katoch John Spencer Herb Kaufman Mariane Stefani Paul R. Klatser Lavanya Suneetha D. A. Lopez Richard Truman Murdo Macdonald M. Virmond Celina Martelli Cassandra White Ronald Matthews Diana Williams
427 INTERNATIONAL JOURNAL OF LEPROSY Volume 71, Number 4 Printed in the U.S.A. ACKNOWLEDGMENT
The Board of Directors of the INTERNATIONAL JOURNAL OF LEPROSY gratefully ac- knowledges the financial assistance from special grantors and sustaining members which, with the special donations of certain members, has made possible the con- tinuation of publication of the JOURNAL directly by the International Leprosy Asso- ciation. Without this assistance the official organ of the ILA, so essential to leprosy workers everywhere, could not be published.
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*Aide aux Lepreux Emmaus-Suisse, 9 *Le Secours aux Lépreux (Canada), 1275 Spitalgasse, CH-3011 Berne, Switzer- Rue Hodge, Bureau 12, Montreal H4N land. 3H4, Canada. *American Leprosy Missions, One ALM *Netherlands Leprosy Relief, Wibaut- Way, Greenville, South Carolina 29601, straat 137K, 1097 DN Amsterdam, The U.S.A. Netherlands. *Amici dei Lebbrosi, Fondazione Italiana *Pacific Leprosy Foundation, 115 Sher- Raoul Follereau, Via Borselli 4, 40135 borne Street, Bag 4730, Christchurch, Bologna, Italy. New Zealand. Damien-Dutton Society, 616 Bedford Av- *Sasakawa Memorial Health Founda- enue, Bellmore, New York 11710, U.S.A. tion, Senpaku Shinko Bldg., 1-15-16 Toranomon, Minato-ku, Tokyo 105, *Damien Foundation (DF/APD), 16 Rue Japan. Stevin, B-1040 Bruxelles, Belgium. *Deutsches Aussatzigen-Hilfswerk e. V., Postfach 9062, D-97090 Würzburg 11, Germany.
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