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Leprosy Review

Leprosy Review

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  • Lepromin Test
  • Leprosy in Immunocompromised Individuals
  • Laboratory Tests for the Diagnosis of Leprosy
  • Basic Characteristics
  • Genome, Transcriptome, and Proteome
  • Molecular Identification by PCR
  • Epidemiology and Strain Identification
  • Overcoming Obstacles to Leprosy Research
  • Mouse Footpads and Nude Mice
  • Gene Knockout Mice
  • Nine-Banded Armadillo
  • Genetic Influences on Leprosy in Humans
  • Development of the Immune Response
  • Clinical Presentation, Diagnosis, and Histopathology
  • Immunological Features of Reactions
  • Evidence regarding the mechanisms of leprosy reactions
  • (ii) Type 2 leprosy reaction (erythema nodosum leprosum)
  • Localization of M. leprae to Peripheral Nerves
  • M. leprae Interactions with Schwann Cells
  • Current Therapies and Drug Resistance
  • Molecular Mechanisms of Drug Resistance
  • Development of Drug Resistance in M. leprae
  • Detection of Drug-Resistant Leprosy
  • Future of Leprosy Treatment and Research

The Continuing Challenges of Leprosy

D. M. Scollard, L. B. Adams, T. P. Gillis, J. L. Krahenbuhl, R. W. Truman and D. L. Williams Clin. Microbiol. Rev. 2006, 19(2):338. DOI: 10.1128/CMR.19.2.338-381.2006.

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CLINICAL MICROBIOLOGY REVIEWS, Apr. 2006, p. 338–381 0893-8512/06/$08.00 0 doi:10.1128/CMR.19.2.338–381.2006

Vol. 19, No. 2

The Continuing Challenges of Leprosy
D. M. Scollard,* L. B. Adams, T. P. Gillis, J. L. Krahenbuhl, R. W. Truman, and D. L. Williams
Laboratory Research Branch, National Hansen’s Disease Programs, Louisiana State University-SVM, Baton Rouge, Louisiana 70803 INTRODUCTION .......................................................................................................................................................339 Basic Clinical and Immunopathological Features of Leprosy .........................................................................339 Lepromin Test .........................................................................................................................................................340 Leprosy in Immunocompromised Individuals ....................................................................................................340 Laboratory Tests for the Diagnosis of Leprosy ..................................................................................................342 MYCOBACTERIUM LEPRAE, THE ETIOLOGIC AGENT OF LEPROSY........................................................343 Basic Characteristics..............................................................................................................................................343 Cellular morphology ...........................................................................................................................................343 Growth..................................................................................................................................................................343 Metabolism ..........................................................................................................................................................344 Genome, Transcriptome, and Proteome ..............................................................................................................345 Genome.................................................................................................................................................................345 Transcriptome .....................................................................................................................................................345 Proteome ..............................................................................................................................................................346 Molecular Identification by PCR..........................................................................................................................347 Epidemiology and Strain Identification...............................................................................................................347 EXPERIMENTAL MODELS OF LEPROSY..........................................................................................................348 Overcoming Obstacles to Leprosy Research.......................................................................................................348 Mouse Footpads and Nude Mice..........................................................................................................................348 Gene Knockout Mice ..............................................................................................................................................349 Nine-Banded Armadillo .........................................................................................................................................350 HOST RESPONSE TO M. LEPRAE........................................................................................................................351 Genetic Influences on Leprosy in Humans .........................................................................................................351 Genetic influences on innate resistance to M. leprae.....................................................................................351 (i) PARK2/PACRG ...........................................................................................................................................351 (ii) NRAMP1 ....................................................................................................................................................351 Genetic influences on acquired immune responses in leprosy.....................................................................352 (i) HLA .............................................................................................................................................................352 (ii) Chromosome 10p13..................................................................................................................................352 (iii) TAP ...........................................................................................................................................................352 (iv) TNFA .........................................................................................................................................................352 (v) TLRs ...........................................................................................................................................................352 (vi) VDR............................................................................................................................................................352 Development of the Immune Response................................................................................................................352 Innate immunity..................................................................................................................................................352 (i) Antigen-presenting cells and dendritic cells..........................................................................................352 (ii) Pattern recognition receptors.................................................................................................................353 (iii) C-type lectin receptors ...........................................................................................................................353 (iv) Toll-like receptors....................................................................................................................................353 (v) C receptors.............................................................................................................................................................354 Adaptive immunity: development of cell-mediated immunity...........................................................................354 (i) Protective and destructive effects of cell-mediated immunity .............................................................354 (ii) T-lymphocyte populations .......................................................................................................................354 (iii) Cytotoxic cells..........................................................................................................................................354 (iv) Macrophages ............................................................................................................................................355 (v) Cytokines in leprosy .................................................................................................................................356 LEPROSY REACTIONS............................................................................................................................................357 Clinical Presentation, Diagnosis, and Histopathology ......................................................................................357 Immunological Features of Reactions..................................................................................................................357

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* Corresponding author. Mailing address: Laboratory Research Branch, National Hansen’s Disease Programs, LSU-SVM, Skip Bertman Dr., Baton Rouge, LA 70803. Phone: (225) 578-9841. Fax: (225) 578-9856. E-mail: dscoll1@lsu.edu. 338

VOL. 19, 2006



Evidence regarding the mechanisms of leprosy reactions ............................................................................357 (i) Type 1 (reversal) reactions ......................................................................................................................358 (ii) Type 2 leprosy reaction (erythema nodosum leprosum) ....................................................................359 Evidence from therapeutic trials regarding mechanisms of reactions........................................................359 MECHANISMS OF NERVE INJURY .....................................................................................................................360 Localization of M. leprae to Peripheral Nerves...................................................................................................361 M. leprae Interactions with Schwann Cells .........................................................................................................363 Adhesion to Schwann cells ................................................................................................................................363 Ingestion by SCs .................................................................................................................................................363 Effects of SCs on M. leprae ................................................................................................................................363 Effects of M. leprae on SCs ................................................................................................................................363 Immune responses and SCs ..............................................................................................................................364 CHEMOTHERAPY.....................................................................................................................................................364 Current Therapies and Drug Resistance.............................................................................................................364 Molecular Mechanisms of Drug Resistance .......................................................................................................365 Development of Drug Resistance in M. leprae ....................................................................................................367 Detection of Drug-Resistant Leprosy ...................................................................................................................367 PCR-direct DNA sequencing .............................................................................................................................367 PCR-SSCP............................................................................................................................................................368 PCR–solid-phase hybridization.........................................................................................................................368 PCR-heteroduplex analysis................................................................................................................................368 PREVENTION: THE QUEST FOR A LEPROSY VACCINE...............................................................................368 SUMMARY AND CONCLUSIONS..........................................................................................................................371 Future of Leprosy Treatment and Research.......................................................................................................371 ACKNOWLEDGMENTS ...........................................................................................................................................372 REFERENCES ............................................................................................................................................................372 INTRODUCTION Leprosy is best understood as two conjoined diseases. The first is a chronic mycobacterial infection that elicits an extraordinary range of cellular immune responses in humans. The second is a peripheral neuropathy that is initiated by the infection and its accompanying immunologic events, but whose course and sequelae often extend many years beyond the cure of the infection and may have severely debilitating physical, social, and psychological consequences. Both aspects must be considered by clinicians, researchers, and policymakers who deal with persons affected by this disease. Leprosy is not going to disappear anytime soon. Effective multidrug regimens are now used worldwide, and the infection in individuals is curable. However, although the reported number of registered cases worldwide has declined in the last two decades, the reported number of new cases registered each year has remained the same (at 500,000 to 700,000) over the same interval (42, 237). In some countries where leprosy is endemic the number of new cases actually appears to be increasing, while in others decreasing trends are reported. Great caution must be used in reaching conclusions from these observations, however, because they are based entirely on operational data which reflect the intensity of ongoing work more than the extent of any given problem (112). Mathematical modeling of the potential decline in leprosy incidence and prevalence, using various premises regarding efficacy of treatment and prevention, suggests that the disease will remain a major public health problem for at least several decades (259). The precise mechanism of transmission of Mycobacterium leprae is unknown. No highly effective vaccine has yet been developed, and extensive laboratory efforts have not yet produced any practical tools for early diagnosis of clinically unapparent disease. The full genome of M. leprae was among the first to be sequenced, and this new knowledge is beginning to bear fruit. Molecular microbiology has begun to explain, for example, M. leprae’s fastidious nature and predilection for an intracellular lifestyle. Similarly, recent human genetic studies have been highly informative, indicating that immunity to M. leprae is controlled at two fundamental levels: first, genetic determinants of overall susceptibility and resistance to this organism have now been described, and second, a range of HLA-D-related immune responses have been demonstrated among individuals who are infected. Only recently has the probable mechanism of intracellular killing of M. leprae been identified. The regulation of cellmediated immunity to M. leprae by cellular and cytokine interactions continues to be unraveled. The major animal models available are the nine-banded armadillo and footpad infection of normal or immunologically crippled (nu / ) mice. These models, however, are seriously flawed in their ability to recapitulate many aspects of the human disease and are exceptionally slow, difficult, and expensive to employ. Leprosy therefore remains a medical and scientific challenge of the first order, even though support for research on this disease has declined substantially as other conditions have assumed greater global priority. A great deal of important new information has been generated by recent research. Brief, authoritative overviews on progress in leprosy have been published in recent years, notably those of Jacobson and Krahenbuhl (167) and Britton and Lockwood (42). Specialized reviews of narrower scope are cited in the appropriate sections below. Here, we have attempted to provide a critical summary of current knowledge from basic and clinical research, focusing particularly on developments from 1990 to the present.

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Basic Clinical and Immunopathological Features of Leprosy Leprosy presents a wide range of clinical and histopathological manifestations. This great diversity puzzled and frustrated




clinicians and investigators until it was appreciated that this diversity was based on the ability of the host to develop a cellular immune response to M. leprae. The first full formulation of this concept was described by Skinsnes as an “immunopathological spectrum” in 1964 (384). Soon thereafter, a practical classification scheme based on the same principles was proposed by Ridley and Jopling (319), enabling a degree of global uniformity in clinical practice that gave renewed impetus to research on this disease. In the same decade, the discovery by immunologists of functionally and phenotypically distinct T- and B-lymphocyte subsets and their respective roles in cell-mediated and antibody-mediated immune responses revolutionized immunology. Scientists rapidly developed an entirely new set of tools and simultaneously discovered leprosy as a challenging human disease that appeared to be an ideal model with which to examine theories and methods related to cellular immunity in humans. The convergence of these and other factors prompted an extraordinary burst of research on leprosy during the last three decades of the 20th century (355). The five-part Ridley-Jopling classification identifies, at one extreme, patients with a high degree of cell-mediated immunity and delayed hypersensitivity, presenting with a single, welldemarcated lesion with central hypopigmentation and hypoesthesia. Biopsies of these reveal well-developed granulomatous inflammation and rare acid-fast bacilli demonstrable in the tissues; this is termed the polar tuberculoid (TT) (Fig. 1). At the other extreme, patients have no apparent resistance to M. leprae. These patients present with numerous, poorly demarcated, raised or nodular lesions on all parts of the body, biopsies of which reveal sheets of foamy macrophages in the dermis containing very large numbers of bacilli and microcolonies called globi. This nonresistant, highly infected form of the disease is termed polar lepromatous (LL). The majority of patients, however, fall into a broad borderline category between these two polar forms; this is subdivided into borderline lepromatous (BL), mid-borderline (BB), and borderline tuberculoid (BT). Very early lesions may present as relatively nonspecific perineural infiltrates in which rare acid-fast bacilli can be demonstrated, but without sufficient infiltrates to classify them; these are called indeterminate. This classification should be used only when the biopsy sample shows definite diagnostic evidence of leprosy (nerve involvement and acid-fast bacilli), since a diagnosis of leprosy may often have significant impact on a patient’s family, employment, and psychological and social status. In spite of nearly three decades of intensive research into the immunology of leprosy, the mechanism by which M. leprae is able to elicit the entire range of human cellular immune responses has still not been explained. Most clinical immunological inquiries have focused on the “immunologic defect” of lepromatous patients, i.e., their apparently specific anergy to M. leprae. The broad research efforts of recent years have, however, provided an increasingly detailed description of the immunological components in skin lesions across the leprosy spectrum, detailed below under Development of the Immune Response.

Lepromin Test The lepromin test is often the cause of confusion and misplaced diagnostic expectations. The lepromin skin test is not diagnostic of leprosy or exposure to M. leprae. The test response is measured as induration (in mm) 4 weeks after injection and is ideally also evaluated by biopsy and histopathological examination of the test site. This test provides a measure of the individual’s ability to mount a granulomatous response against the mixture of antigens present. Responses to lepromin are not leprosy specific; many individuals who have never been exposed to M. leprae will develop a positive lepromin reaction. Leprosy bacilli, derived from different sources and subjected to different purification procedures, are the basis for different types of preparations used for intradermal skin testing (227). The most frequently used preparation, and the one for which the response is best characterized, is Mitsuda lepromin. This is a suspension of whole, autoclaved leprosy bacilli (357) that is injected intradermally. Early studies used bacilli isolated directly from human lepromatous lesions, but armadillo-derived organisms have been used exclusively since the 1970s. In recent years, Mitsuda lepromin has been distributed for research applications by the World Health Organization. This skin test material is not approved by the Food and Drug Administration and is not recommended or provided for diagnostic use in the United States by the National Hansen’s Disease Programs. Studies are under way to try to identify defined protein antigens that might be useful as diagnostic reagents (37, 94), but none of these has yet been determined to be satisfactorily sensitive or specific for this purpose. Although the response to Mitsuda lepromin is not leprosy specific, a negative response is associated with lepromatous types of leprosy, i.e., with an inability to respond to M. leprae and to eliminate the bacilli. A positive lepromin test (at 4 weeks) is associated with the ability to develop a granulomatous response, involving antigen-presenting cells and CD4 lymphocyte participation and, in leprosy patients, successful elimination of bacilli (121, 299). Lepromin is probably the only widely studied skin test antigen that reflects the ability of an individual to generate a granulomatous response to mycobacterial antigens (as opposed to the 48- to 72-h delayed hypersensitivity response to tuberculin and other skin tests). For this reason, the possibility of genetic influences on lepromin responsiveness has been of interest to geneticists concerned with the inheritance of immunologic aspects of the granulomatous response (8, 30, 107). Leprosy in Immunocompromised Individuals Unlike tuberculosis, leprosy has not been observed to be more frequent in patients infected with human immunodeficiency virus (HIV) in regions where both diseases are endemic (162, 238, 303). It has been suggested that this may be due to the relatively low virulence of M. leprae or that HIV-infected individuals may die before leprosy (with its long incubation time) becomes clinically apparent (238). Nelson (286) has recently urged that investigators explore alternative explanations, however, since the apparent dissociation between the two diseases has continued even as the prevalence of AIDS has increased.

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asm. 1. 2012 by guest FIG. leprae.VOL. 63). This spectrum is the yardstick against which is measured each new hypothesis and discovery regarding immunological mechanisms proposed to be responsible for the wide range of human responses to M. .and eosin-stained sections (magnification. with only occasional lymphocytes. A search of more than 50 fields was required to find the two organisms shown in a cutaneous nerve in the TT sample. Representative fields of each classification are shown in Fite-stained sections in the lower panel (magnification. The well-formed epithelioid granulomatous infiltrates seen in polar tuberculoid (TT) lesions become increasingly disorganized in each successive increment in the scale until they become completely disorganized aggregates of foamy histiocytes. in hematoxylin.000). 19. Immunopathologic spectrum of leprosy. in polar lepromatous (LL) lesions.org/ on January 16. Representative fields from each of the histopathological types of leprosy in the Ridley-Jopling classification are presented in the upper panel. 1. 2006 LEPROSY 341 Downloaded from http://cmr. and organisms are often similarly difficult to find in BT lesions.

immunosuppression with humanized monoclonal antibodies to tumor necrosis factor alpha (TNF. and identification of M.and eosin-stained sections (described above). 205. and the identification of acid-fast bacilli within nerves using the Fite-Faraco modification of the carbol fuchsin stain (70). coinfection with M. 50. leprae is not cultivable in vitro (see Basic Characteristics. and these have been used in epidemiological studies. leprae DNA in clinical specimens using PCR and other molecular techniques. do not reproducibly produce detectable. infection with M. and it is not available in the United States. although it is costly and has not yet been approved or become available as a routine clinical test. leprae. below). 230. Together. 300). but care must be exercised to identify bacilli within nerves because.asm. The occurrence of leprosy reactions in HIV-positive patients with leprosy has been the focus of several reports (18. below). where bacilli may be rare and difficult to find. but since leprosy reactions affect a high percentage of all leprosy patients (see Leprosy Reactions.org/ on January 16. even as HIV infection progresses and circulating CD4 cell numbers decline. CLIN. M. fixed in neutral buffered formalin. In tuberculoid lesions. discussed above) reflects the ability of an individual to develop a granulomatous response to this organism. Treatment of HIV infection with highly active antiretroviral therapy has resulted in the emergence of previously unsuspected leprosy in a small number of reported cases (77. a small number of patients who have been immunosuppressed for renal or heart transplantation have developed leprosy (266. Similarly. leprae may be more prevalent among HIVpositive individuals than has been generally recognized. and these have also responded well to antileprosy treatment. has been the development of methods for the extraction. leprae (46. leprae (the lepromin test. none of these assays has a satisfactory degree of sensitivity and specificity for diagnostic application. and granuloma annulare. leprae with a multidrug regimen (see Chemotherapy. Notably. the differential diagnosis of the granulomatous response commonly includes cutaneous tuberculosis. This suggests that infection with M. the involvement of cutaneous nerves. the slit-skin smear. however. specific circulating antibodies. Interestingly. Similarly. The primary characteristics to be recognized are histological patterns of the host response in hematoxylin. cutaneous infections with other mycobacteria can mimic the florid infection of lepromatous leprosy. avium. and the other mycobacterial pathogens in AIDS. but it does not reflect infection by or even exposure to M. 34. No serologic tests are available for the routine laboratory diagnosis of Hansen’s disease. These patients are presumed to have had minor. The experience with highly active antiretroviral therapy suggests that in leprosy-endemic areas.342 SCOLLARD ET AL. 97).) has resulted in the rapid development of lepromatous leprosy in at least two individuals who were being treated for severe arthritis (355a). In addition. The greatest drawback to the serologic diagnosis of leprosy arises from the fact that patients in whom the diagnosis is most difficult. bacilli are easily demonstrated in the infiltrates of polar lepromatous leprosy. many of these individuals have also developed type 1 reactions. leprae may elicit antibodies cross-reactive with HIV screening assays (13. This technique is reliable only when performed and interpreted by experienced technicians. and examined by an experienced pathologist. unpublished observations). 372). however. REV. but that early leprosy lesions are overlooked when these patients are confronted by the other major. Scollard. Enzyme-linked immunosorbent assays and related immunoassays have been developed to detect antibodies to phenolic glycolipid 1 (PGL-1) of M. It is possible that the very slow growth of M. it is not clear that they are actually more frequent or more severe in HIV-positive individuals. MICROBIOL. 298. comparable to that of non-HIV-infected individuals. life-threatening complications of AIDS. leprae may be more common than has been documented among HIVpositive individuals in leprosy-endemic regions of the world. as noted above with AIDS patients receiving highly active antiretroviral therapy. leprae. An ancillary procedure. Studies of cell phenotypes and cytokines in leprosy lesions in patients with and without HIV infection have found no significant differences between the two groups with respect to these immunologic parameters (136. reviewed in reference 221). It has been used in epidemiological studies. undetected leprosy lesions prior to the administration of immunosuppressive treatment. 230) and notably. Laboratory Tests for the Diagnosis of Leprosy The “gold standard” for the diagnosis of leprosy is a fullthickness skin biopsy sample obtained from the advancing margin of an active lesion. and lack of growth on standard mycobacterial isolation media can be regarded as one laboratory criterion differentiating this organism from other mycobacterial pathogens. below). 329). This is best illustrated by studies following cohorts of infected patients (162. it appears that the restoration of immune function after highly active antiretroviral therapy may be associated with the development of type 1 reactions. A Downloaded from http://cmr. 31. tuberculosis. leprae and HIV appears to have minimal effect upon the course of either leprosy or HIV/AIDS. but it has no diagnostic utility in individual cases. TT and BT patients with moderate to highgrade cellular immunity to M. M. no skin test that enables the diagnosis of Hansen’s disease has been developed. in immunosuppressed individuals. 15. the evidence indicates that broad therapeutic immunosuppression does render individuals highly susceptible to infection with M. This is an invaluable addition to laboratory diagnosis and to studies of the basic microbiology of this uncultivable organism. Although they have some value in population follow-up studies. amplification. can be used for the semiquantitative enumeration of acid-fast organisms in infected skin and is useful in follow-up of patients during and after treatment. They responded promptly to antimicrobial treatment for M. leprae and only small numbers of organisms. leprae is maintained. but that in HIV-positive individuals a degree of host response to M. 298. but did develop type 1 reactions during their recovery. At the other extreme. leprae allows the host immune response to keep pace with this infection to a much greater degree than is the case with M. subclinical and early clinical infection with M. and no laboratories in the United States perform such assays routinely. Intradermal injection of heatkilled M. sarcoidosis. embedded in paraffin. 2012 by guest . The major advance in the laboratory diagnosis of Hansen’s disease in the last 15 years. In contrast to all other experience with mycobacterial infections in HIV-positive individuals.

composed of chains of alternating N-acetylglucosamine and N-glycolylmuramate linked by peptide cross-bridges. 425) (Fig. suggesting a role for PGL-1 in peripheral nerve-bacillus interactions (288). Magnification. bovis BCG. leprae has been restricted to animal models. The cell wall core contains peptidoglycan. Magnification. leprae’s genome and comparative genomic studies with other bacterial genomes have produced insight into the putative genes needed to direct the synthesis of this complex cell wall biopolymer (38). while speculative. Magnification. B. The organisms are seen here within a human nerve. a gene (fabH) involved in meromycolate synthesis. forming. an electron-dense zone around M. The dominant lipid in the cell wall which gives M. 3). and gene Downloaded from http://cmr.000. leprae under the scanning electron microscope. lipomannan. like other mycobacteria. and M. M. tuberculosis. non-sporeforming. 98. which reveals the surface of the organisms. As a result. and a glycosyltransferase gene (pimB) involved in the biosynthesis of phosphatidylinositol.org/ on January 16. shorter beaded or granular shapes are observed when the bacilli are dead or dying. counterstained with methylene blue.asm. forming a capsule-like region. below). leprae DNA is presented below (see Molecular Identification by PCR. leprae are observed in this ultrathin section of the bacilli under a transmission electron microscope. microaerophilic. leprae. THE ETIOLOGIC AGENT OF LEPROSY Basic Characteristics Cellular morphology. but basic chemical studies have concluded that the cell wall is a covalently linked peptidoglycan-arabinogalactan-mycolic acid complex similar in composition to all mycobacterial cell walls (79. avium. smegmatis. M. leprae with the laminin of Schwann cells. It should be noted that much of this comparative work. athymic.000. Growth. M. rod-shaped organisms. leprae. M. and lipoarabinomannan. provides an important framework from which to investigate the authenticity of these putative pathways. Most of the genes necessary to build the peptidoglycan-arabinogalactan-mycolate polymer appear to be present in the M. phosphatidylinositol mannosides. leprae has never been grown on artificial media but can be maintained in axenic cultures in what appears to be a stable metabolic state for a few weeks (414). along with the peptidoglycan layer. Morphology of M. leprae. leprae is weakly acid fast but. approximately 800. M. A suspension of nude-mouse footpad-derived M. Similar approaches with M. tends to cluster. propagation of M. C.VOL. . A few exceptions are two genes involved in polyprenyl-phosphate synthesis (dxs-II and idi). 29. A. when stained with the Fite-Faraco method. Mycolic acids are linked to the termini of arabinan chains to form the inner leaflet of a pseudolipid bilayer. The outer leaflet is composed of a rich array of intercalating mycolic acids of trehalose monomycolates and mycoserosoic acids of phthiocerol dimycocerosates as well as phenolic glycolipids (PGLs). leprae genome and fit a reasonable strategy for its construction. approximately 12. A great deal has been learned about the nature of the mycobacterial cell wall through biochemical and genetic manipulation of cultivable strains such as M. which is linked to the galactan layer by arabinogalactan. Recent studies suggest that PGL-1 is involved in the interaction of M. Internal features of M. 2006 LEPROSY 343 detailed discussion of PCR evaluation of specimens for M. M. The round and oval images seen in the upper portion of this photograph are bacilli that have been cut in cross section. It has been postulated that many of these same molecules together with phosphatidylinositol mannosides and phospholipids are released from the cell wall after synthesis. 2012 by guest FIG. MYCOBACTERIUM LEPRAE. 19. leprae have been meager by comparison. leprae is a nonmotile. it appears as red. Three branched chains of arabinan are in turn linked to the galactan. leprae immunological specificity is PGL-1. 2). Annotation of M. 2. including the armadillo (415) and normal. acid-fast-staining bacterium that usually forms slightly curved or straight rods (Fig. forming the electron-transparent zone.

An outer leaflet is formed by the mycolic acids of trehalose monomycolates (TMM) and mycocerosoic acids of phthiocerol dimycocerosates (PDIMs) and PGLs as shown. phosphatidylinositol mannosides. 414). M. Overall. With the completed sequencing and annotation of M. and phospholipids surrounds the bacterium. A capsule presumably composed largely of PGLs and other molecules such as PDIMs. The plasma membrane is covered by a cell wall core made of peptidoglycan covalently linked to the galactan by a linker unit of arabinogalactan. leprae stored at 33°C in 7H12 medium has been shown to remain viable for weeks. and many standard laboratory practices. metabolic. MICROBIOL. These systems have provided the basic resources for genetic. an improved understanding of M. and acetyl-coenzyme A synthase. tuberculosis suggest that these organisms rely heavily upon lipid degradation and the glyoxylate shunt for energy production. rapidly reduce the viability of this organism (414). Acetate has been lost to M. REV. and antigenic studies of the bacillus. The primary reasons for investigating the metabolic aspects of M. leprae contains a full complement of genes for B oxidation but. leprae. phosphate acetyltransferase. M. In addition to glycolysis for energy production. Schematic model of the cell envelope of M. Early work provided a picture of a bacterium with some basic anabolic and catabolic pathways needed for survival in the host. supporting earlier biochemical observations. compared to M. leprae as a carbon source since only pseudogenes are present for acetate kinase. leprae and M. leprae has many fewer enzymes involved in deg- . CLIN. Acetyl-coenzyme A from glycolysis enters the Krebs cycle. such as incubation at 37°C. 3. 2012 by guest FIG. Growth of M. very few genes capable of lipolysis. However. Annotation of the genome identified genes showing that M. Mycolic acids are linked to the termini of the arabinan chains to form the inner leaflet of a pseudolipid bilayer. and lipoarabinomannan (LAM).344 SCOLLARD ET AL. Downloaded from http://cmr. leprae in mouse footpads also provides a tool for assessing the viability of a preparation of bacteria and testing the drug susceptibility of clinical isolates (364. The viability of M. In this regard. leprae harvested from several different sources is now known to vary greatly. (Reprinted from reference 425 with permission of the publisher.org/ on January 16. 432). Metabolism. producing energy in the form of ATP. leprae’s genome. leprae’s metabolic potential was still lacking. leprae have been to determine whether special media could be formulated to support in vitro growth of the bacilli and to learn more about metabolic pathways that could potentially be exploited for developing new antileprosy drugs. Three branched chains of arabinan are in turn linked to the galactan. genome analysis as well as biochemical studies in M. tuberculosis. lipomannan (LM).) knockout mice (222). are also found in the capsular layer as shown. known to be anchored in the plasma membrane. Lipoglycans such as phosphatidylinositol mannosides. leprae’s metabolic capabilities now exists (43. but a thorough assessment of M. 105. leprae has the capacity to generate energy by oxidizing glucose to pyruvate through the Embden-Meyerhof-Parnas pathway. M.asm.

and short-chain alcohol dehydrogenases and their probable regulatory genes. most amino acids. complete pathways are predicted for synthesis of purines. rendering it unable to make either the membraneassociated or excreted form of mycobactin T. extremely limited quantities of bacterial proteins can be purified for analysis.008 91. compared to six pseudogenes in M. leprae utilizes iron. and 34% of M.000 bpd AL123456 4. The maintenance of these anabolic systems suggests that the intracellular niche that M. 2012 by guest a Data obtained from the Current Data Release (17 October 2003) for the M. One of the most striking features of M. leprae appear relatively unharmed.024 1. While comparative genome analysis provides useful clues to identify deficits in general cellular metabolic potential and cellular composition.fr/TubercuList/).106 1.133 pseudogenes. of pseudogenes No.fr/Leproma/).2 65. metabolism and modification of fatty acids and polyketides.133 45 3 2 2. there is little doubt that M. Transcriptome. Defense against toxic radicals is severely degenerative. Genome. Annotation of M. compared to M. tuberculosis are pseudogenes in M. nucleotides. single-nucleotide polymorphism.6 1 in 3. 105). leprae axenically. leprae (strain TN)a M. it offers only a starting point from which functional studies can proceed. In addition.411. several other genes involved TABLE 1. Redundancy as seen in the M.614 142 1. Because of our inability to cultivate M. those genes that are expressed help define the min- . While genes known to be involved in iron acquisition are not obvious in its genome. leaving a pathogen with very specific growth requirements. In addition. leprae genome (http://genolist. Annotation of M. leprae insert DNA in pUC18 (74). leprae appear to be associated with metabolic pathways. leprae that was originally purified from the skin lesions of a multibacillary leprosy patient from Tamil Nadu. India (TN strain). of stable RNA genes Gene density (bases/gene) Avg gene length (bases) % Protein coding %G C SNP c frequency AL450380 3. leprae’s genome is that it possesses 1. pyrimidines. compared to 3. Genes are present for cytochrome c (ccsAB). There are no intact polymorphic G Crich sequence. and Proteome Genome. biosynthesis of the heme group (hem genes). of protein genes No. leprae versus 4.614 open reading frames potentially encoding functional proteins. Genome size (bp) No. leprae’s genome has thus provided insight into why the leprosy bacillus is an obligate intracellular parasite and has provided the basis for future experimentation to better understand its pathogenicity.133 inactivated genes (genes lost through mutation. nucleosides. c SNP.5 57.8 1 in 24. or pseudogenes). leprae’s genome has revived interest in basic investigations of its metabolic.or major polymorphic tandem repeat-related repetitive sequences in the genome of M.asm. Comparative genomics of M. leprae’s annotated genome contains only 1. tuberculosis Parameter M. tuberculosis. the iron storage bacterioferritin bfrA.VOL. tuberculosis. The result of this massive gene loss leaves M. tuberculosis (72). e Data obtained from reference 114. and ideR. other major problems associated with metabolism for M. leprae’s genome with that of its close relative M. 2006 LEPROSY 345 radative pathways for carbon and nitrogenous compounds than M. leprae.pasteur. cell envelope synthesis. tuberculosis (Table 1). of tRNA genes No.4 Mb for M. leprae.268. tuberculosis) and a major reduction in G C content (58% for M. M. of unknown genes No.000 bpe Downloaded from http://cmr. as discussed above (Metabolism). leprae has undergone an extreme case of reductive evolution (reviewed in references 74 and 104). leprae has found for itself may not contain these compounds or transport systems. Transcriptional analysis of M. and transport of metabolites (74. the anabolic capabilities of M. tuberculosis genome is often lost in M. leprae are that the bacilli have lost anaerobic and microaerophilic electron transfer systems and that the aerobic respiratory chain is severely curtailed. 19.org/ on January 16. with detoxification are pseudogenes or are missing from the genome. This is reflected in the paucity of oxidoreductases. leprae are those involved in gene regulation. For example. leprae’s proteins identified in silico appear to be the products of gene duplication events or share common domains (105). leprae versus 66% for M. as neither katG nor the narGHJI cluster is functional. tuberculosis (Table 1) suggests that M. The largest functional groups of genes in M. tuberculosis (strain H37Rv)b EMBL/GenBank/DDBJ accession no. thereby expanding our knowledge of genes expressed during infection that might otherwise be missed when examining M. In contrast to the reduction in catabolic pathways. a ferredoxin (fdxCD). leprae with less than 50% of its genome encoding functional genes.3 Mb for M.532 3. leprae to generate ATP from the oxidation of NADH. tuberculosis genome (http://genolist. Comparison of M. leprae genes provides a perspective that is complementary to protein analysis by identifying actively transcribed genes. and most vitamins and cofactors. tuberculosis). leprae and only a limited number of proline-proline-glutamic acid and prolineglutamic acid proteins (72). of rRNA genes No. and pathogenic potential. d Data obtained from reference 272. Downsizing of the genome has resulted in the elimination of several metabolic pathways. The genome sequence was generated by sequencing a combination of Lorist6 cosmid library inserts (103) and sixfold whole-genome shotgun sequencing of M. In addition. as most paralogues seen in M. M. oxygenases. Transcriptome.pasteur. 104. With less than 50% coding capacity and 1. in which 90% of the genome encodes functional genes. b Data obtained from the Current Data Release (20 July 2005) for the M. leprae genome (74). a large number of genes apparently have been entirely deleted from the genome. leprae and M. This is reflected by its smaller genome (3. biochemical. leprae’s proteome.007 49. None of the additional 142 genes found only in M.993 open reading frames predicted in M. leprae appears to have a major deficiency in its ability to acquire iron from its environment. and subsequently expanded in and purified from the liver of a nine-banded armadillo provided the source of DNA for sequencing of the M.203 1. M. the iron regulation protein dependent on intracellular iron (432). making it impossible for M.993 606 6 45 3 2 1. a hemogloblinlike oxygen carrier (glbO). The entire mbt operon is deleted.

338. with a small percentage designated unknown genes. Of the two strategies. leprae has become an important model for conceptualizing the minimal gene set needed for obligate intracellular parasitism. Understanding the basis of M. and iron transport and storage and genes associated with virulence (see supplemental Table S1 at http://www. leprae genome in 2001 (74) protein analysis of M. leprae’s ability to cause disease. MICROBIOL. To identify genes transcribed during infection. and transcribes several genes associated with virulence in M. Transcript levels of nine of these potential virulence genes (aceA. including approximately 1. leprae (74). Clark-Curtiss used an alternative genomic screening approach in which M. cell division. coli. and mce1A) were compared in M. The other half of M. virulence. Proteome. leprae has the potential to produce secreted proteins. leprae actively catabolizes fatty acids for energy. leprae expressed in Escherichia coli. suggesting that profiling of M. imal gene set necessary for in vivo survival of this mycobacterial pathogen as well as genes potentially required for infection and pathogenesis as seen in leprosy. For example. leprae proteins. This led to the discovery of a 46-kDa protein from M. In addition. together with their regulatory circuits. aphC. Identifying genes associated with growth and survival during infection should lead to a more comprehensive understanding of M. leprae gene expression in E.5% of the genome. SecA-dependent protein secretion. By comparative genomic analysis with M. Therefore. soda. produces a wide array of secretory proteins. providing powerful probes for antigenic proteins of M.600 open reading frames are scattered among a decaying genome. energy production. leprae promoter activity was possible in E.org/ on January 16. leprae relied primarily on traditional subcellular fractionation of purified bacilli and genomic library screening using immunologic reagents. leprae growing in athymic nude mice have been surveyed using reverse transcription-PCR and cross-species DNA microarray technologies with an M. leprae with immunologic screening and chemical analysis of purified proteins using microsequencing and mass spectrometry to expand our understanding of the cellular location and function of many M. The functional complement of any genome is the proteome. tuberculosis and other known gene sequences. tuberculosis.346 SCOLLARD ET AL. immunologic screening of genomic libraries proved more fruitful and expanded the number of purified and characterized proteins to over 40 (41. produces several proteins involved in iron transport. leprae’s growth. leprae derived from the lesions of multibacillary leprosy patients and from infected nude mouse footpad tissue using quantitative realtime reverse transcription-PCR. it appears that approximately 50% of M. leprae’s open reading frames can be assigned putative functions. above). relA. Annotation of the M. leprae with the purpose of identifying proteins useful for diagnostics and vaccines. The power of this new portrait of M. leprae has lost many metabolic pathways. mouse. tuberculosis microarray (440). leprae genes were examined for expression in E. bioinformatic analyses of metabolic pathways indicated that M. leprae genome indicates that approximately 1. coli (165) and the demonstration that M. storage. Bioinformatic approaches have identified the basic genes necessary for a functional SecAdependent secretory system in M. Early work was driven by efforts to produce vaccines and diagnostic reagents. with the goal of developing strategies for improved treatment and control of leprosy. particularly those involved with catabolic potential (104). 131. Proteins have been identified from major cellular compartments. Much of this work has now been assimilated into a larger framework established from the completion of the M. a group of M. leprae capable of complementing a citrate synthase mutant of E. while genetic complementation in E. most of which have not been studied for immunogenic potential. gene transcripts from M. Purified bacilli from armadillo. with gene remnants making up the remaining 23. which consists of the proteins expressed by a given organism under defined conditions. and immunogenicity has always been the focus of proteomic discovery. including cytosol. A major drawback to immunologic screening was that nonimmunogenic proteins were missed. and a comprehensive list of these proteins and their characteristics was published earlier (248). leprae genome sequence. leprae’s Downloaded from http://cmr. coli had been very successful for identifying genes in other enteric bacteria. Finally.html). leprae’s genes are considered to encode hypothetical proteins. Williams and coworkers (440) have shown that all genes in the SecA-dependent pathway are transcribed during growth of M. 407). Aided by this metabolic picture. and human tissues by definition lack (or are significantly reduced in their concentration of) secreted proteins. coli (356). leprae helps integrate what the bacterium can and cannot do. and cell wall. sigE. leprae were abundant.asm. leprae impacts earlier work involving antigenic analysis of M. 2012 by guest . researchers can now investigate gene expression as it relates to various metabolic conditions either in limited culture with M. leprae in nude mouse footpads and that some 25 proteins with potential for secretion are transcribed (see Transcriptome. Brennan and coworkers have combined subcellular fractionation of M. it appeared that problems associated with either M. Another important element of establishing the proteome of M. It is unlikely that all of M. fbpA. membrane. REV. which include genes involved in DNA replication. esat6. CLIN. sigA. Transcripts were detected for 221 open reading frames. coli or the relatively large evolutionary distance between the two bacteria limited further application of the approach. leprae or by studying gene function in cultivable mycobacteria into which specific mutations have been introduced. utilizes the limited array of sigma factors available. In this way it may be possible to determine the conditions that enhance or suppress M. leprae viability when it is held in axenic culture. intermediary metabolism. A full assessment of these proteins may give rise to important antigens useful for developing muchneeded early diagnostic tests as well as therapeutic and prophylactic vaccines. Prior to the publication of the full DNA sequence and annotation of the M.100 pseudogenes. Gene transcript levels were comparable in the two isolates for all but one of the genes studied (esat6). by virtue of its relatively small gene set and possibly smaller proteome.org/Mycobacterium. leprae genes from animal models such as the nude mouse should be continued. M. Unfortunately. For example. Immunologic screening took advantage of the fact that serum and T cells from leprosy patients or healthy individuals previously sensitized to M. and regulation (in the absence of recognizable genes encoding iron scavengers). leprae proteins that have gone understudied until recently is secreted proteins. M.leprosy-ila. These results support the view that M.

..... leprae is sometimes problematic....................................................... a wide range of molecular tests have been applied to reveal genotypic variation in M........ 109 Blood...........VOL.............. 2012 by guest TABLE 4........... leprae and tracing transmission patterns.... However.... 409 Nasal smears ................... and 348). onset of infection........................ frozen at 80°C indefinitely...Specimens from newly diagnosed............Freshly acquired and processed immediately.............. unfixed (and unfrozen) specimens ....32...... On the basis of extensive assessment of these tests in field studies..... leprae-specific sequences using PCR and identification of the M..... leprae isolates ob- Downloaded from http://cmr........asm..... Genetic markers may hold the key to establishing species..... Automation of PCR-based assays has allowed their implementation in many reference laboratories.. leprae in clinical specimens.... leprae cannot be cultured in vitro............ leprae was highly conserved........ 90............. Many different M.............................. Epidemiology and Strain Identification Understanding the epidemiology of leprosy is a prerequisite for effective control of the disease.................. 436)................... This technique has been applied not only to skin biopsy samples but also to several different types of specimens. Shin et al..................... leprae genes have been utilized in the development of PCR assays for detection of M..... it has been virtually impossible to assess exposure....... but the value of these elements for differentiating possible M...... 297......... Over the last two decades.............68. These tools should be helpful for improving our understanding of the epidemiology of leprosy......................... A polymorphic structure in the polA gene (119) and variation in a GACATC repeat in the rpoT gene (252) have been described..... which could give rise to better diagnostic reagents and potentially a vaccine to improve our chances of managing leprosy.... refrigerated specimens.... Rapid molecular-type assays have been developed for detection of M........... as indicated in Table 2.. RNA analysis using 16S rRNA and reverse transcription-PCR has the added benefit of measuring viability posttreatment (146........ 194 Skin biopsies . PCR-based and reverse transcription-PCR-based techniques have shown a specificity of 100% and a sensitivity ranging from 34 to 80% in patients with paucibacillary forms of the disease to greater than 90% in patients with multibacillary forms of the disease................. reported evidence for diversity among M... 228 open reading frames are expressed during all phases of growth and parasitism. leprae (see Table 3)............... clinical history.. Restriction fragment length polymorphism analysis of M............ This problem is confounded today by the increased prevalence of other mycobacterial infections of the skin...... since the organism is not cultivable............ fixed in 10% formalin for 24 h and paraffin embedded.........17...435 ag36. Teasing apart these intricate relationships should provide important insights into mechanisms of virulence.. the sequence of events that must occur for successful transmission of leprosy is poorly understood... leprae and.......68......... clinical history....... 301 16S rRNA ........Identification of acid-fast organisms when bacilli are numerous but tissue site..... and various aspects of disease progression........................ untreated.... has the ability to provide rapid drug susceptibility results from specimens taken directly from the patient............. such as nerve infection.....62 Ocular (iris) lesions. leprae strains appears to be limited.... Molecular Identification by PCR Definitive identification of M........ 150..... 170 rRNA 16S rRNA .....................143............ leprae genome.............. leprae strains.. 435 Paraffin-embedded skin biopsy samples .............................. 208........................ 318 Noncoding sequences RLEP ... M.... as well as identifying proteins expressed during early infection...... fixed in 70% ethanol and stored at room temperature for up to 2 yr Unsuitable specimens .......54 Gene hsp18..........................To find bacilli that are not identifiable in good-quality Fite-stained sections Biopsies suitable......... frozen in over-the-counter cryopreservative at 80°C....................................... The results of initial studies suggested that the genome of M... 19.. (74) identified several tandem repeats that could prove useful for discriminating M.... Application of PCR for the detection of M.......and strain-specific markers for assessing exposure to M.Biopsy samples from treated leprosy patients...... leprae....... Therefore..150 groEL1... or other circumstances are questionable............. leprae PCR Category Description PCR indicated ..... Cole et al............. bacilli are sparse and tissue site. These assays have been based primarily on the amplification of M. 91... Since M............ leprae isolates using a combination of restriction enzymes and probes and sequencing of the internal transcribed spacer region of the 16S-23S rRNA operon yielded no polymorphic DNA sequences (69... leprae directly from patient specimens using available genetic data (reviewed in references 132..... leprae in human specimens Tissue sample Reference(s) LEPROSY 347 TABLE 3.. Indications and suitable specimens for M. coupled with mutation detection analyses.................... 2006 TABLE 2..................... leprae genes used in the development of PCR assays Template for assay Reference(s) Skin smears ............ or other circumstances are questionable PCR not indicated ......... chiefly in countries with endemic leprosy................ Recently...... as summarized in Table 4.......... 92... or relapsed leprosy patients not yet retreated Suitable specimens . As a consequence.......334 Nerve lesions........ 228)..194............... PCR can provide an excellent adjunct to clinical and histopathological diagnosis of leprosy.........17.146. PCR has thus generated new approaches to the detection and identification of M... leprae DNA fragment...org/ on January 16............. one should not infer from this that any tissue or specimen is suitable for PCR-based detection of M..... In completing the sequence of the M....... fixed in 10% formalin for 24 h........

various saltwater fish. In athymic nu/nu mice. and knockout murine models was established. In addition to the TTC locus. 208. a resource unprecedented in almost 130 years of leprosy research. leprae strains used in the laboratory and successfully grouped identical samples and passage samples tested in blind panels (412). CLIN. lovebirds. A large ( 200 mice) colony of M. snakes (including rattlesnakes). a novel. leprae appears to be unimpeded. the athymic nu/nu mouse footpad allowed the routine culture of large numbers of highly viable M. A new era was entered with description of the mouse footpad model by Shepard in 1960 (363). A protocol of rigorously programmed passage of M. Passage of M. toads. in addition to its immunological significance. “dancing” mice. Mouse Footpads and Nude Mice Shepard’s demonstration of the multiplication of M. rats. An important research emphasis in the National Hansen’s Disease Program laboratories over the past few years has been the production. tadpoles. chipmunks. leprae. some dissemination does occur. The disease appears to have originated in eastern Africa or the Near East and spread with successive human immigrations (272). quantitative. leprae inoculum. Among the 64 possible permutations of different bases at each of these polymorphisms. albino hamsters. Unlike the course in an immunologically intact mouse. the minimal successes reported could have been due to local lepromin-like responses. with radiorespirometry (the oxidation of 14C-labeled palmitic acid) as a measure of viability. an inoculum of a few thousand bacilli grows locally and plateaus at approximately 1 million organisms per footpad.e. leprae in the footpads of Carworth Farms white mice (363) opened new opportunities for investigation into basic immunological mechanisms of host resistance as well as screening of antileprosy drugs and drug combinations and detection of drug-resistant strains of M. The importance of the T lymphocyte in host resistance was revealed in experimental M. leprae for cooler temperatures (4°C for storage. and the cells are engorged with bacilli (61) (Fig. a variety of nonhuman primates. 229. the dark ages. An exhaustive yet incomplete list of animal species tested as models for leprosy begins with rabbits infected by Hansen and includes dogs.348 SCOLLARD ET AL. on a routine (weekly) schedule. leprae-infected athymic nu/nu mice is maintained for this purpose. and the histopathological changes are minor. there is essentially no dissemination of infection. turtles. goldfish.org/ on January 16. cats.asm. pigeons. and provision of viable Mycobacterium leprae for our own researchers and qualified investigators around the world. REV. in silico analysis of the genome sequence indicates that M. tained from several patient biopsy samples in the Philippines. drug evaluation and rudimentary immunology studies became feasible. pigs. The variation in singlenucleotide polymorphism genotype is highly correlated with the geographic origin of the strain.. With these organisms we have confirmed the preference of M. has enabled the harvest. reliable. development of this mouse model was a second major milestone in leprosy research for. chickens. and post1960. rainbow perch. characterization. gerbils. and this has been shown to be correlated with growth in the mouse footpad. Recent studies employing four different variable-number tandem repeat markers have successfully differentiated M. white mice. which usually consisted of fresh or frozen homogenates of nodules and lesions from untreated human lepromas. golden hamsters. Histopathologically. and if observed for a long enough interval. The second impediment in the early attempts to develop a leprosy model in animals was failure to recognize the prolonged growth cycle of M. and analysis of strains from different continents has been useful in predicting the evolution and global spread of leprosy. eels. leprae for experimental use (208. leprae infection in various immunocompromised. however. Furthermore. No serial passage was reported and. 315). black mice. and the basis for subsequent exploration of M. leprae infection was achieved. The M. EXPERIMENTAL MODELS OF LEPROSY Overcoming Obstacles to Leprosy Research Rees (315a) divided experimental leprosy research in animal models into two eras: 1874 to 1960. This confirms previous findings regarding biophysical optima for maintaining M. based on the frequency of TTC repeats located downstream of a putative sugar transporter pseudogene (371). i. In immunocompetent mice. leprae. frogs. 2012 by guest . leprae and not acknowledging its preference for cooler body sites. reaching 1010 or more bacilli per footpad. transgenic. MICROBIOL. leprae. leprae single-nucleotide polymorphism frequency ( 1 per 28 kb) is among the lowest seen for a human pathogen. in hindsight. canaries. leprae infection of neonatally thymectomized or congenitally athymic mice and rats (75. Thus. Far less diversity is seen with regard to single-nucleotide polymorphisms within the genome. evidence of growth in the opposite hind footpad or forefeet is seen. or leproma. paddy birds.” but these were usually abortive or at best inconclusive and unconfirmed findings. of 4 to 6 billion bacilli that are 80 to 90% viable. leprae. One obstacle to the development of an animal model for leprosy has been the poor quality of the M. leprae. only 4 are observed. and only three informative single-nucleotide polymorphisms have been identified. twocolor fluorescence viability staining assay (Molecular Probes BacLight bacterial viability kit) has recently been adapted to provide a rapid ( 1-h). because of the relative indestructibility of even dead M. Europeans and North Africans appear to have introduced leprosy into West Africa and the Americas within the last 500 years. parrots. leprae has several other tandem repeat loci which may provide the genetic diversity necessary for creating a typing scheme capable of answering important questions related to the epidemiology of leprosy. 414) (see Basic Downloaded from http://cmr. There is virtually no disease in these footpads. local footpad multiplication of M. Whereas radiorespirometry measures the metabolic activity of a suspension of M. 26°C to 33°C for metabolic activity) and the rapidly deleterious effects of incubation at 37°C or a single freezing-thawing cycle (414). the infected footpad tissue becomes an enormous foreign body-type macrophage (M ) granuloma. after the Shepard mouse footpad model. and guinea pigs (190). direct-count viability assay that measures the cell wall integrity of individual bacilli (229). Experiments employed a confusing myriad of protocols with widely variant reports of “success. 4). consisting of small granulomas containing a few lymphocytes and very few bacilli.

or cell surface markers. We have examined several strains of KO mice in our studies on M. Gene Knockout Mice The importance of the production of Th-1 cytokine responses in host resistance to intracellular infections. multiple KO. contained epithelioid M and scattered lymphocytes that were not assembled into organized granulomas. dense. experimental M. including severe combined immunodeficiency mice. leprae initially grew to slightly higher levels than seen in wild-type controls. including those unable to produce specific cytokines and chemokines. leprae (7). are continually being developed. M. and granulomas in liver tissue demonstrated a pattern similar to that seen in human leprosy lesions (268) with CD4 T cells distributed throughout the lesion. Perhaps the best characterized is the inducible nitric oxide synthase (iNOS) KO (NOS2 / ) strain. their receptors. 1. which lack both T and B cells (22). B. the M. M isolated from NOS2 / mice are incapable of producing reactive nitrogen products and they cannot inhibit M. and infected phox91 / mice develop a footpad induration similar to that of wild-type mice (Adams. 4. submitted for publication). surrounded by CD8 T cells (D. Hagge et al. Enlarged nude-mouse footpad 6 months after infection with 5 Heavily infected macrophages harvested from mouse footpad (magnification. Cultivation of M. More recently. Numerous targeted gene KO strains are now commercially available. When inoculated into NOS2 / mouse footpads. An invaluable means for studying immunological parameters of host defense is via gene transfer technology in the murine system. Characteristics. The resulting knockout models allow investigation at the level of the direct effects due to the loss of the functional gene as well as the compensatory mechanisms operational in its absence. In addition. However. but thereafter the course of infection was similar. and knockin mutants. the granulomas formed in NOS2 / mice contained large.asm. Scollard. immune modulators. leprae. was confirmed with the recently identified human genetic deficiencies in cytokine receptors (420). 19. leprae infections in cytokine knockout (KO) mice have substantiated the immunological importance of these cytokines across the leprosy spectrum and revealed compensatory mechanisms of host resistance to M. leprae in vitro. although they are fully competent in producing reactive oxygen products (3). leprae. Clinical evidence for an altered course of leprosy in such individuals has not been demonstrated. conditional KO. other immunosuppressed strains of mice and rats have been reported to allow enhanced growth of M.. leprae. 2012 by guest FIG. Growth of M. leprae was enhanced approximately 1 log over that in wild-type mice and a Th-2-type cytokine profile was generated. macrophages from mice that are superoxide deficient due to a defective gp91 subunit of the phagocyte oxidase (phox91 / ) (93) efficiently kill M. in contrast. Overall. focal collections of mononuclear cells. however. In addition.VOL. unpublished).org/ on January 16. in contrast to NOS2 / mice. Gamma interferon (IFN. tuberculosis and opportunistic mycobacteria. above). leprae in mouse footpads. new generations of mice. 107 live M. Th-1-type cytokine expression was significantly augmented in NOS2 / mice. The footpad. including M.000). Overall. and Krahenbuhl. The granulomas formed in wild-type mice in response to M. 2006 LEPROSY 349 Downloaded from http://cmr. leprae. leprae growth and granuloma development in experi- mental leprosy./ ) mice also exhibited enlarged footpads and enhanced cellular infiltration upon infection with M. leprae-infected NOS2 / mouse model exhibits findings similar to those of BT leprosy in humans. A. leprae metabolic activity in vitro. A. leprae infection consisted of only small. the gen- . organized collections of epithelioid cells and lymphocytes which infiltrated the perineurium and destroyed muscle bundles (6).) KO (IFN. although this is most probably due to the protracted course of the disease and the relative avirulence of M. Through a variety of mechanisms a specific gene can be rendered either totally or conditionally inactive (244). Interestingly. including tissue-specific KO.

tumor necrosis factor receptor. and other tissues.org/ on January 16. CD4. which include both diapausic development and polyembrony (394).350 SCOLLARD ET AL. as well as notable involvement of the lips. and CD8 (Adams. The granulomatous response of armadillos to M.140 days) for effective challenge studies with armadillos currently limit the utility of armadillos as vaccine models. and they may be a source Downloaded from http://cmr. and armadillos are useful models for studying both susceptibility and the variable resistance to leprosy that may result from treatment or vaccination (185. whereas bacillary growth was augmented in mice deficient in IL-12. skin. peripheral nervous system. leprae-laden macrophages throughout the liver. Immunoregulation in these mice is being studied further by additional “conditional” approaches. Little evidence for M. did not reach the enormous levels seen in the T-cell-deficient mouse models. and armadillos have been the hosts of choice for in vivo propagation of M. Intravenous inoculation with 108 to 109 bacilli regularly results in 10. Surveys conducted over the last 30 years on more than 5. Armadilloderived lepromin (lepromin-A) can be used effectively to index the cell-mediated response of individual animals and to classify their type of leprosy according to the Ridley-Jopling scale. 188. leprae for more than 30 years. the infection of peripheral nerves in the armadillo constitutes a unique model of lepromatous nerve involvement in humans (350. 413). The nine-banded armadillo (Dasypus novemcintus) is the only immunologically intact animal that regularly develops fully disseminated M. leprae growth in these KO footpad models. However. tongue. nasal mucosa. The ecological relationship between humans and armadillos with M. With high burdens of bacilli in their reticuloendothelial tissues. leprae. Like humans. leprae infection is found among armadillos elsewhere in the U. 214. vaccination. leprae (413). the number of armadillos and duration of time required (up to 1. In addition to the armadillo’s value as a source of organisms and an immunological model.nih. Thus. leprae could resist infectious challenge (213). Nine-Banded Armadillo Armadillos were originally adapted to captivity in order to study their unusual reproductive traits. and nerve injury. gonads. spleen./ model. including the IFN.gov/BLAST) is likely to rapidly expand the availability of new immunological probes and reagents for use with armadillos and will advance their use as models for resistance. Studies with these animals could potentially benefit our efforts to generate more efficacious antituberculosis and antileprosy vaccines (29. bone marrow.nlm. In 1973 Kirchheimer showed that 80% of the armadillos sensitized with heat-killed M. armadillos can yield gram quantities of M. leprae with BCG. leprae (185. However. 187–189). unpublished data). leprae. both lepromin-positive and lepromin-negative armadillos can resist challenge with M. 215). below). Kirchheimer and Storrs began experimenting with armadillos to potentially exploit their cool body temperature (30 to 35°C) and showed that armadillos are uniquely susceptible to M. the Human Genome Consortium completed the sequencing of the armadillo genome as part of a comparative genomics initiative. Subsequent vaccination studies with BCG demonstrated effective protection of armadillos against M. Recently. Armadillos are the most common modern representatives of Xenathra. and leprosy was present in Texas and Louisiana prior to the arrival of armadillos. and armadillo IgG reacts well with protein A or G. The reactions of individual animals can range from polar lepromatous to polar tuberculoid.000 animals confirm that the infection is present among armadillos in Arkansas. equivalent to rates observed for BCG in several human vaccination trials. Unfortunately. and only a few reports relate finding the infection among animals in Central or South America. infected armadillos constitute a large reservoir of M. Approximately 65% of all armadillos experimentally inoculated with M. Mississippi.. that M. CLIN. lymphotoxin . leprae will develop a fully disseminated infection. Free-ranging armadillos are exposed to a number of atypical mycobacterial species in the environment and may develop nonspecific antibody responses cross-reactive with antigens shared by M. eral features exhibited by the IFN. and Texas. eyes.ncbi. Armadillo immunoglobulin M (IgM) is highly cross-reactive with human IgM. Wild nine-banded armadillos in the south central United States are highly susceptible natural hosts of Mycobacterium leprae. 2012 by guest . Histopathological examination of infected animals typically reveals heavy infiltration of M./ model were similar to those of BB-BL leprosy. However. leprae infections. and lymph nodes. respiratory instillation and percutaneous and intraperitoneal inoculation are also known to be effective (413). TNF. leprae. The availability of extensive sequence information on the armadillo (http://www. Armadillos only recently expanded their range into the United States. leprae is histopathologically identical to that seen in humans. range. leprae over a span of about 18 months. Growth of M. lungs. nose. the issue has received only scant attention in other countries. 351) (reviewed in Mechanisms of Nerve Injury. Studies in other KO mouse models are currently under way. MICROBIOL. These immunoregulatory mechanisms may be crucial in the manifestation of the unstable borderline areas of the leprosy spectrum. but 90% of the animals that exhibit signs of systemic dissemination will eventually succumb to their leprosy (181). In 1968. Infected armadillos exhibit few discernible clinical signs. compensatory mechanisms are able to limit bacillary growth. Louisiana. leprae over the course of infection. REV. leprae (216. The armadillo is the only animal model that can demonstrate effective protection against M. even though these cytokines and cell types are important for the expression of cell-mediated immunity. leprae from their tissues with high purity and are selected most commonly for propagation purposes (182–186). leprae in this region remains unclear. leprae in mice deficient in interleukin-10 (IL-10) was similar to that seen in immunocompetent mice. Lepromatous armadillos develop the very large burdens of bacilli needed to obtain M. 217). such as treatment with competitive inhibitors to create a second KO or restoration of the KO in infected mice. a family of mammals that diverged from the rodent-primate tree in the Cretaceous period.000-fold increases in the number of M.asm.S. However. It is important to note. Intravenous inoculation promotes the most rapid and severe infections. armadillos can upgrade and downgrade their response to M. however.

429).org/ on January 16. If innate resistance is insufficient and infection becomes established. 19. 2006 LEPROSY 351 of infection for some humans in this country.asm. The various genes and loci listed are discussed in the text under Genetic Influences. The idea that at least two different genes might control the human immune response to leprosy was proposed in the 1970s (89) and supported by subsequent investigations (1.VOL. 239). The impact of armadillo leprosy on humans is difficult to measure. leprae probably occurs through a skin or nasal route. These results were confirmed by a second analysis of Brazilian families with one or more persons affected by leprosy. 5. Nonetheless. Although the precise function of human NRAMP1 has not been definitely established. murine NRAMP proteins influence pathogen viability and/or replication within macrophages by transporting iron and other divalent cations across the phagosomal membrane (139). Functionally. Located at a single locus on mouse chromosome 1. Such acquired immunity is mediated primarily through the function of T lymphocytes. leprae. i. leprae. One of the most extraordinary advances in the understanding of leprosy has been the identification by Mira and colleagues (262) of a locus within the gene PARK2/ PACRG that is associated with overall susceptibility of human populations to M. Infection by M. LEPRAE Genetic Influences on Leprosy in Humans Before Hansen’s discovery of the leprosy bacillus in 1874. influencing the degree of specific cellular immunity and delayed hypersensitivity generated by the infected individual. the specific locus identified codes for the synthesis of a ligase in the ubiquitin-proteasome pathway of intracellular protein degradation (454). overall susceptibility/resistance to the infection. HOST RESPONSE TO M. Among Louisiana residents developing leprosy. and the degree of risk attributable to armadillos is quite low. An appreciation of the role of immunity in the different clinical and pathological manifestations of leprosy as well as subsequent advances in the field of immunology provided a foundation for focused inquiries concerning the genes that might influence susceptibility to M. PARKIN was named for its association with an early-onset form of Parkinson’s disease. an increased likelihood for lepromatous-type leprosy was reported (408). leprosy was widely regarded as an inherited disease. below). and casecontrol studies have yielded conflicting results (45. PACRG. Genetic influences on innate resistance to M. understanding the actual impact of armadillos on human infection will likely require the evolution of better molecular techniques that can track transmission. and the cells listed are discussed in the succeeding sections. The specific locus is a promoter region of PARK2 and a coregulated gene. Two-stage model of genetic influence on human immunity to M. In their initial association scan of a linkage peak identified through a genome scan of a Vietnamese patient population. thereby affecting antigen presentation to lymphocytes and the resulting immune response (278. operating at two levels (Fig. Leprosy remains rare in the United States. A convincing body of evidence now exists to indicate that different genes do influence the human immune response to M. The first evidence of a genetic determinant of overall susceptibility or resistance that might relate to leprosy was the demonstration by Skamene and colleagues of a gene controlling susceptibility and resistance of mice to intracellular pathogens (383). leprae. However. is highly homologous with the mouse gene. Functionally. is a manifestation of innate resistance mediated by cells of the monocyte lineage. The first level. 427). leprae (112). the human gene. leprae. leprae. This is the first example of the use of positional cloning to identify a human gene associated with FIG. but among Mexicanborn patients who presented in Los Angeles and who had lived in areas where they could have been exposed to armadillos. located on chromosome 2q35. regardless of the subtype of this disease. 2012 by guest susceptibility to an infectious disease (48). the investigators identified a locus within this gene that was highly associated with leprosy (leprosy per se). Recent work has revealed some mechanisms by which this pathway regulates the processing of protein antigens within macrophages. located on chromosome 6q25-q27.. Based on its function in mice it was termed natural resistance-associated macrophage protein 1 (NRAMP1) and is now designated SLC11A1. Evidence from studies of twins with leprosy and of family clustering of cases continued to suggest that some inherited influence was a factor in susceptibility to this disease. remains to be determined. and the finding that a locus within this gene is also associated with susceptibility to leprosy is very unexpected. The exact mechanism by which this gene influences overall susceptibility to leprosy. Downloaded from http://cmr. (ii) NRAMP1. this gene was initially designated BCG. armadillos are a large natural reservoir and can be an effective vehicle for exposure to large numbers of M. by mechanisms that are not yet defined. and genetic defects in different components of the type 1 cytokine pathway that affect human resistance to mycobacteria have been reviewed by van de Vosse and colleagues (420). no association with armadillo contact was found (110). however.e. and perhaps in other locations across the animal’s range (415). in cooperation with antigen-presenting cells (see Adaptive Immunity. 5). A number of anecdotal reports have associated handling armadillos with individuals’ developing leprosy. Subsequent studies have also suggested . An association of this gene with overall susceptibility to leprosy was first reported in a study of families with multiple cases of leprosy (2). genetic influence is expressed at the second level. The association of genes involved in both innate and acquired immunity to leprosy has recently been reviewed by Marquet and Schurr (249). (i) PARK2/PACRG.

org/ on January 16. leprae (87). leprae. MICROBIOL. regulation of expression of TNFA. and other aspects of resistance to M. The ability (or inability) of an individual to develop a granulomatous response to an intradermal injection of killed M. 195. (iv) TNFA.. leprae (the Mitsuda test) have also been reported (108. the nasal mucosa or skin abrasion. 2012 by guest . Uptake of the bacilli by DC and subsequent local production of cytokines and chemokines could regulate inflammation and manipulate the ensuing course of the adaptive cell-mediated immunity into a Th-1 or Th-2 response to M. several genetic studies have reported associations of TNFA alleles with different types of leprosy. A genomewide linkage scan of 244 families in southern India revealed significant linkage of a series of microsatellite markers on chromosome 10p13 with susceptibility to leprosy (377).asm. especially in the promoter region. Advances in the technology of molecular genetics and in mathematical methods of interpretation of the data have extended these investigations far beyond the capabilities of the earlier techniques. no evidence convincingly demonstrated an association of the lepromatous response with any other HLA-D loci.is generally associated with resistance to M. another allele was associated with tuberculoid disease (335). An effective innate immune response in combination with the low virulence of the leprosy bacillus may underlie resistance to the development of clinical disease. where they are joined to MHC class I molecules for antigen presentation. Evidence from studies of leprosy patients indicates that TLR2 controls the production of cytokines.352 SCOLLARD ET AL. 265. plays a major role in nonspecific inflammation and innate resistance and is also one of the most powerful stimulants of cell-mediated immunity. located on chromosome 12q12. REV. (vi) VDR. that NRAMP1 may be associated with different leprosy types in some populations. located on chromosome 6p21. but because this gene is located so close to other HLA genes. leprae bacilli (151). several of these serotyping studies suggested an association of HLA-DR2 and -DR3 with tuberculoid (paucibacillary) leprosy. These have been reviewed by Sergeantson (358) and Ottenhoff and de Vries (296). the clinical. Most of the patients in these families had tuberculoid (paucibacillary) leprosy. 108) but not in others (152). (ii) Chromosome 10p13. For example. and it is not clear whether the loci that were identified are associated with overall susceptibility to leprosy or only with the tuberculoid type of leprosy. Genetic influences on acquired immune responses in leprosy. these promoter polymorphisms are of great interest as possible modulators of the degree of host response and therefore of clinical types of leprosy. After earlier studies suggested that polymorphisms of the human vitamin D receptor gene (VDR) were associated with susceptibility to tuberculosis (323). 223). The latter study also found this allele to be protective against leprosy per se. a study of leprosy patients indicated that different alleles of this gene were associated with tuberculoid and lepromatous leprosy (325). Early studies using serotyping techniques attempted to find associations between leprosy and human leukocyte antigens (HLAs) in major histocompatibility complex class II. leprae. TNF. against the overall likelihood of acquiring leprosy of any type. Binding to this receptor leads to the activation of monocytes and influences the function of both CD4 and CD8 T lymphocytes (153). in a Brazilian population. (i) HLA. interpretation of the results has been difficult and the significance of this finding is uncertain and awaits more detailed studies.25(OH)2D3. e. human Toll-like receptors (TLRs) are cell surface molecules that play an important role in the recognition of pathogens. Tumor necrosis factor alpha. Dendritic cells (DC) likely play a key role in modulating the early innate immune response to M. Several polymorphisms of this gene have been identified. The VDR gene. and expression of this cytokine is also increased locally in skin lesions in these manifestations of leprosy (see Leprosy Reactions. encodes an intracellular receptor protein which binds the active metabolite of vitamin D. Because of the wide range of influence of TNF. an association of one allele with lepromatous leprosy was observed (326). leprae. 336). lie within the MHC class II region between HLA-DP and -DQ (388). 1 . TAP proteins transport peptides to the endoplasmic reticulum in antigen-presenting cells. experimental. DC have been found to be very effective presenters of M. The TAP2 gene has been associated with tuberculoid leprosy (306). and in the absence of an adaptive immune response. produced primarily by macrophages and causing activation of macrophages and T cells. and genetic evidence suggests that the TNFA gene is involved in a complex manner in the regulation of human immune resistance to M. Overall. Although some studies indicated an association of HLA-DR2 with both tuberculoid and lepromatous leprosy. The host defense events that operate early in infection during the indeterminate phase are perhaps the least understood aspects of the immunology of leprosy. (iii) TAP. and induction of nitric oxide synthase (33).are elevated in patients with resistant (tuberculoid) disease and with type 1 reactions. leprae (35.e. possibly through its influence on the expression of major histocompatibility complex (MHC) class II molecules. Because activation of TLRs results in the release of several chemical mediators of immunity. The TNFA gene is located in the MHC class III region on chromosome 6p21. Subsequent molecular genetic studies have borne out many of the early suggestions that the HLA region does play a determining role in the response to M. i. leprae antigen (242. The transporter associated with antigen processing (TAP) is a protein composed of two polypeptides. (i) Antigen-presenting cells and dendritic cells. TLR genes also exert an important influence on the early events in specific immune responses (154). leprae invasion of the host.. 196. but DC Downloaded from http://cmr. cell signaling. Development of the Immune Response Innate immunity. TAP1 and TAP2. possibly due to the frequency of different polymorphisms in different populations or to methodological differences in the studies. Together. 273).g. Colorfully named after their counterpart in Drosophila melanogaster. leprae. Associations of some TNFA alleles with the strength of skin test responses to M. Functionally. (v) TLRs. In leprosy.on cellular immunity. leprae (the Mitsuda skin test) has been linked to NRAMP1 in some studies (8. In an Indian population. Thus. MHC class I and II expression was downregulated in monocyte-derived DC infected with M. serum levels of TNF. the DC may be the first cell to encounter the bacilli. below). Their respective genes. At the site of M. CLIN.

Mannose-capped lipoarabinomannan can modulate several effector functions of mononuclear phagocytes. binds carbohydrate moieties on a variety of pathogens (9). of which TLR2-TLR1 heterodimers. In addition.(417). leprae-reactive. and M. leprae expressed PGL-1 on the cell surface. leprae 19-kDa and 33-kDa lipoproteins could activate monocytes and monocyte-derived dendritic cells through TLR2. also called CD209). 2006 LEPROSY 353 stimulated with M. 400. tuberculosis as well as M. with complement receptors and the mannose receptor playing a minor role (400). tuberculosis. 19. Langerin (CD207) is a C-type pattern recognition receptor expressed by Langerhans cells. The cytokine profile present in the lesion also appeared to be correlated with TLR function: Th-1-type cytokines were generally associated with TLR1 and TLR2 activation. Interestingly. the primary mycobacterial ligand for DC-SIGN was mannose-capped lipoarabinomannan (220. and fucose (219). Another C-type lectin is the dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN. Langerhans cells found in the epidermis of leprosy lesions coexpress high levels of CD1a and langerin (161). specific cytokines could regulate the TLR through two independent mechanisms in vitro. leprae by DC (336). pathogen-associated molecular patterns displayed on many microorganisms are recognized by pattern recognition receptors expressed on immune cells at the sites of initial exposure.VOL. including TNF.org/ on January 16. It is expressed primarily on cells of the myeloid lineage. prostaglandin E2. Engagement of DC-SIGN may also inhibit TLR signaling (422). 14. and IL-10 and anti-IL-12 have been reported to inhibit the lymphoproliferative response following presentation of M. TLRs are phylogenetically conserved transmembrane proteins that contain repeated leucine-rich motifs in their extracellular domains. as well as M activation for microbicidal capacity (5). Again. leprae membrane-specific CD8 cytotoxic T cells (212). TLR2 homodimers. Langerin is necessary for the formation of Birbeck granules. In studies using M. (iii) C-type lectin receptors. which activates transcription factors such as NF-KB to induce cytokine production. 242. 422). they were highly susceptible to killing by M. N-acetylglucosamine. leprae. (ii) Pattern recognition receptors. leprae membrane antigens upregulated MHC class II and CD40 ligand-associated IL-12 production (242). and a major mycobacterial ligand is likely lipoarabinomannan (304) which. Mammalian Toll-like receptors are crucial for the recognition of microbial pathogens by M and dendritic cells during innate immunity. Macrophage-derived DC have been shown to be even more efficient antigen-presenting cells. and masking of DC-expressed PGL-1 with specific antibody upregulated both the proliferative response and IFN. It has also been reported that uptake of mycobacteria via the mannose receptor does not elicit a respiratory burst (20). and on some subsets of dendritic cells. Administration of recombinant cytokines such as granulocyte-macrophage colony-stimulating factor (201) and IL-2 (199) into LL lesions has been shown to induce an infiltration of Langerhans cells into the sites. and nitrite production (5. Both IL-12 and IL-10 are produced by DC. PGL-1 has exhibited immunosuppressive properties. contains terminal mannose caps on the arabinose side chains of the molecule (38). regardless of whether the biopsy sample was taken from healthy skin or a lesion.production by T cells stimulated with M. a receptor belonging to the C-type lectin superfamily. DC-SIGN is expressed on dendritic cells and also recognizes pathogens via the binding of mannose-containing structures (422). Higher levels of CD1 DC are found in TT lesions than in LL lesions (379). Langerhans cells are a subset of DC that initiate immune responses in the skin. A second category of pattern recognition receptor is comprised of Tolllike receptors. possibly through the inhibition of IL-12 production (290) and the induction of IL-10 (125). although not on monocytes. DC infected with M. the pentalaminar endosomal structures found exclusively in Langerhans cells. via modulation of TLR expression or by affecting TLR activation. Some investigators have proposed that virulent mycobacteria may subvert DC function via DC-SIGN by suppressing DC maturation. This suggests that whole bacilli may suppress the interaction of DC and T cells. TLRs have been found to be necessary for the optimal production of IL-12 (39). a glycolipid component of the mycobacterial cell wall. In contrast. Exogenous carbohydrate ligands are endocytosed via langerin and transported to the Birbeck granules for processing. and TLR4 appear to be significant in the recognition of mycobacteria. DC-SIGN was shown to be the major receptor on DC for the bacilli. especially mature M . One class of pattern recognition receptor contains the calcium-dependent or C-type lectins. LL patients have significantly fewer Langerhans cells in the skin. in vitro studies showed that the M. which bind specific carbohydrate moieties on pathogens and facilitate internalization for antigen processing and presentation. 2012 by guest . The cytoplasmic signaling domain is linked to the IL-1 receptor-associated kinase. Langerin may play a role in the uptake of nonpeptide mycobacterial antigens (161). The mannose receptor (also called CD206). patients with TT lesions have increased numbers of Langerhans cells in the lesions. and Th-2-type cytokines were associated with inhibition of activation. furthermore. suggesting an active infiltration of these cells to these sites. (iv) Toll-like receptors. a proinflammatory cytokine responsible for the induction of Th-1-type immunity. The antigen presented was likely arabinomycolate. A third class of receptor important for mycobacterial uptake contains the complement receptors. Engagement of these receptors can trigger release of antimicrobial products which can exert a preliminary assault on the pathogen as well as signal expression of costimulatory molecules and production of cytokines which induce the adaptive immune system. leprae-infected DC (151). During the innate immune response. as well as TNF. The M has been shown to play a role in uptake of virulent mycobacteria (342). 59). 24. Langerin oligomerizes as trimers at the cell surface and possesses a single calcium-dependent carbohydrate recognition domain with specificity for mannose.. a cytokine important in cellular activation and granuloma formation but also implicated in the Downloaded from http://cmr.asm. compared to uninfected controls or TT patients (133). Examination of leprosy biopsy samples has revealed that monocytes and dendritic cells in tuberculoid lesions expressed Toll-like receptors TLR1 and TLR2 much more strongly than those in lepromatous lesions (223). CD1a-restricted T-cell clones derived from leprosy patients responded to antigen presented by Langerhans cell-like DC. on virulent strains of M. Ten TLRs have been identified.

leprae. uptake of PGL-1. There are two distinct groups of CD1 molecules.B (35) and decreased production of IL-12. LL patients are not immunocompromised hosts and are not prone to cancer or the opportunistic infections that afflict persons with immunodeficiency diseases. particularly through dendritic cells (discussed above). Kang and Chae (193) first noted the correlation of a C-to-T substitution at nucleotide 2029 of TLR2. long-term consequences. thus. Upon examination of leprosy patients. However. Schlesinger and Horwitz (343) established that complement receptors 1 and 3 on the surface of monocytes and CR1. tissue destruction associated with leprosy reactions. Neither NK cells. M . 196) compared to wild-type cells. was facilitated by complement component C3 (343). Lysis of target cells by cytotoxic T lymphocytes is mediated by perforin and cytotoxic granules such as granzyme B. The collateral damage to tissues caused by the granulomatous inflammation accompanying cellmediated immunity may have serious. leprae or M. leprae. (b) CD1-restricted T cells. Recent work has substantiated an important role for TLRs in the recognition and subsequent immune response to M. Mycobacterium-reactive double-negative T-cell lines derived from a skin lesion of a leprosy patient responded to subcellular fractions of mycobacteria in the presence of CD1-expressing antigen-presenting cells (378). this mutation was shown to be associated with a defective activation of NF. The protective aspects of cell-mediated immunity in paucibacillary leprosy are largely defined as controlling the multiplication of organisms. with no overt signs of disease. CD4 cells were distributed throughout the lesion. and maturation that lead to restriction or elimination of the pathogen. Downloaded from http://cmr. and CR4 on M are key mediators of phagocytosis of M. TT lesions exhibited mostly CD4 helper cells with a CD4 /CD8 ratio of 1. Lipoarabinomannan-depleted soluble cell wall fraction did not induce detectable T-cell proliferation. activation.. Although the CD4 /CD8 ratio in normal peripheral blood is also 2:1. (iii) Cytotoxic cells. protection probably occurs early. In vitro and in vivo studies have indicated an important role for the CD1 system of mycobacterial lipid antigen presentation in immunity to M. there was a strong upregulation of CD1 cells in the granulomatous lesions of patients with TT leprosy or reversal reaction (324). thus. such as injury to peripheral nerves. deep antigen-binding pocket that is designed to accommodate the hydrocarbon chains of lipids. and both are capable of lysing M. displayed a CD4 /CD8 ratio of 0. proliferation in. However. this is one mechanism whereby pathogenic mycobacteria can elude the toxic oxygen radicals which can be generated during phagocytosis. b. is found in human systems but not in rodents. leprae. 149. and unlike TT lesions. even those classified with paucibacillary disease and having high levels of cell-mediated immunity. Interestingly. Upon exposure. there appeared to be a preferential migration into. 2012 by guest . comprised of CD1s a. a marker for dendritic cells. and T cells lysed lipoarabinomannan-pulsed monocytes in a CD1b-restricted manner. LL lesions. and e. indicating a strong correlation between CD1 expression and cell-mediated immunity in leprosy. IL-2. perforin is released by cytotoxic T cells and forms pores in the target cell membrane. and TNF. they proposed that these CD8 suppressor cells may serve to downregulate M activation and suppress cell-mediated immunity. Uptake via complement receptors does not elicit a respiratory burst (see below). All have evolved to present lipid and glycolipid antigens rather than peptides. In addition. (a) MHC-restricted CD4 and CD8 cells. as evidenced by the prolific local footpad multiplication of the bacilli in neonatally thymectomized (315) and congenitally athymic (75) mice. 192). CD8 and CD4 T cells can function as class I. which resulted in the change of Arg to Trp at amino acid residue 667. administration of granulocyte-macrophage colony-stimulating factor. Interestingly. Subsequently. It has been estimated that 95% of persons are resistant to leprosy. In contrast. and human CD1 molecules present nonpeptide components of mycobacteria to specific CD1-restricted T cells.asm. Individuals with clinical leprosy. CD1 molecules bind ligands via hydrophobic interactions in a structurally unique. in contrast. Using monoclonal antibodies which could distinguish T-cell subsets. These cells may play a role in mediating the M localization. There is no test currently available to reliably detect exposure to M. possess living organisms in their tissues.and class II-restricted cytotoxic T cells. Granulysin is another defensive antimicrobial protein used by T lymphocytes and is expressed in tuberculosis (393) and leprosy (293). a serine protease located in cytotoxic T cells and NK cells (369). Recognition of purified lipoarabinomannan from M. Cells of the T-cell lineage play an essential role in resistance to M. upon stimulation of cells expressing this mutation with M.org/ on January 16. T cytotoxic cells were numerous in TT lesions. where it activates caspases and leads to target cell death. respectively. c. CLIN. (ii) T-lymphocyte populations. IFN. leprae. The immunological anergy associated with LL is specific for the antigens of M. leprae.6:1. (i) Protective and destructive effects of cell-mediated immunity. CR3. These cells were also CD83 . a cytokine which can promote dendritic cell activation. with the lepromatous form of leprosy. REV.. a role for the recently described Foxp3-expressing CD4 CD25 regulatory T cells (159) in the various forms of leprosy has yet to be determined but may prove critical in the development of LL. leprae. MICROBIOL. a major surface glycolipid of M. the CD8 T cells were distributed throughout the lesion rather than at the periphery. Lipoarabinomannan also induced these T cells to secrete large amounts of IFN. leprae antigens. allowing granzyme B to enter the cell. whereas CD8 cells were stationed at the periphery in the TT lesion (268). Group II contains CD1d of humans and CD1 of rodents. Adaptive immunity: development of cell-mediated immunity. (a) T cells. few CD1 cells were found in LL leprosy lesions. Upon contact with the target cell. By immunohistological staining. leprae or to diagnose preclinical infection. to LL leprosy patients induced infiltration of CD1 cells into the lesions (332). leprae was restricted by CD1b. Group I. In leprosy lesions the presence of granulysin correlated with the polar forms of the disease and was observed more frequently in TT skin lesions than in LL skin lesions. (v) C receptors. or retention of selected cells in the various types of leprosy lesions in that cells of the T helper/ memory phenotype outnumbered the naive phenotype 14-fold in TT lesions. leprae-infected M (65.9:1 (269). the authors found that the CD4 cells present were primarily of a naı phenotype and the CD8 cells were predominantly ¨ve of a suppressor subset.but increased generation of IL-10 (195.354 SCOLLARD ET AL. Perforin was equally distributed in cells across the spectrum.

these relatively nontoxic bacilli can multiply in M to over 100 organisms per cell (145). leprae. leprae in vitro (307). this may offer important insight into the mechanisms that underlie the broad spectrum of human immune response in this disease. (b) Natural killer cells. Lysis of M. Khanolkar-Young et al. are subsequently generated. Moreover. TLRs have also recently been shown to be important in the differentiation of monocytes into macrophages capable of antimicrobial functions or dendritic cells having primarily antigen-presenting capabilities (224).VOL. where they may be responsible for the subsequent local clearance of the bacilli (199). including hydrogen peroxide. leprae can be maintained in a metabolically active state for weeks in M supplemented with IL-10 and cultured at 33°C (122). The implication of these findings is that upon initial M. but lepromatous patients are unable to proceed to the adaptive response seen in tuberculoid patients. phagosomes harboring M. leprae fused with secondary lysosomes. possibly due to a downregulation of superoxide generation by PGL-1 (58). leprae. hydroxyl radical. 2012 by guest . elevated levels of nitrates were measured in the urine of leprosy patients undergoing reversal reactions. Two important antimicrobial pathways by which M can inhibit or kill invading pathogens are the generation of reactive oxygen intermediates and of reactive nitrogen intermediates. Ex vivo and in vitro data from mice have demonstrated that the long-term intracellular presence of live M. Other reactive oxygen intermediates. leprae. leprae is supported in normal mouse M . However.asm. leprae-infected mice (145. and these levels decreased upon high-dose prednisolone treatment (344. as detected by immunohistochemistry. (210). but not dead. activated M from NOS2 knockout mice could not kill M. a marked decrease in circulating NK has been reported when patients were undergoing erythema nodosum leprosum (ENL) reactions (see below) (160). the stable end product of the nitrosylation of tyrosine residues in proteins by peroxynitrite (345). NK cells appear to be recruited to LL lesions injected with IL-2. It was found that iNOS and nitrotyrosine were expressed in borderline leprosy patients both with and without the reversal reaction. In the absence of an effective adaptive immune response. If confirmed and expanded. leprae (373) and. Thus. including M. NK cells exert spontaneous nonMHC-restricted cytotoxicity against a variety of neoplastic and pathogen-bearing target cells. such as L-monomethylarginine or aminoguanidine. however. more importantly.org/ on January 16. Because detection of iNOS by immunohistochemistry does not necessarily indicate actual production of reactive nitrogen intermediates. being a key player in both the afferent and efferent limbs of the immune response. 345). In addition. This situation reversed when the ENL reaction subsided. is only a weak stimulus of the M oxidative burst (155). M. the cells differentiated into DC-SIGN M but not CD1b dendritic cells. leprae stimulation of monocytes via TLRs. leprae. a respiratory burst ensues in which there is a great increase in the consumption of oxygen catalyzed by NADPH oxidase and the production of superoxide. leprae-infected M target cells may contribute to protection in leprosy as an adjunct to the ongoing attempts at intracellular killing or inhibition by IFN. are derived from the terminal guanidino nitrogen of L-arginine by a high-output. especially their ability to become activated upon stimulation with IFN(373. when peripheral blood monocytes from LL patients were stimulated in such a manner.-activated M can drastically inhibit or kill M. The M also plays an important role in the host’s defense against M. had no detrimental effect on bacterial metabolism (4). on monocytes isolated from TT patients induced differentiation into both (DC-SIGN ) M and CD1b dendritic cells. which are more able to cope with the bacilli than the previous host cells. 373). This pattern of expression was likewise observed in leprosy lesions. leprae (6). 375. the levels of iNOS subsided over the course of prednisolone treatment (233). The role of reactive nitrogen intermediates as a M effector mechanism in humans is somewhat controversial as it has been difficult to get in vitro-cultured cells to generate high levels of nitrite in as consistent a manner as in murine cells (430). Upon phagocytosis of microorganisms by M . leprae also possesses a superoxide dismutase (406) and expresses both SodC and SodA by reverse transcription-PCR (440). at the site of disease in patients infected with intracellular pathogens. In contrast. This model. Although the viability of M. they share many characteristics with cytotoxic CD3 T cells. leprae-infected macrophages (66) and Schwann cells (392). M. 19. M. These findings confirmed the demonstration that in normal M . found that iNOS was highly expressed in the lesions of tuberculoid leprosy patients and was increased to even higher levels during the reversal reaction (see below). While NK cell numbers in the blood were similar across the leprosy spectrum. leprae. leprosy bacilli appear to be well equipped to handle antimicrobial reactive oxygen intermediates generated by the host M . Furthermore. M. (iv) Macrophages. lesions have also been stained for nitrotyrosine. Antigen processing and presentation and monokine secretion are three major functions of the M in the afferent stage. IFN. The cytotoxicity of NK cells and their more active IL2-stimulated lymphokine-activated killer (LAK) cell lacks antigen specificity but is directed against M. that in activated M . and singlet oxygen. These toxic oxygen products are an important antimicrobial defense mechanism of phagocyte cells. 2006 LEPROSY 355 nor dendritic cells expressed granulysin. The M is the primary host cell for M. inducible form of nitric oxide synthase (iNOS) produced by activated M . In vitro. The primary efferent function of M is killing this intracellular pathogen.-activated M . both tuberculoid and lepromatous patients may generate similar innate responses to M. The ability of activated murine M to inhibit M. which enables the study of M from the actual site Downloaded from http://cmr. Methods have recently been developed to isolate granuloma M from the footpads of M. especially against extracellular pathogens. (a) Mechanisms of macrophage killing of M. 376). and although CD3 . leprae metabolic activity is dependent on the generation of such reactive nitrogen intermediates. M. leprae released from these heavily infected M after lysis by T cells would be phagocytized by newly arrived activated M . using mycobacterial antigen. phagosome-lysosome fusion was blocked by live. and subjected to another round of attack by powerful antimicrobial mechanisms. Reactive nitrogen intermediates. as activated M cultured in the presence of competitive inhibitors of the enzyme. leprae impaired the afferent and efferent functions of the infected M . leprae. using antibodies against iNOS. several studies have shown that iNOS is expressed. primarily nitric oxide. Activation of TLR2.

respectively (277). Most studies classified patients only as tuberculoid (T) or lepromatous (L) and did not stratify results within the borderline portion of the leprosy spectrum. However.org/ on January 16. transforming growth factor. 381. These M. 281) (281) Downloaded from http://cmr. TNF.asm.or TNF. (391). The studies noted above have examined biopsies from wellestablished lesions. GM-CSF. minimal or no increase in expression. leprae-infected athymic nu/nu mice indicated that the M were phenotypically indistinguishable from normal peritoneal M except that they contained enormous numbers of M. leprae-infected nu/nu mouse footpad granuloma target M are cocultured with fresh uninfected effector M . 448) / (263) / / (263. Furthermore. they could kill the target cell-derived M. respectively (16. 448) (113. especially those involving tumor models. This functional differentiation has offered an attractive hypothesis to explain the differences between tuberculoid and lepromatous responses to M. This work is now being extended to evaluation of M. cultures of peripheral blood mononuclear cells stimulated in vitro (PBMC). Recent evidence from our laboratory. suggests that M may play a role in cell toxicity in leprosy lesions (145). 285) (285) (285) (263. engulfment. 2012 by guest Specimens for reverse transcription-PCR were frozen biopsies (skin lesions). 113. leprae. 281. 448) (381. Moreover. Overall. but did not require concomitant IFN. Killing of the bacilli in this system was not a rapid process but required 3 to 5 days of coculture for optimal effect. different methods of quantification. these studies have generally revealed a predominance of IL-2. the effector M acquired the bacilli from the infected target M . 448). if the effector M were activated with IFN. The Th-1/Th-2 paradigm. MICROBIOL. however. 285) / / (141) (141) / (281) (391) (391) (391) (391) (391) (381. These are difficult to compare because they have used different designs. (381. The exact mechanism by which the effector M acquired M. histologically consistent with TT or BT disease. has recently been evaluated by Stefani et al. These early lesions. 281. TABLE 5. / . leprae-infected footpad-derived granuloma M were also functionally similar to peritoneal M except for one notable defect: they were refractory to activation by IFN. . 448) / (113) / (113) (381.transcripts in tuberculoid lesions and IL-4 and IFN. Evidence from several reports is summarized to present a consensus of the findings.. TGF. Early. Numbers in parentheses are references. CD4 clones isolated from TT lesions secreted primarily IFN. granulocytemacrophage colony-stimulating factor. 375). also displayed a Th-1-like pattern of cytokine gene expression (Table 5). of infection in experimental leprosy.in lepromatous ones. When normal effector M were cocultured with target M . Current studies are aimed at determining which mechanism(s) a new M may employ to effect cellular turnover in a leprosy lesion.. based on functional discrimination of T-helper cells according to their pattern of cytokine production. in numerous systems. usually present for at least 2 years.-induced augmentation of class II MHC expression or phorbol myristate acetate-induced superoxide production in the M. 380). 380. has allowed determination of culture characteristics. Cytokine gene expression in nonreactional leprosya T-cell clones Skin lesions L T L CLIN. although the studies had different designs and different methods of quantification and used different conventions to express their results. leprae.for both microbicidal and tumoricidal activity. leprae from the target M is not yet known. it was dependent on cell-cell contact and production of reactive nitrogen intermediates. leprae and are highly expressed in tuberculoid lesions (123. Circulating leukocytes and T-cell lines from tuberculoid pa- . Initial studies of footpad granuloma M from M. there was no IFN. gene expression profiles consistent with Th-1 and Th-2 patterns. leprae (374. (v) Cytokines in leprosy. Several major studies of local immune responses in leprosy skin lesions have been published (Table 5). reported increase in expression (above controls). REV. 448) (381. Further studies have also indicated that IL-12 and IL-18 promote resistance to M. 281) (141. and different conventions to express their results. In addition. M have demonstrated cytotoxic capabilities using a variety of functions. 448) 448) 448) 448) (263) (141. Most studies have grouped patients only into tuberculoid or lepromatous groupings. and it is not clear whether variations in cytokine production correspond well with the various degrees of cellular immunity represented in the borderline portion of the leprosy spectrum. lepraeinfected gene knockout mice. (381. using an in vitro system in which M. however. These results provide further support that M. whereas a CD4 clone from an LL lesion produced predominantly IL-4 (381).production. asserts that Th-1 and Th-2 cells promote a cellular and humoral immune response.356 SCOLLARD ET AL. and IFN. leprae is a potent modulator of M effector functions and that its influence is largely restricted to the microenvironment of the granuloma.. and induction of apoptosis or necrosis. single-lesion leprosy was usually found histologically to be consistent with TT or BT disease in the one study of these lesions. leprae-engorged granuloma M . cytokine production. and cell surface phenotypic markers by flow cytometry of these granuloma-derived cells. Positive results were obtained for the cytokines indicated. including direct lysis. or T-cell lines and clones established from PBMC of leprosy patients. in patients with only a single lesion. and CD8 clones isolated from LL patients likewise generated large amounts of IL-4 (328). PBMC T L Gene Single lesion T IL-1 IL-2 IL-4 IL-5 IL-8 IL-10 IL-12 TNFIFNTGF GM-CSF MIP a (113. (381. 381. The immunologic activity within earlier lesions.

but the role they might play in these reactions is unclear. leprae (200). studies of cytokine gene expression in leprosy lesions thus far have given us a more detailed description of the immunological parameters of the polar types of leprosy. Type 2 reactions. In summary. All types of leprosy reactions are believed to be immunologically mediated.. 226. 305). and in the United States. Two major clinical types of leprosy reactions occur. leprae exposure in vitro did not elicit IFN. leprae in vitro have also generally been found to produce a Th-1 cytokine pattern (Table 5). Clinical Presentation. Lucio reactions may be accompanied by a profound anemia.VOL. also known as erythema nodosum leprosum. IFN. LEPROSY REACTIONS Reactions are acute inflammatory complications often presenting as medical emergencies during the course of treated or untreated Hansen’s disease. 352). Diagnosis.in circulating mononuclear cells of lepromatous patients. The factors precipitating reactions and Downloaded from http://cmr.in vitro with leukocytes from tuberculoid patients (95). and severe cases may require intensive wound care and debridement comparable to that used in the management of severe burns. together they may affect 30 to 50% of all leprosy patients (28. as severe nerve injury may develop rapidly. and some degree of neuritis with sensory and motor neuropathy. leprae antigens have been associated with some of these (100. but many patients experience multiple recurrences over several months. extremities. because it is associated with high morbidity and mortality. but these are poorly understood in spite of a substantial body of detailed descriptive information.asm. and myositis may also accompany this reaction. leprae. clinical and histopathological evidence indicated that the immunity in the lesions had increased (upgrading) or decreased (downgrading) (320). no clinical or laboratory tests can accurately predict who is most likely to develop a reaction or when it might occur. M. 2012 by guest . confirming and supporting the original concept that tuberculoid lesions are manifestations of delayed hypersensitivity and cellular immunity and that lepromatous ones result when immune recognition occurs (as indicated by antibody production) but the host is unable to develop cellular immunity to M. paralysis. intradermal injections of IL-2 generated apparent increases in cell-mediated immunity within the skin lesions (199) and resulted in increased levels of antibodies to M. the human immune response to M. These patients experience an abrupt onset of crops of very tender. These reactions have been associated with the presence of high levels of cryoglobulins. and M. However. 19.e. Type 1 reactions usually develop gradually. severe. necrotizing vasculitis occurring primarily in patients of Mexican ancestry (96). and BT). leprae. mechanisms. The cause(s). They are also known as reversal reactions. but did not reverse the specific nonresponsiveness of circulating leukocytes to M. the addition of IL-2 reversed this in most (but not all) of these patients’ peripheral blood mononuclear cells (291). while leukocytes and T-cell lines from lepromatous patients generally produce a Th-2 cytokine pattern (263. leukocytes from approximately 40% of all patients in one such study produced a mixed Th-0 cytokine profile. orchitis. causing sensory and motor neuropathy (Table 6). without predilection for existing lesions (Table 6). above). consultation with the National Hansen’s Disease Program is recommended. leprae antigens (198). 2006 LEPROSY 357 tients stimulated by M. some of the cutaneous lesions may ulcerate. malaise. i. and IL-4 (263).org/ on January 16. Because M. leprae and other pathogens (see Genetic Influences on Leprosy in Humans. arthritis. and deformity. leprae may not correspond entirely with the Th-1/Th-2 model. Fractionated M. The different types of reactions appear to have different underlying immunologic mechanisms. The Lucio phenomenon is an acute. occur in multibacillary patients (LL and BL). Systemically. However.e. for both clinicians and basic scien- tists. 285). or trunk. these patients often also experience fever. but did not enhance systemic cellmediated immunity to M. In severe reactions the lesions may ulcerate. these studies have not yet revealed the mechanisms by which the cellular immune response is so extraordinarily titrated to produce the entire leprosy spectrum. alternatively. leprae antigens have also been found to stimulate IFN. leprae infects peripheral nerves. Some of the answers to these questions may also be found in studies of the genetically inherited ability to respond to M. erythematous nodules that may develop on the face. and their natural course may last for many weeks. It is possible that some of the patients whose cells produced the Th-0 pattern were in the borderline portion of the leprosy spectrum (BL or BT). Research in this area continues on the premise that the answer will be found in an understanding of the complex immunoregulatory mechanisms of cytokine production and inhibition. Experimental immunotherapy with intradermal inoculation of cytokines has provided additional information about their roles in immunological events within leprosy skin lesions (197). and Histopathology Type 1 reactions occur in patients in the borderline portion of the spectrum (i. These reactions present as induration and erythema of existing lesions.. These reactions require intensive inpatient management. this complication is rare. and the factors that initiate them are unknown. because early observations suggested that after the reaction had subsided. BL. but the mechanisms responsible for each type of reaction remain poorly understood. BB. IL-2. The natural course of type 2 reactions is 1 to 2 weeks. In severe type 2 reactions. Iridocyclitis and episcleritis. the inflammation associated with reactions is a medical emergency. and the evidence regarding type 1 reactions reviewed here is based on studies of upgrading reactions.. Both short. In lepromatous patients. Although type 1 and 2 reactions together affect 40 to 50% of all patients at least once in the course of their disease. Characteristically.and long-term intradermal administration of IFNresulted in an influx of mononuclear cells and an increase in the CD4/CD8 ratio in the lesions. Immunological Features of Reactions Evidence regarding the mechanisms of leprosy reactions. and treatment of these reactions remain highly problematic. frequently with prominent acral edema and often with progressive neuritis. Downgrading is rarely seen in the current era of antimicrobial treatment. with subsequent loss of sensation. endothelial cells are unusually heavily infected and may be seen in various stages of degeneration. Fortunately.

330. 449) 1 (233***. reviewed in reference 270). 135). some also measured protein levels of selected cytokines. malaise Polymorphonuclear cell infiltrates in lesions 24 h old Days to weeks Thalidomide. leprae antigens. only about 15 to 30% of the patients at risk (i. experimental intradermal inoculation of IL-2 or of IFN. Early functional studies of lymphocytes demonstrated increased lymphocyte proliferation in response to M. 274*. tender nodules on face.. Patients at risk Onset of reaction Cutaneous lesions Neuritis Systemic symptoms Histopathological features Course (untreated) Treatment BL. 274*. 449) 1 (274*) 1 (274*.org/ on January 16.asm. Type 1 reactions have been observed to follow immunization with other mycobacteria in some circumstances (433. 449) 1 (449) 1 (274*) 1 (274*. Subsequent immunophenotyping studies revealed that the number and percentage of CD4 T cells are increased in reacting skin lesions (267. 332). including IL-1. mRNA in T-cell clones from lesions. Such a decline has been documented. The possibility that genetic factors might predispose some patients to develop type 1 reactions has not yet been examined (see Genetic Influences. 233). it has no benefit in the treatment of type 1 reactions.did not precipitate type 1 reactions (198. for indicators of inflammation such as neopterin and iNOS (114. above). 274*. **. in reacting skin lesions. or trunk.. These observations suggest that in addition to the nonspecific anti-inflammatory effects of prednisolone. 389. and systemically. increases in expression of the genes for several proinflammatory cytokines. 449) 1 (274*. TABLE 6. IL-2. Most studies of skin lesions assessed mRNA by reverse transcription-PCR. these studies have not been able to clearly differentiate which changes observed are consequences of reaction and which (if any) reflect initiating events. different methods of cytokine measurement. 416). IL-12. although it is not certain that this is the case. Substantial evidence now indicates that type 1 reactions are the result of spontaneous enhancement of cellular immunity and delayed hypersensitivity to M... cytokines and chemokines assessed by immunostaining. BT Gradual. 449) 1 (449) 1 (233***. The observation that expression or level of most of the proinflammatory cytokines is increased in both types of reactions highlights the difficulty in determining whether an increase in any cytokine reflects a causative immunologic event underlying the reaction or is a consequence of the intense inflammation occurring in the reaction. 423**. and IL-13 (21. mRNA in peripheral blood mononuclear cells. During type 1 reactions. BB. IL-12. ***. 330. but the causes and mechanisms of this enhancement remain poorly understood. 452). Cytokine gene expression changes during type 1 and type 2 reactionsa Category and cytokine Type 1 reaction compared to BT or BL (reference[s]) Type 2 reaction compared to BL or LL (reference[s]) Circulating TNFIFNIL-1 IL-2 IL-2 receptor IL-4 IL-6 IL-8 IL-10 IL-12p40 Cutaneous TNFIFNIL-2 IL-12p40 IL-4 IL-10 IL-6 IL-8 MCP-1 RANTES iNOS Peripheral nerve TNFa 1 (337) 1 (274*. 233. 115. in serum and in circulating leukocytes. often severe Fever. “overnight” Numerous erythematous. and TNF. often severe Malaise No specific findings Weeks or months Corticosteroids LL. for example. 285*) 1 (274) 1 (274*) 1 (389*) 2 (284) 1 (285*. 389. (i) Type 1 (reversal) reactions. 337*) 1 (274*. 389*) 1 (337) 1 (274*) 1 (416) 2 (284. 353. 274*) Not increased 1 (274*) 1 (274*) 1 (218***. REV. and different criteria for determination of an increase in gene expression. However. 389*) 1 (21. have now been documented in several studies (Table 7). 449) 1 (274*. without relationship to prior lesions Frequent. This activation is present both locally. The pattern of cytokine expression has suggested to many investigators that type 1 leprosy reactions represent a spontaneously enhanced Th-1 response. The events or conditions that trigger type 1 leprosy reactions are unknown. notably. over a few weeks Increased erythema and induration of previously existing lesions Frequent. although thalidomide was reported to inhibit TNFin several experimental conditions (410). 274*) 1 (218***) 1 (218***) 1 (233) 1 (21) _ (25. BL Sudden. it may have some inhibitory effect on the underlying immunologic mechanisms of these reactions.358 SCOLLARD ET AL. In addition. 2012 by guest TABLE 7. 269.e. Downloaded from http://cmr. as well as for cytokines and receptors more indicative of immunologic function such as soluble IL-2 receptor. corticosteroids the earliest events in the development of reactions are therefore unknown. Measurement of soluble IL-2 receptor levels in patient sera found high levels in type 1 reactions when the patients first presented for treatment and found that these levels declined steadily during treatment (416). 449) Not done Not done Not done Not done This table summarizes the results of many studies that employed a variety of study designs. and various environmental factors have been considered but not confirmed to be associated with the onset of these reactions. IL-6. . Clinical studies have determined that the serum levels of some of these cytokines decline during the course of successful prednisolone treatment of type 1 reactions but show little or no reduction during the treatment of type 2 reactions. leprae antigens in vitro during type 1 reactions (26. and several of the reports have not clearly distinguished immunological from inflammatory phenomena. BB. Notably. IL-10. BL. 389*) 1 (337) 1 (274) 1 (416) Not increased 1 (274) 1 (274*) 1 (389*) 1 (21. IFN. *. extremities. IFN. MICROBIOL. and BT) are affected. Comparison of clinical features of type 1 and type 2 leprosy reactions Parameter Type 1 Type 2 CLIN.

. Reduced levels of proinflammatory cytokines have been observed in peripheral blood monocytes from patients treated with corticosteroids for type 1 reactions (245). 349). both of which are potentially significant effects in the context of type 2 reactions. Early studies noted that thalidomide inhibited the IgM response (361).. 418). 283). The only stimulus known to initiate a type 2 reaction is the intralesional injection of IFN. Corticosteroids are the mainstay of the treatment of type 1 reactions. and some mycobacterial constituents have been identified in some of these complexes (322). 421. Antimicrobial therapy should be maintained throughout treatment for both types of reaction. high doses are often required. with mixed results (260. sometimes because they do not respond well to thalidomide. Corticosteroids. The four patients who did not develop the reaction had BL or subpolar LL types of leprosy. Immunoglobulin and complement deposition have been demonstrated in the skin lesions.VOL. in such patients. and more often because the drug is highly restricted or unavailable. Th-2 cytokine re- sponses. leprae is greatly reduced but is not altogether absent. Since these agents act at different points in the development of an immunological response and some information is available concerning their mechanisms of action. leprae may suggest that.. and their management is often difficult and perplexing. Other studies have identified possible evidence of cellular immune activation in type 2 reactions. Evidence from therapeutic trials regarding mechanisms of reactions. The observation that type 2 reactions developed in patients who had the most profound inability to develop cell-mediated immunity to M. and a strong polyclonal antibody response with high levels of circulating immunoglobulins. Type 2 reactions occur in patients with poor cellular immunity to M. and at the concentrations used clinically it has also been observed to enhance the production of IL-2 (360). For type 2 reactions. IL-8. Based primarily on histological evidence. and reduced cell-mediated immunity.(410). Cyclosporine has been used to treat type 2 reactions. TNF. In contrast.e. leprae. but other studies have suggested that alterations in cytokine levels are not always observed in patients who do receive good clinical benefit from anti-inflammatory treatment (12. and IL-12 (Table 7) (449). thalidomide is the treatment of choice (405). in which the ability to develop cell-mediated immunity to M. whereas the reaction may recur severely in the postpartum period in women with lepromatous leprosy (101. treatment is largely empirical and is often suboptimal. In experimental immunotherapeutic trials. Cyclosporine is a potent suppressor of cellular immunity. multiple intradermal injections of IFNover 6 to 12 months elicited this reaction in 6 of 10 lepromatous patients studied. abundant bacilli (i. and it is also known to promote apoptosis in neutrophils (19). antibody production. The effects of thalidomide are strikingly different over a wide range of concentrations. 275. The factors that trigger type 2 reactions are even more poorly understood than the immunologic mechanisms of the reaction itself. because they cannot generate cell-mediated immunity.(332). 2006 LEPROSY 359 (ii) Type 2 leprosy reaction (erythema nodosum leprosum).e. It is not clear. all 6 of these had polar LL disease. a review of these trials is of interest in trying to understand the immunological mechanisms causing reactions (Table 8). indicating that cellular immune activation is occurring locally.) The anti-inflammatory properties of clofazimine are useful in treating type 2 reactions also. consistent with this hypothesis. Reactions are poorly understood. The demonstration of immune complexes within clinical lesions in other diseases has also been difficult and problematic. in spite of the flurry of interest in the apparent inhibitory action of thalidomide on TNF. The great difficulties encountered in treating reactions provide an illuminating example of the importance of understanding the mechanisms of disease: i. Pregnancy appears to have an inhibitory effect on type 2 reactions. however. due to their general anti-inflammatory effects. fever. however. were observed in ENL lesions. observations that have long dominated thinking about this reaction. Corticosteroids are widely used for the treatment of these reactions (235. The acute lesions are characterized by a neutrophilic infiltrate superimposed upon a chronic lepromatous pattern. since the cause(s) and mechanisms of reactions are poorly understood. Wemambu and colleagues proposed that ENL represents an Arthus-like phenomenon mediated by immune complexes (431).asm. and still other anecdotal evidence has suggested that the type 2 reaction is more frequent and severe among children who develop leprosy at the onset of puberty (81). Other infections or viral illness. and the drug might well have been forgotten entirely had it not been so valuable for the treatment of these reactions. i.org/ on January 16. Anecdotal reports suggest that in some women. The mechanism by which thalidomide exerts this strong anti-inflammatory effect in type 2 reactions is not clearly understood even today.e. the effect of IFN is to activate immunologic mechanisms that. neither circulating nor fixed immune complexes have been reproducibly demonstrated in ENL lesions. with the attendant risk of serious side effects (397). Thalidomide was determined to be extraordinarily effective in the treatment of type 2 reactions before its teratogenic properties were recognized (367). 236). and serum complement is decreased in these patients. and psychological stress have all been invoked.. and the immune complex theory of ENL is thus neither proved nor disproved. are instead channeled into pathways that lead to enhanced humoral immune activity. however. the severity of a type 2 reaction fluctuates during the menstrual cycle. the type 2 reaction was the only clear indication for the use of thalidomide. and IL-10 mRNAs and sustained expression of IL-4 and IL-5 mRNAs.. all cytokines associated with neutrophil chemotaxis. Increases in mRNA levels for these cytokines have also been observed in biopsies of skin lesions. are highly effective clinically for both type 1 and type 2 reactions. 2012 by guest . blocking the transcription of IL-2 and Downloaded from http://cmr. immunization. A number of other immunosuppressive agents have therefore been tested for their effect on reactions in the search for additional regimens that might be effective individually or at least offer a steroid-sparing effect. However. increases in the expression of IL-6. 404). (For decades. but no convincing evidence has supported any of these. that this treatment has a significant effect upon the underlying mechanisms of either type of reaction (282. intradermal injections of IL-2 in lepromatous patients did not elicit a type 2 reaction (198). sometimes for prolonged periods of time (40). Notably. antigen) in cutaneous and peripheral nerve lesions. including increases in circulating IFN. 19.

116. better results. although the effect of higher doses has not yet been studied. Neuropathy in leprosy arises not only from the infection of peripheral nerves by M. no data available. although its precise mechanism of immunosuppression is unknown (27).in patients with type 2 reactions as well as inhibit TNF. Methotrexate has recently been reported to be effective when used in combination with corticosteroids in patients whose type 2 reaction could not be controlled with corticosteroids alone (202). Effects of immunosuppressive agents on leprosy reactionsc Agent Primary mechanism(s) of action (reference[s]) Effect on type 1 reactiona (reference[s]) CLIN. 421) (243) / (82. IL-2. 421) (246) (225. REV. clinical inhibitors of TNF. 287. or posterior tibial neuritis or ocular damage in the case of facial nerve involvement. 2012 by guest a b c ND. 304). The overall beneficial effects of corticosteroids on both types of reaction are probably due largely to their anti-inflammatory mechanisms. 211. several other cytokines by interfering with the calcineurindependent translocation of the nuclear factor of T-cell activation (NFAT) to the nucleus (27. however. When followed by prednisolone.. through the development of anesthesia. Infection of peripheral nerves is the sine qua non of leprosy. and other cytokines have had minimal effects on reactions in most cases.asm. it is quite possible that the immunological events that fuel reactions simply run their course and abate independently of the anti-inflammatory treatment itself.. T-cell-related mechanisms may be involved in the pathogenesis of severe or recurrent ENL. has been observed to produce a reduction in circulating levels of TNF. infection of peripheral nerves is a unique property of M. suggests that such T-cell functions may not be a major feature of most type 2 reactions. . thus possibly providing a steroid-sparing regimen in the treatment of this reaction. In summary. has shown no benefit in type 2 reactions in one small study (47). and some evidence has suggested that the inhibition of TNFmay not be the major beneficial effect of thalidomide in ENL (276). treatment of choice. inhibiting proliferation of T and B cells (27. Similarly. Effect observed only in combination with prednisolone. leprae. pentoxifylline has not provided a clinical benefit comparable to that of thalidomide (82. 341) 0 (166) ND b (275.360 SCOLLARD ET AL. However.and other cytokines (321. median radial. 147) Inhibits TNF. Azathioprine is a purine antagonist and is well documented to inhibit lymphocyte proliferation. not beneficial. This remains to be confirmed in controlled studies. / . Mycophenolate mofetil.and T-cell prolif- eration that blocks the production of the guanosine nucleotides required for DNA synthesis (27). Azathioprine alone has not been assessed in the treatment of type 2 reactions but has also been reported to be useful in combination with prednisolone in the management of intractable type 2 reactions that do not respond well to prednisolone alone (243). exact mechanism unknown (27. 362) Antimetabolite inhibiting lymphoid and myeloid proliferation (56) Inhibits IL-2 and other cytokines (27. MICROBIOL. highly beneficial. 276. and the mechanism(s) underlying nerve injury in leprosy is very poorly understood (354). 276). it has been found to provide results comparable to those with prednisolone alone in the treatment of type 1 reactions (246). Effect on type 2 reaction (reference[s]) Thalidomide Methotrexate Cyclosporine Azathioprine Pentoxifylline Mycophenolate mofetil Corticosteroids Multiple dose-related mechanisms: inhibits TNF. however. 331. suggesting that the broad immunosuppression associated with this agent did not interfere with some of the basic mechanisms underlying the reaction. This has not been evaluated in a controlled study. leprae but also from the inflammatory and immunologic responses to this pathogen. beneficial. 147) Block transcription factors AP-1 and NF. The evidence that cyclosporine is beneficial in some severe cases of type 2 reaction may indicate that the mechanisms underlying these reactions are not homogeneous. Pentoxifylline. and potential crippling deformities of fingers and toes due to ulnar. like thalidomide. inhibits lymphocyte proliferation. 418. inhibit synthesis of many proinflammatory cytokines (203. an inhibitor of B. 404) (202) / b (64. peroneal. This neuropathy is often devastating to the patient’s health and well-being. 250). . MECHANISMS OF NERVE INJURY Among bacterial pathogens. inhibits IgM response (333. 331) 0 (47) (276) Downloaded from http://cmr. 250) Purine antagonist. . 395) Blocks guanosine nucleotides. paralysis. 276. The mechanism of the remarkably beneficial effect of thalidomide on type 2 reactions remains unexplained. There is no convincing evidence to date that the anti-inflammatory treatments used actually interrupt the underlying immunological processes. 147.mRNA in skin lesions (275. but many clinical details regarding the frequency and extent of nerve injury have only recently been described. Studies of the pathogenesis of neuropathy in leprosy have been severely hampered by the lack of good experimental models and because . conflicting results. TABLE 8. Although corticosteroid and thalidomide treatments greatly alleviate suffering and mitigate nerve injury. in which cyclosporine was of little benefit. 367. stimulates IL-2. Azathioprine alone did not provide superior results in this study.org/ on January 16. This agent has not been tested in type 1 reactions. but the broader experience. . 211. 283. 275) ND (283) / (82. 261. 0.B. inhibition of lymphocyte proliferation by several potent antimetabolites has had little or no consistent effect in the treatment of either type of leprosy reaction.

the nerves of all types of patients are at risk of chronic inflammation and fibrosis that becomes the final common pathway of injury. A model illustrating this hypothesis of localization of M. This has been extrapolated to imply that M. 316. Four related aspects of nerve injury in leprosy must be considered in understanding the pathogenesis of neuritis in leprosy: localization of M. If. the infection of peripheral nerves has been understood to be an ascending neuropathy originating in sensory cutaneous nerves and traveling proximally to involve larger nerve trunks carrying mixed sensory and motor fibers (327). and blindness. paralysis. appears to be the major target of M. leprae binding to Schwann cells. leprae to nerves may involve the vascular endothelium. 6 (347). sometimes long after they appear to be otherwise cured of infection. leprae to peripheral nerves. Ever since autopsy dissections in the 1890s (85. In patients with advanced leprosy. leprae initially binds to exposed Schwann cells in the dermis and then moves proximally within the nerve. even though the Schwann cells and macrophages of their peripheral nerves are more heavily infected with M. however. 2012 by guest .org/ on January 16. Recent advances in the study of nerve injury in leprosy have been most prominent in five areas: improvements in clinical sensory testing with monofilaments. leprae infects nerves from the outside in. infection of Schwann cells. In addition. 179. 19. first aggregating in epineurial lymphatics and blood vessels and then entering the endoneurial compartment through its blood supply (347. as noted below. leprae in peripheral nerves. In vitro. and the likelihood of developing such interventions is correspondingly decreased. potentially resulting in anesthesia with paralysis of intrinsic and extensor muscles of the hands and feet. binding to endothelial cells. the more sensitive assessments of sensory loss have also demonstrated that nerve function impairment occurs independently of reactions (419). on the other hand. recognition that the mechanisms of localization of M. Using this method. which may exacerbate neuritis. and binding to Schwann cells. leprae (163. Involvement of the facial nerve leaves patients at risk of corneal anesthesia. leprae to Peripheral Nerves The first essential step in leprosy neuritis is the localization of M.asm. leprae in these patients leads to injury to adjacent nerves. reproducible measurement of sensory function using calibrated Semmes-Weinstein monofilaments has been a major advance in the study of nerve injury in leprosy (231). Thus. Such testing can clearly identify loss of protective sensation before it results in ulceration and other injury and can identify early neuropathy. and the remainder of this discussion of nerve injury will concentrate on mechanisms related primarily to injury resulting in anesthesia and. most probably because the granulomatous inflammatory response to M. and the other aspects have suffered especially from the inherent difficulties in obtaining material for investigation. 180). ultimately..VOL. and development of greatly improved Schwann cell culture models for in vitro studies of the consequences of M. “swimming like fish up a stream” (209). However. which followed affected peripheral nerves from the skin lesion to the spinal cord. leprae to peripheral nerves. Overt sensory and motor neuropathies that prompt patients to seek medical attention often occur earlier and more intensely in those patients whose lesions contain few bacilli (BT and TT types). recent evidence also indicates that a large percentage of patients experience neuropathic pain (142. direct examination of immunological parameters in biopsy samples of affected nerves in leprosy patients. a 5-year follow-up study of nerve function impairment in over 200 patients has demonstrated that nerve injury continues to be a problem even after the infection is treated and cured (316). 351). entry into the endothelium. we have observed a similarly brisk and heavy Downloaded from http://cmr. 339). Accurate. although some reports have suggested some preference for nonmyelinating Schwann cells (311). processes that accelerate the physiologic resorption of bone and result in loss of digits. but this improvement was not evident after 1 year (385). 2006 LEPROSY 361 biopsy of the most actively inflamed sites of affected peripheral nerve trunks is not possible. the principal support cell in the peripheral nervous system. a study of prophylactic treatment with prednisolone has shown that it reduces nerve function impairment that is measurable at 4 months. exit from endothelial cells into the endoneurium. The Schwann cell. Localization of M. The affected limbs are then at high risk of injury or mutilation. and inflammation. then there are several potential sites of intervention. that is otherwise often overlooked. In contrast. Although localized anesthesia is a serious and well-known consequence of leprosy. M. leprae infection. it is now evident that early nerve function impairment occurs earlier in lepromatous than in tuberculoid patients (258. lepromatous patients often develop overt neuropathy more slowly. then this is the only opportunity for preventive or therapeutic intervention. the ability to clinically evaluate different degrees of neuropathy and correlate this with responses to intervention has very important implications for both clinical and basic research on the mechanisms of nerve injury in leprosy. untreated infection. 396). abrasion. with subtle functional impairment. If several steps are required for the ultimate entry of M. This has contributed to advances in the prevention of disability in leprosy (419). After prolonged. Of these. immunologic responses. leprae to peripheral nerves is presented in Fig. Little study has been done concerning the mechanisms of neuropathic pain in leprosy. leprae. The armadillo appears to provide a model for some aspects of leprosy neuropathy in humans. 130). but also has posed major limitations as an experimental animal. identification of the molecular mechanisms of M. This view of the pathogenesis of infection of peripheral nerves raises significant implications with respect to both understanding the process and possible points of preventive or therapeutic intervention. Although neuritis and neuropathy have often been studied and discussed in the context of leprosy reactions. both myelinated and nonmyelinated Schwann cells are infected by M. leprae into Schwann cells.g. recent studies of peripheral nerves in experimentally infected armadillos have suggested that M. With these sensitive methods. leprae enters nerves exclusively via the single step of direct binding to exposed Schwann cells in the dermis. since biopsy of affected nerves is seldom clinically indicated and is otherwise unethical. e. the infection of Schwann cells is the most obviously remarkable and has received by far the greatest experimental attention.

colonization of the epineurium (e) occurs when bacilli (red) localize in cells in and around blood vessels (blue).asm. leprae into peripheral nerves are postulated to be unrelated to specific immune function. REV. Downloaded from http://cmr. leprae. 6. extending through it into the interior of the nerve. leprae via blood vessels.362 SCOLLARD ET AL. A cutaneous nerve with three fascicles is represented here to illustrate the proposed steps in the pathogenesis of infection of peripheral nerves by M. The resulting accumulation of bacilli within and around endothelial cells greatly increases the likelihood that bacilli will be available for circulation through the endoneurial vessels which branch off the epineurial ones. Proposed model of infection of peripheral nerve by M. Once inside. leprae. (B) Entry of M. It is possible that this is enhanced by drainage of bacilli through the lymphatics (green) that are intertwined with the blood vessels of the epineurium (lymphatics are here illustrated only at the lower end of the drawing). 2012 by guest FIG. however. (C) If no effective immune respose develops . (A) Initially. the subsequent pathogenesis of neuritis in leprosy probably depends in large part on the patient’s immune response to M. Although these initial events in the localization and entry of M. MICROBIOL. The mechanisms responsible for entry into the interstitial space of the endoneurium remain to be determined. CLIN.org/ on January 16. represented here with concentric layers of myelin surrounding axons. leprae into the endoneurial compartment proceeds along blood vessels from foci on and within the perineurium (p). bacilli are available for phagocytosis by Schwann cells (SC).

Recent studies have demonstrated that M. are at considerable odds with well-documented clinical and histopathological observations. and L1) (144). possibly contributing to the variety of conflicting reports in the literature (264. however. a major protein in the M. In vitro studies of ingestion of M. leprae on SCs. The effect of M. M. transforming growth factor 1. optimal conditions (highly viable bacilli and cooler cultivation temperatures) have not been used in most studies of this interaction. transcript levels for all but one of these genes. Using 2 laminins as a probe. Some investigators. smegmatis. First. interpreted to be a contact-dependent mechanism dependent on PGL-1. and even appeared to favor SC survival rather than apoptosis (311). NCAM. N-cadherin. 2012 by guest (e. therefore. The significance of these observations with respect to clinical nerve injury remains uncertain. leprae was used. SCs apparently provide an environment suitable for the preservation and proliferation of M. Ingestion by SCs. leprae on the SC has been the subject of many studies in vitro. After M. In M. leprae is an indolent. bacilli proliferate within macrophages and Schwann cells. and a variable degree of nerve function is preserved until late in the course of the disease. (Reprinted from reference 347. also using a rat SC-axon coculture system. M. 279. Secondly. leprae also appears to have no effect on intact.org/ on January 16.) . leprae in a laminin-2-dependent pathway. but did alter SC expression of a small number of genes examined (those for glial fibrillary acidic protein. leprae Interactions with Schwann Cells Adhesion to Schwann cells. human SCs express Toll-like receptor 2 both in vitro and in vivo. chelonae. This suggests that the ability to bind 2-laminins is conserved within the genus Mycobacterium. that encoding N-cadherin. However. appears to be involved in the invasion of SCs by M. leprae to the myelinated SC surface is sufficient to induce rapid demyelination of these cells.VOL. leprae specifically binds to -dystroglycan in the presence of the G domain of the 2 chain of laminin-2 (310). ICAM. lepromatous leprosy). including M. the functional significance of these alterations remains to be determined. however. leprae with SCs may be mediated by more than one of its cell surface molecules. patients with untreated lepromatous leprosy may have billions of bacilli in their bodies but show little or no demyelination. In contrast to these observations. leprae has not been observed to cause SC loss (144). Therefore. a granulomatous response follows at sites of infection near epineurial and endoneurial vessels and Schwann cells. This results in perineurial inflammation and thickening (proliferation) and an increasing bacterial load both in the epineurium and in the endoneurium. PGL-1. leprae (398). M. mature SC-axon units. evidence clearly indicates that this mechanism of binding to the SC surface via 2-laminins is not unique to M. 392). as described in recent studies using primary denervated rat SC cultures and SC-neuron cocultures (144) (Fig. leprae has also been demonstrated to bind specifically to laminin-2 in the basal lamina of SC-axon units (288). Other mycobacterial species. leprae appears to persist and grow within SCs in human nerves. M. indicating that the association of M. leprae. leprae inhibited adhesion to SCs. however. and M. However. this includes injury to axons in the vicinity of the granulomas. 308. viable M. 398).asm. rapid Downloaded from http://cmr. Granulomatous inflammation is also potentially injurious to adjacent tissue. well-adapted intracellular pathogen. however. leprae cell wall fraction (ML-LBP21) that binds 2 laminins on the surface of SCs has been identified (370).g. while those directed against both surface and cytoplasmic protein epitopes did not show any such effect (67). leprae. (D) If effective cellular immunity and delayed hypersensitivity do develop (e. Antibodies directed against the polysaccharide and lipid components of M.g. leprae adheres to the SC surface. This immunologically elicited inflammation eliminates nearly all of the bacilli in the epi. tuberculosis. leprae proceeded normally but was minimal when live M. These investigators also identified SCs that had undergone apoptosis in biopsies of human lesions. leprae organisms that have already been ingested by Schwann cells may be relatively protected from immunologically mediated destruction and able to maintain a persistent infection in these cells for a longer time. 2006 LEPROSY 363 infection of both cell types (144). These conclusions. tuberculoid leprosy). Importantly. a component of the M. leprae to the Schwann cell (SC) have been elucidated (310. have described rapid demyelination following adherence of M. leprae by a human Schwannoma cell line (ST8814) found that several protein kinases were essential for ingestion but that cyclic AMP-dependent kinases were not (11). suggesting that viable M. Rambukkana and colleagues. leprae. lepraederived lipoprotein to TLR2 on SCs has been reported to result in apoptosis (294). and binding of an M. leprae viability and temperature of peripheral nerves) (414). leprae cell wall (311).. Phenolic glycolipid 1 of M. thus suggesting a mechanism for demyelination of nerves in leprosy (for a review. Infection of SCs with whole. resulting in impaired nerve function.and B-cell-deficient (Rag1 / ) mice led these authors to conclude that attachment of M. Effects of M. In these studies. 7). 19. Other studies have also demonstrated the ability of myelin P0 to bind M. After ingestion the SC appeared to be incapable of destroying this intracellular parasite when cultures were maintained at 33°C (optimal conditions for M. Studies using highly viable suspensions of nude-mousederived M. leprae-infected nerves. leprae have demonstrated that the viability of the bacilli in rat SCs is comparable to that previously described for bacilli within macrophages in vitro and that survival of this organism within SCs is greater at 33°C than at 37°C (144). 382. have reported exclusive infection of nonmyelinating cells in vitro (309). see reference 309). Since M. This survival within SCs in vitro is consistent with the long-standing histopathological observations that M. however. have been shown to express an 2-laminin-binding capacity (247) and these species readily interact with the ST88-14 Schwannoma cell line. However. This is where most bacilli are found in diagnostic biopsies of tuberculoid lesions. it is slowly ingested. leprae interferes with normal endocytic maturation. leprae to SCs in the absence of immune cells. Similar findings in T. Effects of SCs on M. Notably. Several potential mechanisms of binding of M. varied less than twofold. axons are not badly damaged for a long time.and perineurium and also stimulates perineurial fibrosis and thickening.. acidification of vesicles containing irradiated M.

leprae for 48 h. however. Infected cultures were obtained by exposure of primary Schwann cells to M. and infected SCs are able to associate normally with axons in vitro. selected peripheral nerves in all forms of leprosy undergo demyelination. leprae does not appear to induce SC death. M. leprae organisms bind to SCs via several binding molecules on the surface membrane. 7. After cultivation for 12 days at 33°C. tuberculoid disease). produce measurable changes in transcription of some of the genes that have been examined thus far. M.e. because nerve function impairment occurs more rapidly and more severely in patients with a strong cellular immune response (i. Long-term cultures of human SCs also express MHC class I and II. resulting in treatment failures and resistance levels reported to be as high as 40% in some areas of the world (446. nerves may be heavily infected with only mild to moderate impairment of function. 232). M. and viable organisms appear to be able to interfere with normal endocytic maturation and thus. ultimately leading to the emergence of dapsone-resistant leprosy. however. leaving the patient with an insensate. B. Infected SCs are also able to process and present antigen to T cells. All rights reserved. and thus may become targets of immune responses.org/ on January 16. leprae. Finally. Isolated human SC cultures appear to be able to process and present M. In summary. Understanding the many undeciphered mechanisms underlying nerve injury and applying available clinical and rehabilitation tools to prevent and minimize nerve injury are among the greatest challenges and highest priorities in leprosy research today. Without effective treatment. The end result is a demyelination neuropathy (164. REV. leprae has a unique ability to infect peripheral nerves. many such nerves will become completely nonfunctional. 365). M.. leprae antigens to CD4 T cells (387). As a long-term consequence of these and other. Immunologically driven inflammation is probably responsible for much of the clinically apparent nerve injury. Fortunately. Once they have gained access to the endoneurial compartment. probably. © 2002 by the Infectious Diseases Society of America. 399). A.) stroyed in infected nerves. with potential killing mechanisms. probably entering via their vascular endothelium by mechanisms not yet determined.364 SCOLLARD ET AL. Long-term monotherapy with dapsone resulted in poor compliance in many areas. These cells process and present M. use of rifampin alone or in combination with dapsone for the treatment of dapsone-resistant leprosy led to the rapid development of rifampin-resistant or- . The bacilli are ingested by SCs. paralyzed hand or foot. Although rifampin proved to be a powerful antileprosy drug. ICAM-1. leprae and some of its protein and peptide antigens to MHC class II-restricted CD4 T cells and are efficiently killed by these activated T cells. dapsone (diaminodimethyl sulfone) was introduced as standard chemotherapy for leprosy and was used worldwide for treatment of both multibacillary and paucibacillary forms of the disease. SCs are ultimately functionally impaired or de- Current Therapies and Drug Resistance In the 1950s. by apoptosis or otherwise.asm.and medium-weight neurofilaments (340). Ultimately. Myelinating Schwann cells. The infected SCs were highly susceptible to killing by CD4 cytotoxic T-cell clones derived from leprosy patients. they were seeded onto embryonic rat neurons. Nonmyelinating Schwann cells. In lepromatous patients. and this has been associated with abnormal phosphorylation of high. leprae is able to survive for a limited time at 33°C. Immune responses and SCs. leprae-infected SCs. Within SCs in vitro. unknown mechanisms. CHEMOTHERAPY Downloaded from http://cmr. the immune response may also be directed at M. 2012 by guest demyelination (if it occurs in clinical leprosy) is a late manifestation of the disease rather than an early one (295). Axonal atrophy and accompanying demyelination have also been described in leprous nerves. The infected Schwann cell cultures were induced to myelinate and cultured for 30 days at 33°C. as discussed above. Infection does. with minimal immunological response to M. Segmental demyelination is seen adjacent to areas of infection. and CD80 surface molecules involved in antigen presentation. additional antimicrobial agents such as rifampin and clofazimine were developed and introduced for the treatment of leprosy (44. (Reprinted from reference 144 with permission. FIG. CLIN. Transmission electron micrographs of Mycobacterium leprae-infected rat Schwann cell (SC)-neuron cocultures. MICROBIOL. The limited evidence available indicates that the immunological mechanisms operating within nerves are similar to those which have been described in much more detail in the skin.

232. 19. Multidrug therapy has been very practical and successful for treatment of both multibacillary and paucibacillary leprosy (171. several investigators have identified multidrug-resistant strains of M. These recommendations arise from efforts to reduce the resources allocated to leprosy in developing countries and are the subject of considerable debate. see also http://www. Currently the World Health Organization recommends counting lesions to distinguish paucibacillary from multibacillary disease. 280. 175. 2012 by guest ganisms (137. 2006 TABLE 9. Significant differences were noted in the rates of relapse in multibacillary patients followed at a referral center (9%) versus field clinics (3%). a World Health Organization committee recommended that patients with a single lesion be treated with a single combination dose of rifampin (600 mg). Other drugs with antileprosy activity were also evaluated. 171. 314 156. even with these powerful drug combinations.who . inhibits DNA-dependent RNA polymerase Bactericidal. classifying the lesions on the Ridley-Jopling scale. Current treatment recommendations in the United States have been summarized elsewhere (276a. and minocycline (100 mg) (312. depending on several operational factors and on the duration of follow-up. 118. 120. although shorter than 10 years in duration. and drug resistance still occurs.000 person-years was observed among multibacillary patients given fixed-duration (2-year) multidrug therapy. in both the southern India and Philippine studies.04/1.000 person-years).int/lep /romfaq3. With or without the development of drug resistance. competitive PABA antagonist Weakly bactericidal. less than five lesions being classified as paucibacillary and five or more lesions as multibacillary. is a 6-year follow up of 47. Molecular Mechanisms of Drug Resistance Dapsone (4. significantly greater for paucibacillary patients (1. These recommendations. and during the last 20 years recommendations concerning treatment duration and drug combinations have changed several times. b PABA. longer duration of treatment with multiple antimicrobials are recommended in the United States and most developed countries (349).8/1. rifampin. which revealed an overall relapse rate of 0. leprae (reviewed in reference 434). 441 126.org/ on January 16.asm. leprae isolates from biopsied samples of leprosy patients were resistant to various concentrations of dapsone. 129. inhibits DNA gyrase Bactericidal. 349). 176. Only dapsone is approved specifically for the treatment of leprosy by the Food and Drug Administration. 171. 176. 444.61/1. and the overall number of registered cases worldwide has fallen dramatically (171.276 patients in the Chinese national program (63).000 person-years in patients treated until smear negative (134).int/lep/romfaq3. 177). inhibits ribosomal protein synthesis Bactericidal. inhibits DNA gyrase Bactericidal. 174 a Modified from reference 349 with permission from Elsevier. a 16-year follow-up of patients in Karigiri. India (292). unclear but binds DNA of mycobacteria Bactericidal.000 person-years was observed among multibacillary patients who had received multidrug therapy. 359 44. www. 175. Clofazimine proved to be only weakly bactericidal against M. In the longest study to date. further complicating this assessment. p-aminobenzoate. The initial recommendation for patients with multibacillary leprosy was to give daily dapsone and clofazimine with monthly rifampin and clofazimine for 2 years or until the skin smear was negative. 169. and Lockwood has provided a rigorous evidence-based discussion of the treatment of leprosy (234). In addition.000 person-years) than for multibacillary patients (0. In southern India. 178. 401 106. Optimal diagnostic evaluation employing skin smears or biopsies.who. 175. Ofloxacin and minocycline have been added to the drug arsenal for the treatment of leprosy (126. is structurally and functionally related to the sulfonamide . A recent report demonstrated that 19% of 265 M.4-diaminodiphenyl sulfone) is a synthetic sulfone. Antimicrobial agents for leprosy a Agent Routine dose (per day) Antibacterial mechanismb LEPROSY 365 References Dapsone Clofazimine Rifampin Minocycline Ofloxacin Perfloxacin Clarithromycin 100 mg 50 mg 600 mg 100 mg 400 g 800 mg 500 mg twice Weakly bactericidal. 177). Since 1998 they have also recommended treating multibacillary patients for only 12 months and paucibacillary patients for only 6 months (reviewed in reference 349) (Table 9). underscoring the advice that such patients require longer treatment (128). 176. Relapses occurred 14 to 15 years after release from treatment and again were more frequent in persons with a high initial BI. Downloaded from http://cmr. 445).000 person-years. ofloxacin (400 mg). 317. A 10-year prospective study in the Philippines (55) observed an overall relapse rate equivalent to 2. 447).htm).7/1. 403. the number of newly registered cases has not fallen consistently. leprae and therefore was not suitable as monotherapy for leprosy (169. In addition. The reported extent of relapse varies greatly. inhibits ribosomal protein synthesis 10. 172. a relapse rate equivalent to 20/1. 225. Because of the very slow growth of this organism. as well as diagnostic criteria. a relapse rate equivalent to 0. However. leprae and to improve treatment efficacy. 172. To overcome the problem of drug-resistant M. and conservative. 241.23% were resistant to more than one drug in the mouse footpad susceptibility assay (102). Importantly. 138 57. have been modified several times since 1981. but this regimen remains very controversial. or clofazimine and 6.73/ 1.VOL. 171.000 person-years. The largest study. the World Health Organization recommended multidrug therapy for leprosy in 1981. 402 99. relapse occurs in some cases even after multidrug therapy. reduced to 10/1.htm). follow-up of at least 10 years is necessary to obtain a reasonable assessment of relapse. higher rates of relapse have been observed in patients with a high bacterial index (BI) ( 4) at the time of diagnosis.

A single dose of 1. leprae is unknown. it has an anti-inflammatory effect. it is thought to be similar to that of erythromycin. coli dihydropteroate synthase. It displays significant bactericidal activity against M. Clofazimine attains high intracellular levels in mononuclear phagocytic cells. LysPhe insertion Asp410Asn Asp410Asn. Specific mutations within the highly conserved p-aminobenzoic acid binding site of E. 241 R. the relationship between dapsone resistance and the dihydropteroate synthase of M. 241 437 241. 359). Rifampin (3-{[(4-methyl-1-piperazinyl)-imino]-methyl}rifamycin) is the key bactericidal component of all recommended antileprosy chemotherapeutic regimens. 178. leprae (225). Missense mutations within codons 53 and 55 of the sulfone resistance-determining region of folP1 result in the development of high-level dapsone resistance in M. 437 Ofloxacin/gyrA a b 241 51. 241. CLIN. Clofazimine [3-(p-chloroanilino)-10-(p-chlorophenyl)-2. Substitutions within codon Ser425 have been shown to be the most frequent mutations associated with the development of the rifampin-resistant phenotype in M. 173). Dapsone has also been shown to target the folate biosynthetic pathway of M. its metabolic elimination is slow. Comparison of the deduced primary structures of -subunit proteins from several bacteria to that of M. 437. daily administration of 500 mg of clarithromycin kills 99% of viable M. 241. leprae (Table 10). Pro55Leu Thr53Arg Thr53Ile Pro55Arg Pro55Leu Gly89Cys Ala91Val 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 11 (20) 33 (60) 1 (2) 1 (2) 1 (2) 1 (2) 12 (40) 1 (3) 2 (7) 4 (13) 3 (10) 8 (27) 1 (14) 6 (86) 51 51 156 241 241 158 156 241 51. 441). It is highly lipophilic and appears to bind preferentially to mycobacterial DNA (171). However. result in the development of dapsone resistance (80).200 mg can reduce the number of viable bacilli in a patient’s skin to undetectable levels within a few days (232). leprae genome sequencing project indicated that M. Through surrogate genetic studies with M. 403. a key enzyme in the folate biosynthesis pathway in bacteria. Binding of the drug to DNA appears to occur principally at base sequences containing guanine. Rifampin resistance in M. and targets dihydropteroate synthase. coli is the -subunit of the RNA polymerase encoded by rpoB (156. by acting as a competitive inhibitor of p-aminobenzoic acid (317. Leu427Pro Ser416Cys His420Asp His420Tyr Ser425Leu Ser425Met Ser425Met. Lysophospholipids appear to mediate the activity of clofazimine in some gram-positive bacteria (83). of isolates (%)b Reference(s) Rifampin/rpoB R R R NC NC R R R R R R R NC R NC R R R R NC R Gly401Ser. New evidence from the M. leprae. 437 439 439 241. 256. Mutations within drug target genes associated with drug resistance in Mycobacterium leprae Drug/target gene Drug susceptibilitya Mutation(s) No. 251. Percentage (rounded to the nearest whole number) of isolates in each drug-resistant group that contain a specific mutation.366 SCOLLARD ET AL. leprae is low. 52. The target for rifampin in mycobacteria and E. explaining clofazimine’s preference for the G C-rich genomes of mycobacteria over human DNA. 2012 by guest Dapsone/folP1 191. REV. Although the mechanism of action of this antibiotic against M. MICROBIOL. it is unclear whether this mechanism of action is operational in M. . Clarithromycin is a semisynthetic macrolide that differs from erythromycin in its methyl substitution at the number 6 position of the macrolide ring (reviewed in reference 424). leprae in humans (127. 403. drugs. leprae shares six highly conserved functional regions common to this enzyme in bacteria.9% by 56 days. His420Asp Gln407Val Phe408/Met409. 441). TABLE 10. 368). 191. it is not difficult to understand why only a few cases of clofazimine-resistant leprosy have been reported over the years (84. primarily due to missense mutations within codons of a highly conserved region of the rpoB gene referred to as the rifampin resistance-determining region (280. resistant phenotype. as determined by mouse footpad or radiorespirometry (Buddemeyer) drug susceptibility analysis. smegmatis. 441 156 51 156 241 Downloaded from http://cmr. leprae has been established (439). 156. leprae also correlates with missense mutations within this region of rpoB (156). Since clofazimine may act through several different mechanisms.asm. Leu427Val Ser425Phe Ser425Trp Thr53Ala Thr53Ala. leprae within 28 days and 99. encoded by folP.org/ on January 16. not confirmed by either assay.10dihydro-2-(isopropylimino)phenazine] is a substituted iminophenazine with antimycobacterial activity for which the mechanism has not been fully elucidated (175). and the incidence of resistance to it in M. Mycobacterial resistance to rifampin correlates with changes in the structure of the -subunit of the DNA-dependent RNA polymerase. NC. leprae (Table 10). In patients with lepromatous leprosy. leprae demonstrated that M. leprae possesses two folP homologues (folP1 and folP2) (74).

leprae. ribosomal protection by a soluble protein. leprae activity (175. blocking the binding of aminoacyl-tRNA to the mRNAribosome complex (402). 84. and missense mutations within this region have been found in ofloxacin-resistant strains of M. The first rapid drug-screening assays for M. and very slow. Conventional drug susceptibility testing of M. leprae. 176). paucibacillary leprosy). leprae have been found in many parts of the world (49. 2006 LEPROSY 367 which acts by inhibiting protein synthesis by binding to the ribosome. leprae genes in surrogate hosts. which act by inhibiting protein synthesis. and these mutants are further enriched in a population by inappropriate or inadequate drug therapy. leprae (Table 10). The molecular mechanism of minocycline resistance has not been studied in M.VOL. leprae Lacking direct evidence for the mechanisms of M. leprae are unknown but appear to be relatively low. leprae isolate to standard antileprosy drugs. drugresistant isolates of M. the assay can be designed to be species specific. leprae in the hind footpads of mice by the method described by Shepard and Chang (364). 176). Resistance to tetracycline may be mediated by three different mechanisms: an energy-dependent efflux of tetracycline brought about by an integral membrane protein. Rates of clofazimine resistance in M. leprae from clinical specimens relies on the ability to cultivate M. and ofloxacin-resistant mutants . leprae gyrA is highly homologous to that of M. however. However. 52. with each footpad receiving 5 103 organisms. the frequency of dapsone-resistant mutants in a population of M. our current understanding is based on studies carried out in M. 424. Indeed. Mutations within a highly conserved region of gyrA. leprae). Since untreated multibacillary patients can harbor large bacterial loads ( 1011 M. 51. providing direct evidence for the presence of a particular pathogen in the specimen being tested. leprae is estimated to be 10 6 and the frequency for rifampin or ofloxacin resistance is estimated at 10 7 to 10 8 (158. the quinolone resistance-determining region. the frequency of drug-resistant mutants in a population is primarily inferred from studies with M. and its activity is additive when it is combined with dapsone and rifampin. This method requires the recovery of a sufficient number of viable organisms from a patient to inoculate the footpads of 20 to 40 mice (depending on the number of drugs to be tested). 2012 by guest Detection of Drug-Resistant Leprosy Leprosy presents a very special problem for the detection of resistance because of the inability to culture M. several protocols based on genotypic identification of drugresistant mutants have been developed. Development of Drug Resistance in M. In addition. and in a recent study no mutations were observed within the 23S rRNA gene in clarithromycin-resistant M. which may enhance cell wall penetration (126. very expensive. heteroduplex analysis. similar to the line probe assay (Table 11). Inappropriate therapy (noncompliance or inadequate therapy) for these patients has the potential to enrich the subpopulations of drug-resistant M. leprae were developed based on radiorespirometry techniques (BACTEC and Buddemeyer) and have been used successfully to identify new antileprosy drugs (117).or ofloxacin-resistant organisms. presumably due to its lipophilic property. leprae. tuberculosis. 86. Molecular assays for resistance would simplify susceptibility testing and provide a means for monitoring resistance globally. 19. Sequencing of the PCR amplicon is the most definitive of all of the nucleic acid-based mutation detection protocols because it detects the actual nucleotide changes in the target gene in which mutations associated with antibiotic resistance are found. While this assay gives definitive information pertaining to the susceptibility of an M. and solidphase reverse hybridization analysis. Ofloxacin (4-fluoroquinolone) is a fluorinated carboxyquinolone that has moderate anti-M. or enzymatic inactivation of tetracycline. PCR-direct DNA sequencing has been used to identify rifampin-. the use of these techniques for drug susceptibility testing in leprosy is limited by a stringent requirement for very large numbers ( 107) of viable organisms from each patient. are associated with the development of ofloxacin resistance in most resistant strains of mycobacteria (53.. but in other bacteria it appears to inhibit DNA replication by inhibiting the DNA gyrase. This has not yet been established to be the case with M. Tetracyclines bind reversibly to the 30S ribosomal subunit. Downloaded from http://cmr. single-strand conformation polymorphism analysis. leprae is attributable to chromosomal mutations in genes encoding drug targets. tuberculosis (280) and other bacteria and limited studies with M. leprae. dapsone-. PCR-direct DNA sequencing. tuberculosis. To reduce the number of organisms needed and to minimize the time required for drug susceptibility testing of M. Minocycline (7-dimethylamino-6-demethyl-6-deoxytetracycline) is the only member of the tetracycline group of antibiotics to demonstrate significant activity against M. leprae axenically. 401). it is cumbersome. 280). skin biopsy specimens from leprosy patients) using PCR amplification and detection of mutations associated with drug resistance within these DNA fragments by direct DNA sequencing.asm. these mutations occur spontaneously as a result of errors in DNA replication. 102). Clarithromycin resistance in bacteria and mycobacteria appears to be due to a decrease in binding of the drug to ribosomes and is associated with missense mutations within the 23S rRNA gene (257. The mechanism of action of minocycline against M. leprae. leading to the spread of one or more resistant phenotypes. Results are available after 6 months to 1 year. Minocycline is bactericidal for M. 428). leprae is unknown but is thought to be similar to that of all tetracyclines. The quinolone resistancedetermining region of M. it is feasible that a patient could contain up to 105 dapsone-resistant organisms and thousands of rifampin. leprae’s resistance to most of the antileprosy drugs. leprae cannot be cultivated in vitro. leprae primarily because this drug has only recently been used widely (in the treatment of single-lesion. These techniques are based on the amplification of specific DNA fragments from crude biological specimens (e. The mechanism of action of ofloxacin on M. For example. leprae is unknown. and resistant strains have not been identified.g. a tetramer containing two A-subunits (GyrA) and two B-subunits (GyrB) (99).org/ on January 16. Because M. leprae strains (450). From these studies one can predict that drug resistance in M.

The amplified PCR product is then hybridized to a set of DNA capture probes which have been immobilized at specific sites on a Biodyne C membrane. biotinylated fragment of the M. leprae. unique heteroduplex profiles are observed for susceptible and resistant genotypes. The resultant hybrids are detected by chemiluminescence using a streptavi- din-peroxidase conjugate. PCR-SSCP. leprae (158). when analyzed by electrophoresis. tuberculosis from sputum specimens using a universal heteroduplex generator (UHG) (438). When the heteroduplexes are separated by electrophoresis on polyacrylamide minigels. building a foundation for our understanding of the causes of immunopathogenesis and protective immunity. The DNA fragment patterns observed with SSCP are highly reproducible and yield profiles unique to specific mutations. 437. leprae in skin biopsy homogenates of lepromatous leprosy patients (437). leprae in human specimens (156. Additionally. leprae drug resistance PCR-based assays Assay Target gene Reference(s) CLIN. A PCR heteroduplex-based assay was initially developed to detect the presence of drugresistant M. provide enhanced mutation detection over standard heteroduplex detection. leprae and other mycobacterial pathogens continue to be elucidated. PCR-DNA sequencing gyrA folP rpoB gyrA rpoB folP folP rpoB 51. leprae (Table 11). 52. The immobilized capture probes are small DNA fragments that are homologous to short segments of the rifampin resistance-determining region of the rpoB gene from a rifampin-susceptible strain of M. PCR-UHG requires approximately 6 h to complete and uses 6% precast nondenaturing Tris-borate-EDTA minigels and a nonradioactive detection format. An initial PCR step with a biotinylated and an unlabeled primer produces an 83-bp. leprae genome and bioinformatic processing of the data have changed the way we investigate potential antigens for new vaccines. Given the potential for developing an effective vaccine for leprosy based on these new tools. leprae. 251. cytokines. a synthetic 141-bp sulfone resistance-determining region DNA fragment that contains several base pair mismatches flanking codons that are associated with dapsone resistance. leprae or specific mutant strains. 241. 134. REV. the rifampin resistance-determining region target was amplified by PCR and the double-stranded PCR products were heated to dissociate them into single strands and then separated by denaturing gel electrophoresis under stringently controlled temperature conditions. As discussed above. or quinolone resistance-determining region of M. The stringency of the hybridization reaction is designed so that the PCR product will only bind to probes with 100% sequence homology. PREVENTION: THE QUEST FOR A LEPROSY VACCINE Vaccinology has grown from an empirical science with little in the way of biological understanding of events to a highly structured science drawing on detailed immunological studies at the cellular and molecular levels of both the host and the infectious agent. leprae (Table 10). 241 191. PCR-direct DNA sequencing can be performed in a well-equipped diagnostic laboratory with either manual or automated DNA sequencing systems and requires approximately 1 to 2 days to obtain drug susceptibility results directly from clinical specimens. TABLE 11. called SSCP profiles. the current strategy for controlling leprosy is based on the implementation of effective drug regimens set forth by the World Health Organization. DNA strands from rifampin-susceptible organisms migrate at a rate proportional to their molecular weight and conformation and give a reproducible SSCP profile. PCR–solid-phase hybridization. The DNA sequence of these PCR products is then determined and examined for the presence of mutations previously associated with drug resistance. the debate continues as to whether there is a need for a vaccine in the overall strategy to control or eradicate leprosy. 157. recent reports have shown that relapse rates of 16 to 39% among multiibacillary patients with high BIs are appearing 4 to 10 years after completion of 2-year multidrug therapy (128. Gels were then stained to observe DNA fragment mobility patterns. Acknowledging these clinical realities requires an objective assessment of current control strategies and reminds us that other intervention strategies may be necessary to eradicate leprosy. Target genes for M. A PCR–solid-phase hybridization assay has recently been developed for the detection of rifampin-resistant M. Improved strategies may require new application of old tools. 241. To accomplish this. Enhanced mutation detection occurs because large areas of unmatched nucleotides (bubbles) in the newly formed duplexes greatly affect the mobility of the resultant DNA fragments.asm. 157).368 SCOLLARD ET AL. Annotation of the M. recent epidemiological data suggest that this strategy appears to have had little effect on reducing the annual incidence of new cases of leprosy. 2012 by guest . Unfortunately. A similar approach has been used to develop a PCR-UHG assay to detect the presence of dapsone-resistant M. the resultant heteroduplexes form unique structures which. PCR-heteroduplex analysis. such as special Downloaded from http://cmr. A PCR-single-strand conformation polymorphism (SSCP) assay has been developed to detect rifampinresistant M. The assay requires PCR amplification of the sulfone resistance-determining region of folP1 and the mixing of these PCR products with a universal heteroduplex generator (UHG-DDS-141). leprae rifampin resistance-determining region. 439 51. These assays are based on PCR amplification of the appropriate target DNA directly from skin biopsy specimens using oligonucleotide primers that are specific for the rifampin. The Ser425Leu mutation in the B-subunit of the RNA polymerase is the most frequently detected mutation associated with rifampin resistance in M. The cells. and regulatory pathways active in the host’s immune response to M. sulfone. When UHG-DDS-141 is denatured by heat and slowly annealed to denatured sulfone resistance-determining region PCR products from M. The genotype of the test organism is determined by the capture probes which hybridize.org/ on January 16. MICROBIOL. 441 51 157 439 437 158 PCR-SSCP PCR-heteroduplex PCR UHG-heteroduplex PCR hybridization of M. 168).

bovis BCG. Zodpey and coworkers showed an overall vaccine efficacy of 54%. Three relatively recent vaccine trials have studied the hypothesis that combining BCG with killed M.org/ on January 16. household contacts. French-Danish BCG Glaxo. Two recent case-control studies.1) Close contacts (3. w.VOL. scar Yes No No 80 (72–85) 56 (27–74) 54 (35–68) 65 (50–75) 49 (1–74) 390 76 302 Malawi Total population (0. evidence is available that antileprosy vaccines can provide various levels of protection as well as limited beneficial therapeutic effects. In the Brazilian study. helping curb the emergence of drug resistance.1) Total population (1. Fine (111) has reviewed many of the issues surrounding the curious variability seen with BCG vaccination for tuberculosis. leprae. Used as a therapeutic measure for leprosy control.5) Placebo Unvaccinated 411 240 Modified from reference 23a with permission of the publisher. but should also include basic research designed to develop tests for early diagnosis as well as pre. provide clear evidence that BCG protects against leprosy.85) BCG Glaxo with prior BCG scar. Experience with BCG vaccination for leprosy remains enigmatic in that levels of protection vary from 20 to 80% (Table 12).and postexposure vaccines for leprosy. programs designed to improve drug distribution and treatment compliance. 19. leprae-specific antigens are able to improve the efficacy of BCG. suggesting that an effective vaccine for leprosy.4) HHC d of active BB. vaccines are similar to drugs in that they may be applied as prophylactic (preexposure) or therapeutic (postexposure) measures. heat-killed. HK. Vaccines. appears to have its greatest effect on the disease form (lepromatous) most likely to transmit M. From the standpoint of disease control. 2012 by guest 60 140 India Burma a b Total population (unknown) Children ages 0–14 (5. These results are not unexpected considering BCG’s variable efficacy against tuberculosis (71). exposure to environmental mycobacteria. the investigators argue that their results suggest that neonatal BCG vaccination may have an important impact on transmission of leprosy and that environmental mycobacteria may not impact vaccine efficacy. He speculates that factors such as biological differences in BCG strains. d HHC. and LL patients (16. leprae improves vaccine efficacy. leprae within the community. even in countries with the highest prevalence. c CI. 4 batches HK M.asm. The vaccine studied most in leprosy is M. While most of these concepts remain hypothetical. in India (455) and in Brazil (78). The earliest of the three studies (76) was conducted in Venezuela and concluded that the pro- .8) General population (unknown) Saline Unvaccinated Saline Yes? No Yes Yes Yes Yes Yes Yes? 48 (34–59) 42 (1–66) 34 (14–50) 71 (54–81) 31 (3–51) 65 (47–78) 30 (10–38) 20 (12–28) 23 Downloaded from http://cmr. with the greatest protective effect seen for multibacillary leprosy (68%). 2006 TABLE 12. have a potential added advantage by producing a relatively long-lived immunological memory component. confidence interval. 2 batches Yes 204 Papua New Guinea India India Total population (5. A prophylactic vaccine should also protect against both drug-susceptible and drug-resistant strains. with or without HK M. and ineffective boosting against reinfection with or reactivation of tuberculosis may give rise to the observed variability in protection seen with BCG vaccination. like BCG. ICRC bacillus 2 BCG strains. Accordingly. Because leprosy is a relatively uncommon disease entity. a postexposure vaccine could improve a patient’s response to multidrug therapy by hastening a cure and potentially reducing the incidence of relapse cases. leprae BCG Japan. The primary reason for testing this hypothesis is to determine whether M. however. an effective prophylactic vaccine for leprosy could break transmission by conferring upon recipients immediate as well as extended protection from infection with M. leprae M. at least in the Amazon region of Brazil.000 person-yr) Placebo Vaccinationb Randomized LEPROSY 369 % Efficacy (95% CI)c Reference Uganda Venezuela Malawi Child contact (3. BL.3) Unvaccinated BCG without prior scar No scar Same cohort with restrictions of no early lesion at start Placebo with BCG scar BCG Glaxo BCG with prior scar(s) BCG Glaxo. It is likely that some or all of these factors may play a role in the variability seen in BCG vaccine efficacy against leprosy. case-control studies have been particularly useful in obtaining information concerning vaccine efficacy. Summary of estimates of efficacy of BCG and other vaccines against leprosy a Country Study population (incidence per 1. leprae/BCG Japan/Mix BCG BCG BCG HK M.

either as recombinant BCG overexpressing one or more antigenic proteins or in a prime-boost scenario where antigen is given first in an adjuvant (priming) and then followed by BCG vaccination to boost the initial response. suggesting that a killed mycobacterial vaccine containing M. among scar-positive individuals. In addition. ICRC alone and BCG plus HKML gave approximately the same level of protection (64 and 65%. attenuated BCG vaccine in this population and setting. no advantage was observed by including heat-killed M. animal models in which to test the efficacy of a new vaccine are limited to the mouse and armadillo. significant clinical improvement does result (88. Finally. In addition. 271).asm. Since many indeterminate cases self-heal and tuberculoid leprosy in humans involves nerve damage (not seen in mice).. patients vaccinated with Mycobacterium w demonstrated reduced bacillary indices over time compared to patients receiving only multidrug therapy for 12 months. An important finding in this study. Proteins of interest can be prioritized based on potential B-cell or T-cell epitopes. the proteins selected for further study can be purified as recombinant proteins for an endless supply of the protein for immunologic and vaccine studies (255. REV.org/ on January 16. So. While the immunogenicity of various candidate proteins can be tested in vitro using human cells. leprae challenge to show that benefits for leprosy control through vaccination for tuberculosis are not lost in the process. These tools can identify proteins with special features. tuberculosis (73). that cytokine treatments using IFN. In contrast. The model does. 386. tective efficacy of BCG was proportional to the number of doses (i. in the double-blind. respectively). Interestingly. Shepard’s mouse footpad model is the gold standard for leprosy vaccine studies (366). Recalling the results of earlier vaccine trials with BCG reminds us that it is a fairly potent vaccine for leprosy in many settings. new vehicles for delivery of protein antigens have been identified from research on recombinant DNA over the last 20 years. Both vaccines are nonliving preparations. Less is known about the application of leprosy vaccines for therapeutic purposes. such as unique or shared amino acid sequence homologies with proteins of M. Fortunately. 2012 by guest . A number of reports using Mycobacterium w as immunotherapy suggest that when given as adjunct therapy to multidrug therapy. leprae (74) and M. (88) observed significant improvement in both clinical and histopathological assessments of lesions in patients receiving Mycobacterium w plus multidrug therapy. the incidence of type 2 reactions was similar in controls and vaccinated patients. however.370 SCOLLARD ET AL. CLIN. New approaches to identifying genes from completed M. While many major compounds of M. the only reliable source of M. the vaccine trial done in southern India showed no positive effect of vaccinating BCG scar-positive individuals. while the controversy continues over what elements of a leprosy vaccine are superior in a particular setting. enable screening out of vaccines Downloaded from http://cmr. leprae genome sequences are being applied using standardized bioinformatics tools (253).led to upgrading of LL and BL lesions and were accompanied by a high incidence of ENL (332). 453). neuritis was not increased with vaccination in patients with or without reactions. the number of BCG scars) and that protection against leprosy was not improved when heat-killed M. was that patients in the vaccine group experienced a higher percentage (30%) of type 1 reactions compared to the control group (10%). Supporting this reversal of priming and boosting with BCG are three studies using different protein vaccines for tuberculosis that have shown favorable protective responses in animal models (36. noted above. leprae was combined with BCG. purified proteins have been available in very limited quantities and of poor quality by contemporary standards. Since BCG is given at birth in many countries. this model for defining vaccine efficacy has important limitations. leprae genome. Much of this technology is being used to create vaccines to be administered in concert with BCG.e. but enhanced protection over that with BCG alone was seen in individuals receiving BCG plus HKML (140). 206. leprae (HKML) along with BCG. Application of this strategy for designing leprosy vaccines should not present any unique hurdles now that M. Another important piece of the puzzle that is propelling investigators to search for new molecules with vaccine potential is the completion of the genome sequences of M. 254. This contrasts with the experience. The infection that develops in the mouse footpad may best be described as limited multiplication at the site of infection and may be somewhat analogous to indeterminate or tuberculoid leprosy in humans. Accordingly. 442). De Sarkar et al. a second BCG vaccination gave further protection against leprosy (about 50%) over a first BCG vaccination. leprae antigens was highly purified bacilli originating from infected armadillo tissues. the standard prime-boost schedule for leprosy and tuberculosis vaccines would likely be reversed. MICROBIOL. Similarly. a finding also reported in the earlier study (453). In addition to newly available search algorithms for genes of interest. Additional arms in the southern India vaccine trial included two atypical mycobacteria. Newly designed vaccines for tuberculosis that may utilize BCG altered through genetic engineering or through a prime-boost strategy may or may not provide better protection against leprosy than is afforded by current BCG vaccines. 289. although strict associations between bioinformatics tools and antigenic epitopes remain underdeveloped. making them difficult to use for vaccine development. leprae have been discovered and studied in detail using this material. Mycobacterium w (207) and ICRC bacillus (313). corroborating findings from an earlier study (453). For example. tuberculosis or other mycobacterial species. Prior to the complete sequencing of the M. and the presence of specialized peptide signatures suggesting their cellular location and possible secretion across the cell membrane. certain secreted proteins are almost surely lost upon purification of the bacilli from infected armadillo tissues. leprae antigens of interest can be cloned and expressed in large quantities. there is little or no controversy over the positive effects of vaccination to reduce leprosy incidence. and both were superior to BCG alone with respect to percent protection at the second and third resurveys of the trial. leprae cross-reactive antigens is as effective as a live. controlled trial done in Malawi (204). However. These findings underscore the challenge of producing an effective postexposure leprosy vaccine with the purpose of increasing cell-mediated immunity without inducing harmful sequelae. newly configured vaccines for tuberculosis should be tested for efficacy against M. In contrast to the increased incidence of type 1 reactions following vaccination with Mycobacterium w.

vaccine efficacy must be tested in humans following appropriate safety and potency trials. without an effective vaccine. None of these can yet explain the remarkable spectrum of cellular immune responses to this organism in human subjects. since nerve injury may progress even after completion of chemotherapy. and that type 2 reactions (ENL) correspond to an enhancement of the Th-2 type of response. M. leprae associated with antimicrobial resistance. Thus. Elimination of an infectious disease requires a highly effective vaccine. Implementing treatment of Downloaded from http://cmr.VOL. The armadillo genome has been partially sequenced and is being annotated. defined groups of patients.asm. with its inherent biases. since there are several unresolved discrepancies in these associations. and this organism has been shown to be able to synthesize far fewer proteins than the other major human-pathogenic mycobacterium. Genetic influences on immunity to M. employing advanced molecular tools. developing one was the central focus of leprosy research during the 1980s and early 1990s. although recent studies have shed light on the mechanisms of localization of M. Finally. and others function at the level of acquired immunity. Future of Leprosy Treatment and Research Global elimination of leprosy has been the overarching goal of laboratory research and health policy for nearly 20 years. however. and DNA analysis to detect these mutations is likely to replace the mouse footpad technique. With elimination still in mind. These findings are not yet very satisfying. the number of new cases of leprosy identified annually worldwide has probably not changed over the last two decades. probably due to a complex mixture of social. The availability of knockout mice deficient in selected immunologic abilities will enable dissection of the roles of different cytokines and T-cell subsets in the response to this infection. and several genes possibly influencing adaptive immunity have also been described. These trials must be large due to the relatively low incidence of leprosy in most settings. This has not been successful thus far. including the following. This was not accomplished by 2000 or 2005 and does not appear likely. but the difficulties of implementation of a leprosy vaccine also pose profound challenges to the use of such a vaccine if one is developed. BCG provides a low but measurable degree of protection against M. 2006 LEPROSY 371 with little or no protective efficacy against a challenge with live M. pathology. depending on the classification of the disease. specific vaccine has yet been developed. With the advent of a better understanding of the molecular nature of M. tuberculosis. although the number of registered cases has declined as a result of treatment and removal from registries. the new molecular ability to assess its ability to transcribe and synthesize various proteins in response to different environments and stresses will likely provide valuable information about its mechanisms of pathogenicity in the near future. A variety of mechanisms of innate and adaptive immunity have been identified and postulated to play a role in the development of cellular immunity in leprosy. Reviewed above are major advances that have been made in the basic understanding of leprosy since 1990. Several effective antimicrobial agents are now available to treat leprosy. This is the major focus of the most concerted laboratory research efforts today. PCR analysis of tissues for M. this will soon enable investigators to identify and synthesize immunologically relevant cytokines and probes for use in this animal model. Molecular methods can now identify several mutations in M. Even if this should become technically feasible in the near future. Major advances have been made in experimental models of leprosy. leprae is now known to be killed by reactive nitrogen compounds.org/ on January 16. A major frontier in clinical leprosy is the prevention of disability by active and persistent attention to nerve function impairment. leprae still cannot be cultivated axenically. Reactions in leprosy remain poorly understood. The mechanisms of nerve injury in leprosy remain poorly understood. Vaccines that are effective in the mouse model could then be tested in the armadillo. The challenge for the research and public health systems is to unite the political will and financial resources to test one or more of the new vaccines for leprosy. The medically conservative approach to treatment recommends using multiple agents (multidrug therapy) for several months or years. The World Health Organization has recommended much shorter treatment protocols. SUMMARY AND CONCLUSIONS Based on operational data. treatment. in order to try to interrupt transmission with treatment as early as possible. 2012 by guest . leprae. leprae and the human response to infection. immunology. and no information thus far indicates what triggers reactions or why they affect some patients but not others. which continues to be an obstacle for assessing vaccines as disease management tools in leprosy. Mutations in the M. M. 19. One leprosy susceptibility gene has been identified. and this infection is curable. leprae. however. leprae by Schwann cells. leprae genome that are associated with resistance to several of the drugs used against this pathogen have been identified. This disease poses major challenges to our understanding in microbiology. economic. an animal that manifests most of the characteristics of human leprosy. new vaccines will become a reality. The genome of M. Immunopathological studies have generally found that type 1 (reversal) reactions correspond to an up-regulation of Th-1-type immune responses. leprae has been sequenced. leprae DNA now provides a valuable means for identifying this organism. and biological factors that cannot be resolved in the laboratory alone. and these have been the subject of much controversy among physicians treating patients with leprosy. Within macrophages. although M. No infectious disease has been eliminated using drug treatment alone. the current primary goal is early diagnosis. requiring continued emphasis on basic research and clinical management in the field. this concept faces enormous challenges to verification before treatment of asymptomatic individuals can be recommended. leprae to nerves and on the molecular mechanisms of binding and ingestion of M. but no highly effective. Therapeutic vaccine trials could be more focused and evaluated in smaller. leprae in humans appear to operate at two levels: some mechanisms act at the level of overall susceptibility. and prevention. although vaccine efforts continue.

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