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Copyright X 1988, American Society for Microbiology
Leprosy
ROBERT C. HASTINGS,* THOMAS P. GILLIS, JAMES L. KRAHENBUHL, AND SCOTT G. FRANZBLAU
Laboratory Research Branch, Gillis W. Long Hansen's Disease Center, U.S. Public Health Service,
Carville, Louisiana 70721
least 1961 (232). The relationship between leprosy in wild medians at the wrist, common peroneals at the knee, and
armadillos and the disease in humans is not clear. There posterior tibials at the ankle) are affected in bacteriologically
have been antecdotal reports of leprosy in humans following progressive disease. There is a characteristic pattern of
contact with armadillos (132, 230). On the other hand, most sensory loss due to dermal nerve fiber involvement in
human leprosy occurs in areas (e.g., India) in which arma- advanced lepromatous leprosy which affects cooler areas of
dillos do not exist. If a relationship exists between leprosy in the body surface (194). Despite the widespread involvement
wild armadillos and that in humans, it seems to be quantita- in bacteriologically progressive LL, the patient has remark-
tively minor. ably few symptoms other than those caused by the bacterial
mass and the accumulations of macrophages required to
Clinical Aspects contain them. Histopathologically, lepromatous lesions are
characterized by massive collections of macrophages con-
Leprosy predominantly affects the skin, peripheral taining large numbers of acid-fast bacilli and often containing
nerves, and mucous membranes. The clinical features of the large amounts of lipids which create a foamy appearance on
disease can be grouped into three parts depending on mech- hematoxylin and eosin staining. These foamy macrophages
anism: (i) features due to bacterial proliferation, (ii) features may occupy 90% of the dermis. The dermal foam cell
develop from indeterminate leprosy; it can also develop from Current U.S. recommendations for therapy are 6 months
BL disease by upgrading (by increasing the immune re- of rifampin plus dapsone daily and then dapsone alone until
sponse of the host). Similarly, BL leprosy can develop from 3 years after disease inactivity for indeterminate and TT
indeterminate leprosy or by downgrading from BT. patients. BT patients are treated the same, except dapsone is
continued until 5 years beyond disease inactivity. BL and
Reactions LL patients are treated with rifampin plus dapsone daily for
So-called reactions in leprosy are clinically apparent, at least 3 years, and dapsone is then given alone for the
immunologically mediated inflammatory conditions occur- remainder of the patient's life.
ring during the course of the disease in about 50% of The rate of clearance of bacilli from a patient on effective
patients. These manifestations of leprosy are due to the chemotherapy is a function of the host's immune response.
immunologic response of the host to the bacilli. They are It is 0.5 to 1.0 log per year of effective chemotherapy in LL
basically of two types. Type 1 or reversal reactions are and increases progressively as the disease is more tubercu-
generally agreed to be a result of delayed hypersensitivity loid. The nature of the chemotherapy, e.g., bactericidal or
and affect patients with borderline to tuberculoid leprosy. bacteriostatic, does not affect the rates of clearance of
They are characterized by edema and erythema of preexist- bacilli.
In approximately 50% of leprosy patients, management
CELL WALL STRUCTURE AND ASSOCIATED found to be active antigenic components of mycobacteria.
ANTIGENS OF M. LEPRAE The most notable of the cell wall-associated glycolipid
molecules of M. leprae is phenolic glycolipid I (PGL-I)
All pathogenic microorganisms have evolved characteris- which has been shown to be species specific and immuno-
tics which provide survival advantages in potential hosts. genic during infections with M. leprae (20, 255). Brennan
These may be as overt as the production of a potent, and co-workers established the chemical structure and im-
tissue-destroying exotoxin or as subtle as modulating the munologic specificity of PGL-I through a series of elegant
immune response elicited in the host against the invading studies which have been reviewed in detail elsewhere (18,
pathogen. Intracellular bacteria, such as M. leprae, gener- 69). The general structure of PGL-I can be described as a
ally fall into the latter category, since their survival depends trisaccharide moiety composed of 3,6-dimethyl-p-D-glucose
upon maintaining a stable environment in phagocytic cells (1- 4) 2,3-dimethyl-a-L-rhamnose (1-*2) 3-methyl-a-L-
(particularly macrophages) of the infected host. Two major rhamnose linked to a phthiocerol lipid core through a phe-
areas of study central to the understanding of host-parasite nolic group. The terminal sugar, 3,6-dimethyl-p-D-glucose,
interactions are bacterial metabolism and physiology, in- constitutes the major immunodominant region of the trisac-
cluding cell wall structure-function relationships. Also, re- charide, with the penultimate 2,3-dimethyl-a-L-rhamnose
lated antigenic analysis of the pathogen is helpful in defining completing the composite native epitope (96). PGL-I appears
immunochemical approaches for studying proteins of M. content and by molecular size of the genome indicated that
leprae proved tedious and highly complex. Most of the early M. leprae was significantly different from other mycobacte-
work was performed by immunoprecipitation of protein and rial species (16, 36, 99). Guanine-plus-cytosine content has
other antigens with polyclonal antisera in agarose gels by now been established at approximately 56%, with most other
two-dimensional immunoelectrophoresis. A sophisticated mycobacteria exhibiting guanine-plus-cytosine contents of
scheme for analysis of these profiles was developed, but >60%. Genome size for M. leprae (2.2 x 109 daltons)
little has been learned about the chemical nature of the appears to be smaller than that for most other mycobacteria
antigens contributing to the various immunoprecipitates. A (2.8 x 109 to 4.5 x 109 daltons) with the exception of M.
detailed description of this approach is beyond the scope of tuberculosis H37Ra, which has been reported to be between
this review, but was reviewed recently by Harboe (85). 2.0 x 109 and 2.5 x 109 daltons (16, 36).
More recent developments on proteins of M. leprae have Until recent developments in recombinant DNA tech-
come from studies with murine MAb as monospecific probes niques, major areas of M. leprae genetics and physiology
to identify and characterize the eliciting antigens. The first remained unexplored. Since M. leprae has not been culti-
reported murine MAb to M. Ieprae recognized a 65-kilo- vated in vitro, recombinant DNA methods provide a power-
dalton (kDa) protein which was subsequently found to be ful, indirect approach for studying genes and gene products
associated with the cell wall of the bacteria (73, 74). Four- of the organism. Application of this approach has fostered
and physiologic differences between E. coli and M. lepare. A likewise, a spectrum in the host humoral or CMI response to
similar approach using other host bacteria, such as Bacillus infection. Individuals with TT manifest a strong delayed-
subtilis and appropriate cloning vectors, could be tried to type hypersensitivity (DTH) to M. leprae antigens but pro-
test this hypothesis. Unfortunately, all other potential hosts duce relatively low levels of antibody. At the opposite end of
and related cloning vectors are less well characterized the clinical spectrum (LL), there is a potent humoral anti-
compared with E. coli, limiting their usefulness in the body response but a progressive anergy in CMI to M. leprae
immediate future. Nevertheless, this general approach re- antigens.
mains a potentially powerful tool for studying basic physio-
logical mechanisms operative in a noncultivable microorgan- Immunologic Research
ism, such as M. leprae.
The other significant work in this area was the construc- Goals. As with the study of any infectious disease, the
tion of a genomic library in bacteriophage X gtll by Young's general goals of an immunologist working in the leprosy field
group, which was designed for regulated expression of M. are clearly defined. As a practical measure, tests should be
leprae proteins in E. coli (260). So far, five protein antigens developed to aid in the diagnosis of subclinical disease.
of M. leprae have been expressed in E. coli and analyzed in These tests should also find application in the monitoring of
detail (140, 260). Cloned segments of M. leprae DNA from the course of control measures in endemic areas and in
(94, 96). The "foam" seen in heavily infected macrophages moiety specific for M. leprae, and linked to a protein carrier
characteristic of the lepromatous granuloma is thought to (31, 32, 34, 35, 65, 66), have found widespread application.
contain PGL-I (67, 84). The ability to produce purified Anti-PGL-I antibody is found in virtually all LL patients (23,
preparations of specific M. leprae antigens allowed immu- 35, 255). However, although the number of false-positive
nologists to use state-of-the-art techniques to further explore responses is very low, the response of TT patients and
the immunology of leprosy. contacts is also disappointingly low, thus limiting the use of
this test for epidemiological purposes or for identification of
Clinical Immunology patients with subclinical infection. There is some evidence
that there is a correlation between decreased bacillary load
Lepromin test. The practical clinical immunology of lep- and anti-PGL-I antibody levels after chemotherapy in LL
rosy is at present based almost entirely on the reactivity to (129). Because PGL-I is a major component of M. leprae and
intradermal skin tests with lepromin, a heat-killed suspen- is present in abundance in infected tissue and the blood of
sion of M. leprae obtained originally from homogenized LL patients, detection of this antigen by serological means
human leproma (48, 143) but now prepared from infected could identify infected individuals (254), although this ap-
armadillo tissue. Reactivity to lepromin has no real diagnos- proach may not be feasible at the TT end of the spectrum or
tic value but does establish the immune status of the indi- in subclinical infections.
Mechanisms of Specific Anergy in LL mitogenicity might also suggest that more vital T4 lympho-
cyte-antigen interactions could also be blocked, a concept
Several hypotheses are under investigation to explain the not consistent with CMI anergy in LL being specific for M.
specific anergy in CMI to M. leprae antigens in LL, includ- leprae. However, in the LL lesion, the random distribution
ing a genetic predisposition for LL, clonal deletion of M. of T4 and T8 cells at a 0.5:1 ratio (148) may favor local
leprae-specific T cells, and specific suppression of T-cell suppression of helper cell function and not result in a
function. generalized immunocompromised state. Recently, clones of
Genetic control of CMI in leprosy. Studies of leprosy type T8 lymphocytes isolated from LL lesions were shown to
in homozygous twins showed neither the random concor- mediate major histocompatibility complex class II antigen-
dance expected if there was no predisposition for type of restricted suppression of T4 clones after interacting specifi-
disease nor complete concordance for disease at the TT or cally with lepromin (149, 169).
LL ends of the spectrum, expected if precise genetic control The suppressor cell hypothesis in LL is controversial.
was operating (29, 154). Other attempts have been made to Nath (161) concluded that T-cell suppression in leprosy
explore genetic restriction in leprosy, and evidence is focus- regulates the CMI response in the TT but not LL stage.
ing on the importance of the major histocompatibility com- Moreover, although the suppressor cell hypothesis may be
linked to the specific anergy characteristic of LL, it does not
major role in resistance to a wide variety of obligate and chemotaxis (28) also produced high levels of PGE2 in vitro
facultative intracellular pathogens (122, 166, 167). The (Sibley and Krahenbuhl, unpublished results).
events leading to formation of an epithelioid cell granuloma Although indomethacin blocked production of PGE2 and
and the subsequent limitation and elimination of the local reversed the defective response to IFN-y in our in vitro
bacilli in TT leprosy or a positive lepromin reaction are likely studies (Sibley and Krahenbuhl, in press), it did not reverse
the consequences of such a scenario. Activated macro- the defective response of footpad granuloma macrophages
phages are distinct from normal macrophages by numerous (Sibley and Krahenbuhl, in press), suggesting that prosta-
morphological and metabolic criteria (108, 171), including noid production is not the sole mechanism inhibiting macro-
enhanced production of oxidative metabolites such as super- phage function in vivo in the lepromatous lesion. Of interest,
oxide and hydrogen peroxide which may underlie their a mycobacterial component, LAM, not only blocks T-cell
enhanced microbicidal capacity (167, 168). proliferation (105) but also induces a refractory response to
A fundamental problem plaguing studies of the role of the IFN--y in vitro in mouse macrophages (221) as well as in
macrophage in resistance to leprosy is the inability to human monocyte-derived macrophages (Sibley and Krahen-
routinely quantitate the viability of the uncultivable leprosy buhl, submitted for publication). Defective macrophage re-
bacillus. Thus, macrophage effector function in leprosy has sponse induced by LAM was not associated with PGE2
been addressed indirectly. Although there is some evidence production. These findings again emphasize the probability
leprae as a vaccine preparation. Killed M. leprae afford found in animal tissue. The need to purify the bacterial
protection against experimental challenge with viable bacilli suspensions to rid them of contaminating host tissue debris
in the mouse (216, 217) and armadillo (112) and induction of further reduces bacillary yield.
ability of whole cells to oxidize pyruvate, citrate (243), (46). The extracellular bacilli can also rapidly oxidize palmi-
malate (248), and succinate (240) to carbon dioxide, stimu- tic acid to carbon dioxide (62).
lation of pyruvate oxidation by citrate (243), and finally, Iron. Exochelins and mycobactins, the iron transport
demonstration of a full complement of tricarboxylic acid compounds produced by cultivable mycobacteria, have yet
cycle enzymes (243). Of interest was the apparent proteo- to be detected in M. leprae, probably due to our inability to
lytic inactivation of fumarase in the M. leprae crude ex- cultivate the bacillus. However, exochelins isolated from M.
tracts, an activity which could play a regulatory role. The neoaurum and from an armadillo-derived mycobacterium
presence of the glyoxylate cycle enzymes also suggests the have been shown to mediate iron uptake in M. leprae,
capacity to regulate tricarboxylic acid cycle activity (245). possibly by facilitated diffusion (81, 82). Since iron transport
M. leprae appears to possess an electron transport system was found to be the critical factor in the in vitro cultivation
as suggested by reduced nicotinamide adenine dinucleotide of M. paratuberculosis, there is an obvious interest in the
oxidase activity (100) and the presence of cytochromes (100, possibility of a similar situation with regard to cultivation of
155). In addition, it is conceivable that diphenoloxidase may M. leprae.
participate in electron transport by formation of a quinone Biophysical parameters. As mentioned above, the prefer-
which can undergo reversible oxidation-reduction (155, 185). ence of M. leprae for temperatures below 370C was first
suggested by the observation that the organism was found in
est such technique used continuous drug administration number of agents which can be evaluated. Therefore, a
throughout the experiment (211). While a number of com- number of attempts have been made in the last decade to
pounds are active in this system, bacteriostatic and bacteri- take advantage of the ability to rapidly quantitate temporal
cidal activity cannot be differentiated. This method has the metabolic activity of M. leprae (as described above), con-
advantage of requiring the lowest number of mice and the sidering the inhibition of such activities to be indicative of
least labor. drug activity.
In the kinetic method (208), drug administration is delayed These systems have used intact bacilli suspended in either
until the bacteria have reached the early logarithmic phase of simple axenic media or macrophage cell cultures, require 1
growth, 60 to 70 days postinoculation, and is continued for to 3 weeks of incubation, and rely upon drug action at a locus
approximately 50 to 60 days. Bacillary growth is determined not necessarily directly involved in the metabolic activity
at selected intervals during drug administration and thereaf- being assayed. Relatively simple systems having bacillary
ter. Bacteriostatic drugs cause a growth delay equal to the requirements of 106 to 107 per assay might be candidates for
period of drug administration, while bactericidal compounds clinical detection of secondary drug resistance, using skin
effect a growth delay greater than the period of drug admin- biopsy-derived inocula. Detection of primary drug resistance
istration. In this method, the potential exists for falsely in which only a small percentage of the bacterial population
attributing bactericidal activity to drugs with tissue-reposi- may be resistant would not be possible in these systems and
tories, North Chicago, Ill.) (59). The semisynthetic macro- 9. Barksdale, L., and K. S. Kim. 1977. Mycobacterium. Bacteriol.
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