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CLINICAL MICROBIOLOGY REVIEWS, JUly 1988, p. 330-348 Vol. 1, No.

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0893-8512/88/030330-19$02.00/0
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

OVERVIEW OF LEPROSY ................................................................ 330


Epidemiology ................................................................ 330
Clinical Aspects ................................................................ 331
Indeterminate leprosy ................................................................ 331
LL ................................................................ 331
TT ................................................................ 331

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Borderline leprosy ................................................................ 331
Reactions ................................................................ 332
Patient Management ................................................................ 332
CELL WALL STRUCTURE AND ASSOCIATED ANTIGENS OF M. LEPRAE ................................ 333
Cell Wall of M. leprae ............................................................. 333
Protein Antigens of M. leprae ................................................................ 333
Molecular Biology of M. leprae ................................................................ 334
IMMUNOLOGY ................................................................ 335
Immunologic Research ................................................................ 335
Goals ................................................................ 335
Obstacles to research ................................................................ 335
Major breakthroughs ................................................................ 335
Clinical Immunology ................................................................ 336
Lepromin test ................................................................ 336
Humoral immunity and serodiagnosis in leprosy ................................................................ 336
CMI in Leprosy ................................................................ 336
Mechanisms of Specific Anergy in LL ................................................................ 337
Genetic control of CMI in leprosy ................................................................ 337
Role of the lymphocyte in specific anergy in leprosy ...............................................................337
The Macrophage in Host Resistance to Leprosy ................................................................ 337
Leprosy Vaccine ................................................................ 338
MICROBIOLOGY ............................................................. 339
Metabolism ............................................................. 339
Catabolic activity ............................................................. 339
Amino acid metabolism ............................................................ 340
Nucleic acid metabolism ............................................................ 340
Lipid metabolism ............................................................ 340
Iron ............................................................. 340
Biophysical parameters ............................................................. 340
In Vivo Drug Testing ............................................................. 340
In Vitro Drug Testing ............................................................. 341
ACKNOWLEDGMENTS ............................................................ 342
LITERATURE CITED ............................................................. 342

OVERVIEW OF LEPROSY person. There is evidence that transmission of leprosy can


Leprosy or Hansen's disease is a chronic infectious dis-
occur through
of bacilli (i) intact skin, (ii) inhalation and deposition
onto intact nasal mucosa, and (iii) penetrating
ease caused by Mycobacterium leprae. wounds, such as thorns or biting arthropods. Which of these
mechanisms is most common is unknown. Perhaps the most
Epidemiology popular view is that the leprosy bacilli are expelled from the
The total number of leprosy patients in the world has been nose of a patient with active disease and impact on the nasal
estimated to be about 10.6 million (199). Of these, about 62% mucosa of another individual.
are in Asia and 34% are in Africa (170). Expressed in terms Although the disease is predominantly one of humans,
of the intensity of the disease in a population, i.e., as mean since 1975 it has been demonstrated to be a natural infection
prevalence, the disease is about three times as intense in of wild armadillos (Dasypus novemcinctus) in Louisiana and
Africa as it is in Asia. Texas (236, 237). Spontaneous cases of leprosy have also
Transmission of leprosy is thought to be from person to been described in two mangabey monkeys (78), and experi-
mental leprosy has been transmitted to the rhesus monkey
* Corresponding author. (10). The disease has been present in wild armadillos since at
330
VOL. 1, 1988 LEPROSY 331

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

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due to the immunologic responses of the host to the leprosy accumulations are separated from the epidermis by a clear
bacilli, and (iii) features due to the peripheral neuritis caused zone.
by the first two processes (179). Leprosy always involves TT. Polar tuberculoid leprosy (TT) is the localized form of
peripheral nerves, almost always involves skin, and fre- the disease. In contrast to uncomplicated LL in which
quently involves mucous membranes. The three cardinal bacterial proliferation results in signs and symptoms, much
signs of the disease are skin lesion(s), skin anesthesia(s), and of the clinical picture in TT is due to a combination of
enlarged peripheral nerve(s). bacterial proliferation and the immunologic responses of the
The majority of people effectively resist infection with M. host to the bacilli. Characteristically, TT consists of one or,
leprae even in highly endemic areas. It is now thought that as at most, a few well-circumscribed skin lesions with profound
many as 200 individuals become infected with M. leprae for anesthesia of the skin lesion itself. There may be an enlarged
each overt case which develops and is detected (1). peripheral nerve in the vicinity of the skin lesion(s). Histo-
Indeterminate leprosy. For those individuals unable to pathologically, there are very few, if any, demonstrable
resist infection with M. leprae, the incubation period varies, acid-fast bacilli. There is a dense, well-organized granuloma
but it is usually in the range of 2 to 4 years. The earliest sign consisting of epithelioid cells, surrounded by lymphocytes,
of the disease is usually one or a few hypopigmented skin and containing multinucleated Langhans giant cells. The
macules with minimal sensory loss confined to the lesion. granuloma involves the basal layer of the epidermis in polar
The histopathology of such a lesion may be only that of a TT.
nonspecific, chronic dermatitis with scattered round cell Borderline leprosy. Borderline leprosy encompasses those
infiltrates of the dermis. The presence of acid-fast bacilli or types of the disease between LL and TT. Mid-borderline is
an infiltrate selectively in a nerve bundle in the dermis is rare because it is very unstable. A patient with borderline
diagnostic. Indeterminate leprosy has a variable course. In leprosy can develop clinical, bacteriological, and histopatho-
approximately three-fourths of such patients, the disease logic features of more tuberculoid disease with time, and this
heals spontaneously; some cases remain indeterminate for a is called upgrading. Conversely, developing more leproma-
prolonged period of time, and some progress to one of the tous disease with time is called downgrading. As in TT, the
established forms of the disease. signs and symptoms of borderline leprosy tend to be due to
Established leprosy illustrates a continuous spectrum of a mixture of bacterial proliferation and the immunologic
disease from a localized, self-healing, granulomatous disease response of the host to them. Borderline tuberculoid leprosy
with very few demonstrable leprosy bacilli to a widespread, (BT) resembles tuberculoid disease except that the number
progressive, anergic disease with massive numbers of M. of skin lesions is usually greater, the edges of the skin lesions
leprae. To describe the position of a leprosy patient on this are less well defined, there is a tendency for satellite lesions
spectrum, most centers use the classification of Ridley and to develop near the edges of the larger lesions, and individual
Jopling for research purposes (192). The so-called polar lesions tend to be larger. Damage to peripheral nerves tends
types of leprosy are stable clinically. The so-called border- to be more widespread and more severe in BT than in TT.
line types of leprosy are characteristically unstable clinically This nerve damage is largely on the basis of the immune
and form a continuous spectrum between the two polar response of the host to the bacilli. The histopathology of BT
forms. skin lesions resembles that of TT except that the granuloma
LL. Polar lepromatous leprosy (LL) is the widespread, does not extend up to involve the basal layer of the epider-
anergic form of the disease. Proliferation of M. leprae results mis. The numbers of acid-fast bacilli in lesions vary from
in skin lesions of a variety of types ranging from diffuse undetectable to 1 in every 10 to 100 oil immersion micro-
generalized skin involvement to nodules (called lepromas) in scopic fields. Borderline lepromatous (BL) leprosy resem-
a widespread, usually symmetrical distribution. These skin bles lepromatous disease except that at least some of the
lepromas in advanced LL may contain 1010 M. leprae per g skin lesions are selectively anesthetic and show varying
of tissue. Characteristically, lepromatous skin lesions in- degrees of distinctness in their borders. Peripheral nerve
volve cooler parts of the body surface. This is thought to be trunk involvement (due to the immune response of the host)
due to preferential growth of M. leprae at temperatures is more widespread than in LL, but mucous membrane
cooler than core body temperature (91). With this tempera- involvement (due to bacterial proliferation) is less than in
ture preference of M. leprae, the anterior third of the eye LL. Skin lesions of BL leprosy contain predominantly
(but not the warm, posterior two-thirds), the nasal mucosa macrophages with relatively large numbers of lymphocytes.
(but not the oral mucosa in a nose breather because the oral The numbers of acid-fast bacilli are usually somewhat less
mucosa is not cooled by inspired air), and peripheral nerve than those in an LL lesion of comparable duration, but
trunks as they course superficially (the ulnars at the elbow, substantially more than in a BT lesion. BT disease can
332 HASTINGS ET AL. CLIN. MICROBIOL. REV.

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

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ing lesions and a tendency for the overall disease classifica- must also include controlling reactions caused by the host's
tion to upgrade. Type 2 or erythema nodosum leprosum
lesions have long been thought to be manifestations of an immunologic response to the leprosy bacilli. The aim of such
Arthus type of hypersensitivity reaction. They are seen in anti-inflammatory drug treatment is to prevent or minimize
BL and LL patients and are characterized by the develop- permanent disability and is directed predominantly at con-
ment of crops of tender, erythematous skin nodules and trolling peripheral nerve damage and irreversible eye dam-
fever. Both types of reaction can involve peripheral nerves, age. Inflammatory eye changes occur predominantly in
but type 1 reactions more characteristically do so. Type 2 lepromatous patients and can frequently be managed with
reactions can involve any tissue containing antigens of the topical corticosteroids. Peripheral nerve damage from reac-
leprosy bacillus; therefore, lesions of erythema nodosum tional leprosy can occur throughout the spectrum, but is
leprosum are not confined to the skin, but can involve the particularly prominent in BT disease. In general terms, acute
eye, joints, nasal mucosa, etc. losses in peripheral nerve function are treated with cortico-
Type 1 reactions histologically consist of edema on a BT steroids. More subacute or chronic reactional states can be
or TT background, initially. If the outcome of the type 1 treated with limited courses of relatively high doses of
reaction is upgrading, there may be an early increase in the clofazimine, an antileprosy drug with both antibacterial and
number of lymphocytes. In severe type 1 reactions, case- anti-inflammatory properties. Erythema nodosum leprosum
ation necrosis may occur. Type 2 reactions are characterized is usually completely suppressed with thalidomide, a terato-
by an influx of neutrophils on a BL or LL background. A genic sedative-hypnotic with remarkable immunosuppres-
vasculitis involving arterioles or venules is demonstrable in sive/anti-inflammatory activity in this condition (92).
In virtually all leprosy patients, there is some degree of
about half of the cases in type 2 reactions. irreversible peripheral nerve damage caused by bacterial
Patient Management proliferation or the immunologic response of the host to
these bacilli or both. In some patients, e.g., an indeterminate
Successful management of the leprosy patient consists of case with minor sensory impairment confined to a small skin
controlling the three mechanisms by which the disease lesion on the trunk, this is inconsequential. In others,
causes symptoms, i.e., (i) bacterial proliferation, (ii) the widespread destruction of mixed peripheral nerve trunks can
immunologic response of the host to the leprosy bacilli, and result in widespread skin anesthesia and widespread perma-
(iii) peripheral neuritis caused by the first two processes. nent muscle paralysis involving the face, hands, and feet.
Antibacterial chemotherapy for leprosy, in general, has For patients with muscle paralysis due to irreversible nerve
been both adequate and available since the early 1940s (57). damage due to leprosy, there are a variety of reconstructive
The problems in antibacterial chemotherapy have come from surgery techniques which can frequently restore reasonable
inadequacies in health care delivery systems in leprosy- motor function (17). It should be pointed out that most of the
endemic areas, inadequacies in patient compliance for vari- deformities attributed to leprosy are not caused by the
ous reasons, and the potential for the development of disease itself. Leprosy removes the sensation of pain. The
drug-resistant M. leprae on a large scale. At present, four lack of feedback provided by pain allows the leprosy patient
drugs are used widely: dapsone, rifampin, clofazimine, and a to damage and deform himself (as it does any patient lacking
thioamide, either ethionamide or prothionamide. A variety the feedback of pain). This secondary damage is the most
of drug combinations are used in various parts of the world. disabling, and all of the secondary damage and deformity are
Some centers use monotherapy with dapsone. Many centers preventable. A variety of techniques and principles apply to
utilize the multidrug regimens recommended by the World wound prevention in anesthetic extremities. Perhaps, the
Health Organization (253). For indeterminate, TT, and BT most important principle relates to the care of wounds after
leprosy, for example, daily dapsone is recommended plus they are sustained. Lacking sensation, individuals will con-
rifampin once monthly. The total duration of treatment tinue to use a wounded and infected extremity and subject it
recommended in these so-called paucibacillary cases is 6 to stress in spite of the infection. More than any other cause,
months. For mid-borderline, BL, and LL disease, the World this accounts for the destruction of hands and feet in leprosy
Health Organization recommends daily dapsone, plus daily which, to a large extent, accounts for the stigma of the
clofazimine, with additional clofazimine given once monthly disease. The continued use of an infected extremity leads to
together with once-monthly rifampin. The total duration of osteomyelitis and septic destruction of bones, resulting in
therapy in these so-called multibacillary cases is at least 2 permanent secondary deformities. These can often be pre-
years and preferably until bacilli are no longer demonstrable vented by simply splinting wounded extremities to prevent
by the usual slit-skin smear techniques. their use until the wound has healed (17).
VOL. 1, 1988 LEPROSY 333

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

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those structures of the bacterium involved in the immune to be associated with the outer surface of M. Ieprae (258) and
response of the host during infection. Since metabolic pro- has been isolated from purified bacteria and M. leprae-
cesses of M. leprae are covered below, we summarize here infected tissues in relatively high concentrations (94). Taken
the biochemical data related to the major cell wall structures together, these characteristics suggest that PGL-I may rep-
of M. leprae, and, when appropriate, indicate those mole- resent the M. leprae "capsule" which could function as a
cules which have been shown to induce immune responses in virulence factor providing an important interface between
either humans or experimental animals. parasite and host, critical for maintenance of the parasitic
relationship. The immunogenicity of PGL-I during M. leprae
Cell Wall of M. keprae infection in humans and various other animals has been
firmly established, but further studies need to be performed
Extensive chemical analysis has shown mycobacterial cell on potentially important immune and nonimmune regulatory
walls to be highly complex, lipid-rich, macromolecular struc- functions of PGL-I and related extracellular glycolipids in
tures. While many cell components appear to be unique to relation to intracellular parasitism.
the mycobacteria, the common bacterial structure, peptido- Recently, Brennan and co-workers isolated and character-
glycan, is present in all mycobacteria with only a few minor ized a group of arabinose and mannose-containing phospho-
variations. For example, meso-diaminopimelic acid is found rylated lipopolysaccharides from M. leprae (97). Earlier
in the tetrapeptide allowing cross-linking of the peptidogly- work on similar carbohydrate-rich fractions from mycobac-
can through adjacent meso-diaminopimelic acid residues or teria (204) and M. leprae (141) had established the serologic
through meso-diaminopimelic acid-D-alanine residues (9, reactivity of the arabinomannan component but not the
125, 202). In addition, M. leprae peptidoglycan, unlike that cellular location of the component. Detailed studies by
in other mycobacteria, contains the unusual substitution of Hunter et al. (97) established that the serologically active
glycine for alanine in the tetrapeptide (49-51). While these component, lipoarabinomannan B (LAM-B), is acylated,
substitutions may provide some advantage in pathogenicity contains substituents of phosphatidylinositol, and may be
for M. leprae (e.g., resistance to degradation), it is unlikely membrane associated. LAM-B has proven to be highly
that they affect the adjuvant properties of the muramyl immunogenic in most mycobacterial infections, inducing
dipeptide region found in all bacterial peptidoglycans (238), strong humoral antibody responses. Since LAM-B is a
including that of M. leprae. common antigen among mycobacteria, it cannot be used
Covalently linked to the peptidoglycan is an arabinogalac- effectively for serologic tests to detect early infection with
tan-mycolic acid complex, which constitutes approximately M. leprae. However, there are data to suggest that high
70% of the cell wall mass and forms a major amphipathic levels of anti-LAM-B are correlated with high bacterial loads
region external to the peptidoglycan. The arabinogalactan in leprosy (129). This may make possible the estimation of
polymers consist of linear arrays of D-arabinose-D-galactose bacterial clearance during antimicrobial chemotherapy by
with appendages of D-arabinose extending laterally (2, 5, 7). monitoring serum antibody levels to LAM-B.
The absence of the more common L-isomer of arabinose in
mycobacterial cell walls suggests a heightened resistance to Protein Antigens of M. leprae
degradation by host enzymes, possible contributing to per- Immunochemical information combined with detailed
sistence of cell wall material in host cells upon death of the fine-structure analyses of M. leprae cell walls and associated
mycobacterial cell. Esterified to the terminal arabinose moi- molecules has led to our understanding of a cell wall essen-
eties are mycolic acids which contribute significantly to the tially devoid of proteins and polypeptides. Since proteins are
hydrophobicity of mycobacterial cell walls (8). In M. leprae major structural and functional molecules in all biological
these long-chain fatty acids form two groups, the alpha- systems, the need to elucidate their role in M. leprae has
mycolates and beta-mycolates, and can be used for taxo- been of paramount importance. The major impediment to
nomic purposes to differentiate M. leprae from other myco- studying the proteins of M. leprae stems from the inability to
bacteria (77, 103, 142). culture the bacteria in vitro, limiting workers to bacilli
While peptidoglycan, arabinogalactan, and mycolates con- obtained from tissues of experimentally infected mice, rats,
tribute significantly to the structural integrity of the cell wall or armadillos. These relatively low numbers of organisms
of M. leprae and other mycobacteria, they do not appear to provide only small quantities of purified proteins, severely
constitute major immunogens of the bacteria. Instead, other limiting detailed analysis.
glycolipids (20, 94), glycopeptidolipids (19, 21), and treha- Prior to the application of monoclonal antibodies (MAb)
lose containing lipooligosaccharides (97, 98) have been and recombinant deoxyribonucleic acid (DNA) techniques,
334 HASTINGS ET AL. CLIN. MICROBIOL. REV.

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

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teen nonoverlapping antigenic epitopes have been defined on research in areas as diverse as analysis of metabolic path-
the 65-kDa protein by competitive inhibition studies with ways (36, 102), immunochemical analysis of antigenic pro-
murine MAb (25). The majority of these epitopes were teins (25), and taxonomic studies measuring genotypic relat-
cross-reactive and present on homologous 65-kDa proteins edness of phenotypically similar bacterial species (J. E.
of other mycobacteria, while one epitope, defined by MAb Clark-Curtiss, Abstr. Annu. Meet. Am. Soc. Microbiol.
IIIE9 and IVD8, was specific for M. leprae within the 1987, U-172, p. 117).
context of the 23 mycobacterial species studied. Serologic The initiation of recombinant DNA studies with M. leprae
tests in humans for antibody to this species-specific epitope required two major breakthroughs. The first came with the
have revealed antibody primarily in patients with LL, limit- successful propagation of M. leprae in the armadillo (113),
ing the usefulness of this antigen for detecting early infection providing large quantities of bacteria from which purified
with M. leprae (128). DNA could be obtained ahd analyzed. The second break-
The general scheme of multiepitopic proteins of M. leprae, through came with the establishment of M. leprae genomic
containing both species-specific and cross-reactive B-cell libraries which could be manipulated in cultivable bacteria,
determinants, has been reported by other workers, using such as Escherichia coli, for subsequent analyses of M.
murine MAb (101, 119). A collaborative study for the com- leprae genes and gene products. Libraries of this type are
parison of 20 MAb to M. leprae was recently reported by the composed of small segments of M. leprae DNA (4 to 20
World Health Organization in which M. leprae-specific and kilobases) present in independent recombinant molecules
cross-reactive epitopes were found on proteins with molec- which can be manipulated to meet experimental needs. For
ular weights of 12,000, 18,000, 28,000, 36,000, 55,000 to example, a small segment of DNA from M. leprae, localized
65,000, and 200,000 (56). Antigenic analysis with Mab is only in a plasmid or bacteriophage vector, can be directed by the
a first approximation of the extent and complexity of M. vector to be transcribed. Subsequent translation of the
leprae protein antigens. Moreover, the relevance of these resultant messenger ribonucleic acid should result in an M.
proteins to the human immune response during infection leprae protein(s), the gene(s) for which is encoded in the
with M. leprae remains unanswered. Obviously, immuno- cloned segment of M. leprae DNA. Should an intact protein
genic proteins might provide suitable components for devel- or partial peptide result, the enzymatic or antigenic nature of
opment of vaccines against leprosy, but detailed studies are the molecule can be studied. Theoretically, successful appli-
needed to define the B- and T-cell stimulatory capacity of cation of this approach should make possible the elucidation
these molecules. This work will require relatively large of enzymatic pathways important for M. leprae growth and
amounts of purified proteins which are not attainable from metabolism and, thereby, suggest strategies for in vitro
native purified bacilli. cultivation studies and indicate potential sites for chemo-
Major accomplishments have been realized in the area of therapeutic agents with activity against M. leprae. In addi-
mycobacterial cell wall chemistry and structure. These ad- tion, potentially important immunogenic proteins of M.
vances have not been accompanied by parallel advances in leprae may be produced and tested as vaccines.
our understanding of the biological significance of these Genomic libraries of M. leprae have been established in
same components. In particular, our knowledge concerning the cosmid vector pHC79 and the plasmid vector pYA626 by
the role of M. leprae cell wall and cell wall-associated the Clark-Curtiss group (36, 102). Initially, clones from the
constituents, as they relate to virulence factors of M. leprae, libraries were used to explore the ability of M. leprae genes
remains speculative at best. Further research aimed at to complement known enzymatic pathways in auxotrophic
defining structure-function relationships, using highly de- mutants of E. coli. Complementation with M. leprae DNA
fined and purified components of M. leprae and other was observed for the gltA (citrate synthase) and aroB
mycobacteria, should lead to a more complete understanding (dehydroquinate synthetase) mutations in E. coli (102). Com-
of the pathogenic mechanisms of this group of highly suc- plementation of three gltA mutants was found only when M.
cessful pathogens. leprae DNA was linked to a strong promoter from Strepto-
coccus mutans, suggesting that mycobacterial promoters are
Molecular Biology of M. leprae
utilized only weekly if at all in E. coli. The complementing
DNA fragment specified a 46-kDa polypeptide and probably
Analysis of genome size and guanine-plus-cytosine con- represented the citrate synthase gene of M. leprae. Other M.
tent are two important characteristics of bacteria used in leprae DNA sequences capable of complementation of E.
comparing relatedness among bacterial species. Molecular coli mutants have not been reported, reflecting possible
analysis of purified M. leprae DNA by guanine-plus-cytosine inherent difficulties with this approach due to basic genetic
VOL. 1, 1988 LEPROSY 335

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

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the X gtll library have provided DNA for sequencing of at epidemiological studies. More basic research can be focused
least two M. leprae proteins (18 and 65 kDa), both of which on the immunological mechanisms that contribute to the
have been shown to be immunogenic subsequent to vacci- protective as well as pathologic aspects of host responses to
nation with M. leprae (158, 159). Sequence data of this type leprosy. Ultimately, exploitation of fundamental knowledge
for the 65-kDa proteins of M. leprae, M. bovis BCG, and M. of the immune response to leprosy should allow develop-
tuberculosis have provided detailed amino acid sequence ment of an effective vaccine.
information concerning cross-reactive and species-specific Obstacles to research. Although the leprosy bacillus was
antibody-reactive epitopes (219). Similar work with T-cell identified as a human bacterial pathogen in 1874, until
clones should provide information related to T-cell recogni- recently workers in the leprosy field have made few contri-
tion regions on these and other proteins, allowing for de- butions to the field of immunology and have adopted little in
tailed immunochemical analysis of M. leprae proteins. the way of immunological know-how to their studies. This
Moreover, DNA-derived peptide sequences and subsequent lack of progress has not been due to a lack of effort,
synthesis of these moieties will provide reagents needed to expertise, or dedication by researchers. At virtually every
analyze the complex interactions of antigenic peptides and turn in investigating this disease seemingly insurmountable
class II major histocompatibility complex molecules on obstacles arose that stymied progress. Even 115 years after
antigen-presenting cells. Studies on this type may help its discovery, M. leprae remains uncultivable. Until re-
elucidate the molecular aspects of triggering M. leprae- cently, human biopsies served as the sole source of leprosy
reactive T cells, important in mediating protective cell- bacilli. These crude preparations yielded inadequate num-
mediated immunity (CMI) against M. leprae. bers of organisms, and the lack of reliable methods for
Finally, application of molecular techniques for analysis of quantitating viable organisms prevented standardization of
genomic DNA ultimately may provide new methods for different preparations.
taxonomic purposes and new insight into our thinking about Major breakthroughs. Three major discoveries in the past
M. leprae and related bacterial species. Traditionally, genus, 25 years have allowed leprosy workers in general and
species, and strain differentiation of microorganisms has immunologists in particular to put leprosy research in pace
been based on phenotypic differences observed between with other infectious diseases. (i) The mouse footpad model
bacteria which ultimately are the result of, and consistent for M. leprae infection, developed by Shepard in 1960 (206)
with, the genetic constitution of those organisms. However, offered a means of quantitating the growth of the leprosy
it has been estimated that only 20% of the genomic capability bacillus. Although only a relatively few organisms could be
of a microorganism is represented when applying detailed obtained, this model allowed evaluation of chemotherapeu-
phenotypic analyses (22). Accordingly, major areas of the tic regimens (209) and serves as the basic model for testing
genome, possibly encoding important functional or regula- the efficacy of potential leprosy vaccine preparations. The
tory genes not demonstrable as quantifiable phenotypic use of athymic (nu/nu) mice (33, 39, 117) and rats (60) defined
characteristics, may go undetected. the importance of CMI in host resistance to leprosy and
Obviously, new approaches are needed for studying larger provided models that more closely mimic LL. (ii) In 1971,
areas of all bacterial genomes and particularly noncultivable Kirchheimer and Storrs (113) identified the nine-banded
bacteria, such as M. leprae. Analysis of DNA restriction armadillo as a susceptible host for the growth of M. leprae.
fragments after restriction endonuclease digestion of ge- Armadillos in Louisiana and Texas have subsequently been
nomic DNA (38, 175) and the related approach of restriction shown to harbor a naturally acquired M. leprae infection
fragment length polymorphism analysis hold promise in this (226, 231, 237). The armadillo model provided for the first
area. As we obtain a more complete understanding of the M. time large quantities of leprosy bacilli for immunologic,
leprae genome and its related functional capacity, it is immunoprophylactic, immunotherapeutic, and immuno-
anticipated that most areas of study related to the leprosy chemical studies. (iii) Using armadillo-derived M. leprae,
bacillus will be enriched and, thereby, advance our under- Brennan and his colleagues in Colorado (18, 20, 94, 96, 98)
standing of M. leprae and its basis for pathogenicity. were able to identify in preparations of the leprosy bacillus a
phenolic glycolipid that possesses a unique trisaccharide
IMMUNOLOGY structure that is immunologically specific for M. leprae.
Immunochemical studies and electron microscopy indicate
Active infection with M. leprae is characterized by a that PGL-I is located on the surface of the organism and that
broad spectrum of host response, with great variability in it may accumulate in the tissues of the armadillo in quantities
histopathology and clinical course of infection. There is, equal to half the total weight of the leprosy bacilli present
336 HASTINGS ET AL. CLIN. MICROBIOL. REV.

(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.

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vidual to M. leprae and thus is of prognostic value (189). Sera from individuals with leprosy, including TT patients,
Typically, a positive reaction is biphasic. An early (24 to react strongly with the highly immunogenic LAM isolated
48-h) Fernandez reaction (58) is a DTH reaction (probably to from the cell wall of M. Ieprae (130). In general, these
soluble protein antigens in the preparation) and occurs in TT antibodies cross-react with LAM from M. tuberculosis,
patients as well as contacts or healthy individuals sensitized although specific epitopes for M. leprae LAM are demon-
to M. leprae or cross-reacting antigens from other mycobac- strable with MAb (70). Distinct chemical features for arabi-
teria. A major goal of recombinant DNA research in leprosy nogalactan and peptidoglycan of M. leprae cell wall have
is the production of M. leprae-specific protein antigens that been demonstrated (70, 97). Further work is necessary to
can serve as a reagent for detection of specific DTH in determine whether a routine assay for specific M. leprae
leprosy. antibodies to these cell wall subunits can be developed.
The late (Mitsuda) reaction to lepromin is measured at 21 MAb technology has focused attention on at least five
days and reflects the induction of acquired CMI to M. major protein antigens of M. leprae: those of 65, 36, 28, 18,
leprae, manifested by formation of an organized epithelioid and 12 kDa. Of these, the 65-kDa antigen is the most widely
cell granuloma (143, 189). Positive Mitsuda reactions are studied, including complete expression in the X gtll recom-
seen in the vast majority of contacts and unexposed individ- binant DNA system (259, 260). The 65-kDa protein has been
uals as well as in persons with TT. Weakly positive reactions shown to possess epitopes that cross-react with those of
aid in classification of borderline disease. In LL, no response other mycobacteria (73), but M. leprae-specific epitopes
is seen, indicating the absence of CMI to M. leprae. Even have been found as well (25, 74). With MAb, M. leprae-
after many years of chemotherapy, the lepromin test remains specific epitopes have also been found on the 36- and 12-kDa
negative in LL (156, 232). proteins (101, 115, 119). These specific MAb lend themselves
Humoral immunity and serodiagnosis in leprosy. Because to use in competitive inhibition assays for the serodiagnosis
of the intracellular nature of M. leprae, humoral antibody is of leprosy (114, 225) and may detect a specific antibody
probably not important in resistance but could play a role in response in TT patients.
the pathogenesis of erythema nodosum leprosum reactions
by formation of antigen-antibody complexes (14). LL has
long been associated with a state of hypergammaglobuline- CMI in Leprosy
mia (27, 205), and there is, in general, an inverse correlation
between the anti-M. leprae antibody titer of a patient and the Early pathologists recognized the importance of CMI,
potency of his CMI response to the leprosy bacillus, i.e., a manifested by the epithelioid cell granuloma, in conferring
direct correlation between antibody level and bacillary load. resistance in TT. In addition, DTH appears to underlie much
Demonstration of antibody to specific M. leprae antigens is of the host tissue damage observed in reactional episodes in
not as straightforward. Prior to the availability of armadillo- BT disease (170, 252). As discussed above, the lepromin test
derived M. leprae, organisms extracted from human tissue is uniformly negative in LL patients and remains negative
were used in indirect fluorescent antibody studies, radioim- even after years of successful chemotherapy. An in vitro
munoassays, and crossed immunoelectrophoresis assays (1, manifestation of anergy in LL is demonstrated by the lack of
37, 86, 87), but M. leprae-specific antigens could not be lymphocyte blast transformation in the presence of M.
demonstrated. leprae antigen (75, 160). Early indications that LL was
The discovery that PGL-I is biochemically unique (94, 95) associated with a general suppression of CMI (83, 188, 205)
and immunologically specific (20, 177) for M. leprae reawak- have not held up. The T4/T8 (helper-inducer/suppressor-
ened interest in the serodiagnosis of subclinical leprosy. A cytotoxic) ratio of T lymphocytes in the peripheral blood of
detailed summary of leprosy serodiagnosis can be found in LL patients ranges near the normal value of 2:1 (187). In LL,
the recent review by Gaylord and Brennan (69). The major- there is no evidence for an increased incidence of cancer or
ity of human antibody to PGL-I is of the immunoglobulin M infection with the opportunistic pathogens commonly asso-
subclass (35, 256), and virtually all MAb generated against ciated with the immunocompromised host. For example, the
PGL-I in mice are immunoglobulin M (258). Enzyme-linked clinical course of pulmonary tuberculosis in an LL patient is
immunosorbent assay technology that uses the highly hydro- identical to that in an otherwise healthy individual. The
phobic native PGL-I (35) has been worked out, and attempts absence of specific CMI to antigens of M. leprae in LL has
have been made to standardize (177, 198) and simplify the attracted widespread attention by immunologists interested
procedures (257). Alternatively, hydrophilic synthetic neo- in exploring immunoregulatory mechanisms in a model of
glycoprotein antigens bearing the immunologically distinct nonfatal immunodeficiency disease in humans.
VOL. 1, 1988 LEPROSY 337

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

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plex class II genes. Individuals with the HLA-DR2 or
HLA-DR3 haplotype or both may be predisposed to the TT as yet address the actual mechanisms whereby sensitized
form of leprosy (173, 233, 234), while the incidence of helper T cells fail to respond to antigen. Nor does this
DR2-DQW1 may be increased in individuals with LL (201). hypothesis address the argument of how and why only
Genetic predisposition and as yet unclarified exogenous certain individuals develop a T suppressor cell response
factors such as environment, mode of transmission, and upon challenge with the leprosy bacillus and subsequently
infecting dose should be important considerations in any progress to the lepromatous form of the disease.
widespread vaccine trials for prevention of leprosy. Other groups have developed T-cell clones from the
Role of the lymphocyte in specific anergy in leprosy. At the peripheral blood lymphocytes of leprosy patients by using
lymphocyte level, the presence of T-helper cells specific for autologous antigen-presenting cells to define their reactivity
antigens of the leprosy bacillus is a key characteristic of the to M. leprae antigen. Mostly, antigen-reactive clones, in-
TT end of the clinical spectrum of leprosy. The reason for cluding clones from M. Ieprae-vaccinated volunteers, have
their absence or lack of function in LL remains unclear. been T4 in phenotype (158), some of which proliferated in
Haregewoin et al. (88, 89) appeared to restore T-helper cell response to the 18-kDa M. leprae recombinant protein (260).
response to M. leprae in vitro by supplementing the cell Some T4 clones from a TT patient were shown to react
culture media with the T-cell-amplifying cytokine interleu- specifically with M. leprae, while others responded to other
kin-2 (IL-2). However, others have been unable to confirm mycobacterial antigens (55). As an alternative to autologous
these findings (105, 107, 163) in totally anergic LL patients. blood monocytes as antigen-presenting cells, some workers
Mohagheghpour et al. (153) suggested that lack of specific used Epstein-Barr virus-transformed autologous B cells to
T-cell response in LL may be due to absence of the IL-2 support T-cell clones from TT and BL patients (80, 172, 174).
receptor on these cells. On the other hand, Modlin et al. Clones from the TT patient were all T4, and, although some
(146, 147) have shown T cells bearing IL-2 receptors in the cross-reacted with other mycobacteria, others appeared to
LL lesions themselves, although there were far fewer IL-2- proliferate specifically in the presence of M. leprae 36-kDa
secreting T cells in LL compared with TT lesions. Mohag- protein (174). Interestingly, clones of the T8 suppressor
heghpour et al. (152) have recently reported that M. leprae- phenotype from a BL patient suppressed autologous T4
specific T4 cells are present and respond to M. Ieprae lymphocyte response to M. leprae (172).
antigen, but only after a 48-h "rest" in culture.
Although specific reactivity in TT or specific anergy in LL The Macrophage in Host Resistance to Leprosy
is routinely demonstrated in vitro with peripheral blood
lymphocytes, the reactivity of the cells in the leprosy le- Macrophages participate in the immune response in both
sions, not the blood, is probably more relevant to the CMI afferent and efferent roles. In their afferent role they assist in
response of the host to M. leprae. In a series of studies, regulation of the immune response (4, 131) by interacting
Modlin et al. have investigated the cellular makeup of the TT with T cells in the presentation of antigens or by secretion of
and LL lesion, using immunopathological techniques (149- soluble mediators. Macrophages have been shown to sup-
151). Briefly, their findings indicate that, in contrast to the press T-cell responses in leprosy (164, 196) and may be
near-normal (2:1) T4/T8 ratio in the blood in LL, the relative defective in their ability to present M. Ieprae antigens to
number of helper T cells is markedly lower (0.5:1) in the sensitized T cells (93). Studies have also revealed (191, 239)
lepromatous lesions. The T4/T8 ratio remains approximately that, in LL, macrophages may be defective in the production
2:1 in the lesions in TT leprosy, but more importantly, the of IL-1, the cytokine that can amplify the production of IL-2
cells are arranged in a distinct architecture within the lesion: by T cells.
T4 cells in the centers of the epithelioid granulomas and T8 Regardless of the lack of a clear understanding of the
suppressor cells in the margins. underlying mechanism of defective CMI in LL and the
In a series of studies by Mehra et al. (136, 138, 139), M. possible role of macrophages in anergy, the failure of the
leprae-specific suppressor T cells were demonstrated in the macrophage to kill or inhibit M. leprae is a conspicuous
peripheral blood of LL patients. Immunologically specific characteristic of the lepromatous form of the disease. Its
triggering of the suppressor cells was shown to be elicited by inability to cope with M. leprae is an issue central to
the unique PGL-I antigen of M. leprae (136). The demon- understanding the mechanisms of host resistance to the
strated effects of these suppresor cells in vitro involve leprosy bacillus.
nonspecific suppression of helper T-cell function as mea- Macrophages, activated by lymphokines (gamma inter-
sured by a concanavalin A mitogenic response. Suppression feron [IFN--y], especially) released from sensitized helper T
of a CMI response as potent as that of concanavalin A cells responding specifically to antigen, are thought to play a
338 HASTINGS ET AL. CLIN. MICROBIOL. REV.

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

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that macrophages from LL patients are deficient in their that immunological events occurring in the lepromatous
ability to digest M. leprae (12), not all workers have found lesion do not necessarily represent similar events in other
such deficiencies (52, 197). Our own studies showed that anatomical compartments.
mice with potent populations of activated macrophages were Two recent reports stemming from clinical trials of LL
markedly resistant to footpad infection with M. Ieprae (120, patients treated with IFN--y address local changes at the site
121). In other indirect studies, oxidative metabolism of of injection (106, 165). Local changes included induration
patient phagocytes was tested and found to be normal or due to accumulation of T4 lymphocytes and monocytes and
above normal (68, 76, 133). On the other hand, Nathan et al. enhanced keratinocyte proliferation and la expression, but
(165) and Kaplan et al. (106) showed not only that monocytes there was no obvious evidence of clearance of bacilli. In
from LL patients were deficient in hydrogen peroxide pro- contrast, the reports of Convit and his associates (41, 44)
duction (and presumably microbicidal capacity as well), but suggest that local injection of lepromatous lesions with BCG
also that this defect was reversed by treatment with IFN-y. results in clearance of leprosy bacilli. These findings serve,
These findings are not consistent with studies that showed in part, as the basis for including BCG with a killed M. leprae
no defect in the innate microbicidal capacity towards other vaccine to exploit this immunotherapeutic effect (42).
pathogens of macrophages from LL patients (52). We have Collectively, our in vivo and in vitro studies suggest that
recently demonstrated that activated mouse macrophages any clearance of bacilli from lepromatous lesions as a
have a deleterious, probably microbicidal effect on M. Ieprae consequence of local immunotherapeutic measures (41, 44,
as shown by electron microscopy (220) and marked inhibi- 106, 165) or chemotherapy (190) likely depends on the influx
tion of adenosine 5'-triphosphate (ATP) content and synthe- of new mononuclear phagocytes into the local lesion, rather
sis of PGL-I (186, 224). than activation of the resident lepromatous macrophages.
Because of the T-cell anergy associated with LL, the Even then, these newly arrived macrophages encounter
signals are not produced that would activate the microbicidal local conditions that rapidly restrict their responsiveness to
capacity of the infected macrophages in the lesion. Regard- IFN--y. These findings emphasize that defects in CMI in LL
less, our recent studies (222, 223) show that the M. leprae- likely extend beyond the level of the T cell to include
gorged macrophages from the footpad lesions of infected localized restriction of macrophage-afferent and -efferent
nude (nulnu) mice, a model of experimental LL (33), are functions influenced by lymphokines.
refractory to IFN--y in vitro as measured by four parameters
of activation: microbicidal capacity, cytotoxicity for neo- Leprosy Vaccine
plastic cells, superoxide anion production, and expression of
major histocompatibility complex class II (Ia) antigen. Peri- Perhaps the main goal of immunological investigations in
toneal macrophages from these same mice were fully respon- leprosy is the development of a safe, effective, low-cost
sive to IFN--y. vaccine that can be used in conjunction with other control
Interestingly, a similar refractory response to IFN--y could measures in endemic areas, resulting ultimately in the erad-
be induced in vitro in mouse peritoneal macrophages heavily ication of leprosy. Vaccine research must, of course, take
infected with live M. leprae (L. D. Sibley and J. L. Krahen- into consideration progress made in defining environmental
buhl, Infect. Immun., in press). The development of defec- and genetic factors that determine susceptibility to leprosy.
tive activation was time and dose related, requiring 48 to 72 Two broad approaches are currently being explored, and a
h of incubation with large numbers of bacilli, and was clearly third approach involves the genetic engineering of a leprosy
paralleled by an increase in macrophage production of vaccine.
prostaglandin E2 (PGE2). PGE2 is a potent immune modula- The first approach depends on cross-reactivity between
tor that suppresses macrophage Ia induction by lymphokines M. leprae and other mycobacteria. BCG seems to be the
(227), macrophage tumoricidal capacity (228), and lympho- only other mycobacterium that affords protection against M.
cyte blast transformation (54). Immunosuppressive levels of leprae challenge in mice (215). In large field trials, BCG
PGE2 have been demonstrated to be produced in response to vaccination protected against leprosy in Uganda (24) but was
mycobacterial products (116) or by infection of mice with M. much less effective in Burma (11). Other cross-reactive
intracellulare (53). In support of this hypothesis, Ridel et al. mycobacteria are being actively investigated as leprosy
(191) reported elevated production of PGE2 by human mono- vaccine candidates, including the ICRC bacillus (47) and
cytes from LL patients. Moreover, we have recently dem- "Mycobacterium w." (229).
onstrated that skin biopsy tissue from LL patients that The availability of large amounts of armadillo-derived,
produced high levels of a 235-kDa inhibitor of monocyte purified M. leprae provides the basis for using killed M.
VOL. 1, 1988 LEPROSY 339

TABLE 1. Sources of M. leprae


Host Tissue Incubation
time (mo) Yielda Disadvantages
Armadillo Liver, spleen 18 101o-1013 Expensive; requires specialized facility; highly variable yield;
unused tissue must be frozen; often contaminated with other
slow-growing mycobacteria (180, 181)
Athymic "nude" mouse Footpad 12-18 101o-1011 Requires special isolator, sterile water, feed, and bedding
Human Skin nodule 107-109 Often contaminated with other skin-borne bacteria; low viability
Mouse Footpad 6-12 106 Yield too low for physiological studies
a From Wheeler (242).

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.

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DTH in guinea pigs (135). Human volunteers vaccinated Another major problem in studying the metabolism of
with killed M. leprae have been shown to develop a DTH host-derived microorganisms is that of differentiating be-
response (72). tween host- and bacillary-derived activities. Since host-
A novel approach to vaccination combines killed M. derived enzymes may be adsorbed to the surface of intracel-
leprae with viable BCG and seems to exploit specific, but lular bacilli during tissue homogenization, mere removal of
undefined, immunity to M. Ieprae, with relevant cross-reac- microscopically detectable tissue debris is not sufficient to
tive immunity to BCG. Convit and his colleagues devised establish bacterial activity. Surface treatments, usually in-
this immunoprophylactic-immunotherapeutic approach volving washing with NaOH, have been shown to abolish
based on their observations that local injection of this host-derived activities but may also destroy bacterial surface
mixture into lesions of BL and LL patients induced conver- enzymes (249). Host and bacterial activities can often be
sion to positive lepromin skin test reactivity, clearance of differentiated via electrophoretic migration, substrate spec-
bacilli, histopathological upgrading, and clinical improve- ificity, serology, or the use of differential enzyme inhibitors
ment and decreased suppressor cell activity in some patients (242).
(42, 43, 137). Long-term trials of the BCG-M. leprae vaccine Considering its uniqueness at a molecular level (guanine-
are under way or are being planned in Africa, South Amer- plus-cytosine percentage and DNA homology) (6, 36, 45),
ica, and southern India. one might expect to find a number of unusual or unique
Finally, propagation of M. leprae genes in E. coli (260) metabolic activities in M. leprae compared with other my-
allows potentially unlimited production of M. leprae proteins cobacteria. Unfortunately, some comparative studies have
for immunologic analysis and for use as diagnostic skin test used cultivable mycobacteria which were propagated in vitro
reagents. The contribution of any of the M. leprae-defined and thus have compared an in vivo-grown organism (M.
proteins to a protective response against the invading bacilli leprae) with in vitro-grown bacilli. Since phenotypic expres-
is difficult to assess. However, recombinant proteins of M. sion may be strongly influenced by the growth environment
leprae have been used to provide evidence that human T-cell (203), the results of such experiments do not necessarily
clones recognize the 18-kDa (158) and 36-kDa (174) proteins clarify the phenotypic relationship between M. leprae and
of M. leprae. This suggests that both proteins may be other mycobacteria.
relevant to protection against leprosy. Catabolic activity. M. leprae appears to be metabolically
However, even highly M. leprae-specific pure proteins or competent with regard to catabolic pathways for energy
peptides will probably require incorporation in an adjuvant generation. Glucose is actively transported (109) and, as in
to induce long-term CMI and protection in humans. Few, if most mycobacteria, is oxidized to carbon dioxide (240, 241),
any, potential adjuvants suitable for human use are avail- although rather slowly. Also, as with other mycobacteria,
able. Ingenious plans are under way (15) to engineer a safe, oxidation appears to occur primarily through the Embden-
potent leprosy vaccine consisting of highly immunogenic Meyerhof pathway and, to a lesser extent, via the hexose
BCG containing the appropriate genes of M. leprae, which monophosphate pathway (240). Strongly supporting the ex-
may allow these organisms to express those M. leprae istence of these pathways was Wheeler's demonstration of
antigens associated with a protective immune response. the component enzyme activities (241), although two Emb-
den-Meyerhof pathway enzymes could not be detected in
MICROBIOLOGY another laboratory (155). Also, as with other mycobacteria,
glycerol is oxidized through these pathways. What appears
Metabolism unique to M. leprae is its extremely high level of 6-phospho-
gluconate dehydrogenase production (241), over 100 times
The relative difficulty in studying metabolism in a bacte- the level of the first hexose monophosphate pathway en-
rium which can be propagated only in vivo cannot be zyme, glucose 6-phosphate dehydrogenase. In addition, the
overemphasized. The sources of M. leprae listed in Table 1 ability of the bacillus to oxidize 6-phosphogluconate, but not
all have drawbacks for research. In practice, the armadillo gluconate, suggests that M. leprae may actively scavenge
has served as the source of bacilli for most physiological 6-phosphogluconate in vivo for use as an energy source
studies. However, there are only a few armadillo farms in (245).
the world, which severely limits the number of available Pyruvate produced via glycolosis may be converted to
bacilli. This problem is further exacerbated by the extremely lactate by lactate dehydrogenase (250) or cycled through the
slow growth rate (generation time, approximately 12 days) tricarboxylic acid cycle (243). The existence of an intact
and apparently low overall viability of M. leprae populations tricarboxylic acid cycle in M. leprae was suggested by the
340 HASTINGS ET AL. CLIN. MICROBIOL. REV.

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

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Unlike chlamydiae, M. leprae is capable of generating its
own ATP, as demonstrated by the presence of adenylate cooler regions of the body. In the mouse footpad, M. leprae
kinase (127), incorporation of exogenous phosphate into showed a reduced growth rate when mice were housed in the
ATP (126), and short-term 2,4-dinitrophenol-sensitive in- conditions where the average footpad temperature was 360C
creases in ATP pools immediately following harvest from as compared with conditions maintaining footpad tempera-
armadillo tissue (124). In addition, extended maintenance of tures of 27 to 30'C (207). These in vivo observations have
intracellular pools of such a labile compound under appro- been corroborated by the observation that the optimum
priate conditions (62a) indirectly suggests that some active temperature for in vitro oxidation of palmitic acid by M.
synthesis is occurring. leprae is 330C (62). This activity ceases within 3 days at 370C
M. leprae contains a manganese-dependent superoxide in contrast to a linear response for .1 week at 330C. In
addition, M. leprae reportedly takes up 3,4-dihydroxyphen-
dismutase (123, 244, 251) but appears to be devoid of ylalanine optimally at 34WC (110). Consistent with an optimal
catalase (155, 251), thus suggesting a microaerophilic dispo- pH of 5.8 to 6.5 for slowly growing mycobacteria in general
sition. This is supported by optimal maintenance of intracel- (182), M. leprae reportedly has a pH optimum of 5.8 for
lular ATP at reduced oxygen concentrations, as mentioned respiration (100). As mentioned above, most studies suggest
below. that M. leprae utilizes free oxygen in its energy-generating
Amino acid metabolism. M. leprae is capable of amino acid processes. A recent study on ATP maintenance and PGL-I
uptake (245) and incorporation into protein (109), although synthesis indicated biophysical optima of 330C and pH 5.6
there is a lack of data concerning most individual amino and a reduced but maintained oxygen concentration (62a).
acids. 3,4-Dihydroxyphenylalanine is taken up (3) and oxi-
dized (185), although the nature, uniqueness, and signifi- In Vivo Drug Testing
cance of this activity continue to be a matter of controversy Prior to the demonstration in 1960 of limited multiplication
(183, 242). 3,4-Dihydroxyphenylalanine oxidase activity is of M. leprae in the footpads of immunologically intact mice
accepted by many as a specific taxonomic marker of M. (206), therapeutic agents for leprosy were selected solely on
leprae. Other enzymes of amino acid metabolism which have the basis of empirical results in clinical trials. Treatment with
been detected are glutamate decarboxylase (184) and gam- chaulmoogra oil, based on Burmese folk medicine, was only
ma-glutamyl transpeptidase (218). partially effective and was used until the sulfones were found
Nucleic acid metabolism. Thymidine is taken up by M. to be highly efficacious. Until the development of the mouse
leprae residing in macrophage cell cultures (162) or sus- footpad model, activity against M. tuberculosis had been
pended in axenic culture media (111). Uracil is also taken up considered a valid criterion for initiation of clinical trials in
in axenic medium, as is the pyrimidine precursor orotic acid. leprosy.
In general, purine bases are taken up and incorporated into In the immunologically intact mouse, M. leprae can attain
trichloroacetic acid-insoluble material at higher rates than only a density of approximately 106 bacilli per footpad,
are pyrimidipes. M. leprae appears to be incapable of de regardless of the inoculum dosage (206). This, however, is
novo purine synthesis, as evidenced by lack of incorporation sufficient for detection of growth-inhibitory compounds
of [14C]serine or ['4C]glycine into the purine fraction, and when low inocula (5 x 103 to 1 x 104) are used. Bacilli,
may obtain purines primarily via scavenging mechanisms obtained either from patient biopsies or by animal passage,
(246, 247). Enzymes for interconversion of purine bases are inoculated into one or both hind footpads in a volume of
have been detected in cell-free extracts, and rates of incor- approximately 0.03 ml. Test compounds are usually pow-
poration of purines could be accounted for by the levels of dered and mixed in the diet at concentrations ranging from
phosphoribosyltransferases in cell-free extracts. Of interest 0.0001 to 0.1% (wt/wt). Alternatively, compounds may be
is the relatively high level of adenosine kinase in M. leprae administered by gavage or parenterally, although the repeti-
compared with other host-grown mycobacteria. tion of such procedures over the course of several months is
Lipid metabolism. M. leprae residing within Schwann cells laborious. All of the presently used antileprosy drugs can be
reportedly incorporate acetate into the PGL-I fraction (157). administered in the feed, and ultimately any new useful
Similarly, bacilli residing within macrophages have been drugs should be active orally in humans. Bacillary growth is
shown to rapidly incorporate palmitic acid into PGL-I (186). evaluated by direct microscopy (Ziehl-Neelsen staining)
The latter also occurs in M. leprae residing extracellularly in (213) of footpad homogenates following incubation periods
an axenic medium (63). Other precursors, including acetate, of several months to 1 year.
were not incorporated into PGL-I in this system. Incorpora- Four techniques have been described and utilized in the
tion of 32P into the phospholipid fraction has been reported evaluation of potential antileprosy compounds. The earli-
VOL. 1, 1988 LEPROSY 341

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

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tory or bacteriopausal activity. will await the development of a growth-supporting method-
The proportional bactericidal method (40) involves inocu- ology.
lation of groups of mice with 10-fold dilutions of M. leprae Incorporation of tritiated thymidine into the trichloroace-
and administration of test compounds for 2 months. Growth tic acid-insoluble fraction of phagocytized M. leprae is
of bacilli is determined after 12 months, using most-proba- inhibited by clinical antileprosy agents (162) and has been
ble-number calculations. Only bactericidal agents appear used to evaluate a number of phenazines (145). Results in
active in this system, while inactive and bacteriostatic agents this system have been found to correlate well with the mouse
cannot be differentiated. footpad technique in evaluating secondary drug resistance in
The rapidity of onset of bactericidal activity can be clinical isolates (200). A scaled-down version of this tech-
determined by serial inoculation of mice with patient skin nique (144) reduces the requirement for both bacilli and
biopsy material at time intervals following the initiation of macrophages, making its clinical use more practical. While
drug therapy (210). thymidine is also taken up by extracellular M. leprae, other
Only a relatively small number of compounds have dem- nucleic acid precursors such as hypoxanthine are taken up
onstrated bactericidal activity in these systems, including all more rapidly and are also sensitive to the action of antile-
of the clinically useful antileprosy agents: dapsone (weak), prosy agents (111). Thus, these compounds may ultimately
rifampin and related compounds, and clofazimine, with only prove to be superior substrates when nucleic acid synthesis
rifampin being rapidly bactericidal. Ethionamide and pro- is used in a drug-screening system.
thionamide (212), thiacetazone, some of the fluoroquino- Our laboratory has recently developed three distinct sys-
lones (79, 176, 195), and certain cephalosporins (214) have tems for large-scale screening of potential antileprosy
also shown bactericidal-like activity. An effective therapeu- agents. The incorporation of [14C]palmitic acid into PGL-I of
tic regimen should include at least one bactericidal drug if M. leprae has been shown to be sensitive to clinical antile-
that drug is to be used intermittently, e.g., once monthly, prosy drugs as well as a number of other compounds in both
and therefore such drugs are actively sought. intracellular (186) and extracellular (63) M. leprae. The use
Drug-resistant leprosy bacilli can be detected by the of these systems in parallel has the potential for evaluating
continuous-feed technique (178). High-, medium-, and low- the effect of intracellular residence on bacillary metabolic
level resistance to dapsone are defined as growth in the stability and drug susceptibility. Over 25 antimicrobial
presence of 0.01, 0.001, and 0.0001% drug, respectively (90). agents have been evaluated by their ability to effect an
Primary resistance is usually low level, and patients harbor- accelerated rate of ATP decay in extracellular M. leprae
ing such bacilli would normally be expected to respond to suspended in axenic media (64). Finally, palmitate oxidation
full-dosage dapsone. Testing for secondary dapsone resis- to carbon dioxide, measured by radiorespirometry, was
tance enables the physician to determine whether the failure found to be sensitive to antileprosy agents (62). The use of an
to respond to- therapy is due to true drug resistance of the automatic Buddemeyer-type counting system (26) makes
bacilli or to a lack of compliance by the patient (104). this system the simplest described to date for evaluating
The armadillo (113), athymic (nude) mouse (33), and antileprosy agents. The ability to readily detect activity with
neonatally thymectomized Lewis rat (61) all support exten- approximately 106 bacilli and the precedence for using this
sive multiplication of M. leprae. Athymic rodents have been activity in the rapid drug susceptibility testing of cultivable
used as models for treatment of LL, in which initial bacillary mycobacteria (134, 193, 235) make this assay a strong
loads may be quite high (118). In addition, their use enables candidate for use in the clinical detection of secondary drug
larger inocula to be used when attempting to detect the resistance pending further studies.
presence of resistant or "persister" bacilli in treated pa- These systems have identified fluoroquinolines, minocy-
tients. cline, and some phenazines as having anti-M. leprae activ-
ity, findings consistent with the activity of these compounds
In Vitro Drug Testing in the mouse footpad (71, 79, 176, 195). Perhaps most
interesting is the potent activity in these systems of eryth-
Although the development of the mouse model has made romycin and two new semisynthetic macrolides (S. G.
possible the screening of potential antileprosy compounds Franzblau, N. Ramasesh, E. B. Harris, and R. C. Hastings,
prior to clinical evaluation, the long incubation time, high Program Abstr. 27th Intersci. Conf. Antimicrob. Agents
cost, requirement that the test compound have favorable Chemother., abstr. no. 1368, 1987), Roxithromycin (RU 965;
pharmacokinetics in the mice, and requirement for gram Hoechst-Roussel Pharmaceuticals Inc., Somerville, N.J.)
quantities of test compounds have undoubtedly limited the (30) and Clarithromycin (TE-031, A-56268; Abbott Labora-
342 HASTINGS ET AL. CLIN. MICROBIOL. REV.

tories, North Chicago, Ill.) (59). The semisynthetic macro- 9. Barksdale, L., and K. S. Kim. 1977. Mycobacterium. Bacteriol.
lides have demonstrated markedly superior pharmacokinetic Rev. 41:217-372.
properties. Recent (unpublished) studies in our laboratory 10. Baskin, G. B., B. J. Gormus, L. N. Martin, R. H. Wolf, M.
have shown that, when administered in the feed of mice at Murphey-Corb, G. P. Walsh, C. H. Binford, W. M. Meyers,
0.01% (wt/wt), erythromycin ethylsuccinate and Roxi- and R. Malaty. 1987. Experimental leprosy in a Rhesus mon-
thromycin are unable to inhibit multiplication of M. leprae in key: necropsy findings. Int. J. Lepr. 55:109-115.
11. Bechelli, L. M., P. G. Garbajosa, M. M. Gyi, K. Uemura, T.
the footpad, while Clarithromycin fully suppresses growth of Sundaresan, W. M. Dominguez, M. Matejka, C. Tamondong,
the bacillus. As thermostable, relatively inexpensive com- R. Quagliato, V. Engler, and M. Altmann. 1973. BCG vaccina-
pounds acting at a locus not currently exploited in leprosy tion of children against leprosy: seven-year findings of the
chemotherapy and with proven activity against a variety of controlled WHO trial in Burma. Bull. W.H.O. 48:323-334.
intracellular pathogens, including mycobacteria (13), macro- 12. Beiguelman, B. 1967. Leprosy and genetics. A review of past
lides possess many desirable features for inclusion in multi- research with remarks concerning future investigations. Bull.
drug therapy against leprosy. W.H.O. 37:461-476.
In summary, the currently uncultivable M. leprae contin- 13. Berlin, 0. G. W., L. S. Young, S. A. Floyd-Reising, and D. A.
ues to present enormous challenges in all aspects of leprosy Bruckner. 1987. Comparative in vitro activity of the new
macrolide A-56268 against mycobacteria. Eur. J. Clin. Micro-
research. The bacterium has no readily apparent biochemi- biol. 6:486-487.

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cal lesions with regard to energy generation. However, 14. Bjorvatn, B., R. S. Barnetson, G. Kronvall, R. Zubler, and
further studies are required to determine the complete ana- P. H. Lambert. 1976. Immune complexes and complement
bolic requirements and biophysical optima which should hypercatabolism in patients with leprosy. Clin. Exp. Immunol.
facilitate the development of an in vitro growth-supporting 26:388-396.
methodology. While well-established in vivo systems for 15. Bloom, B. R. 1986. Learning from leprosy: a perspective on
assessing antileprosy drug activity exist, the more recently immunology and the Third World. J. Immunol. 137:i-x.
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Bacteriol. 113:645-651.
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ACKNOWLEDGMENTS ill Livingstone, Ltd., Edinburgh.
18. Brennan, P. J. 1983. The phthiocerol-containing surface lipids
The research reported in this review was supported in part by of M. leprae-a perspective of past and present work. Int. J.
Public Health Service grants from the National Institutes of Health Lepr. 51:387-396.
(AI22492, AI22442, A122007, and NIAID Interagency Agreement 19. Brennan, P. J., G. 0. Aspinall, and J. E. Nam Shin. 1981.
Y01-AI-50001) and by grants from the Victor Heiser Foundation, Structure of the specific oligosaccharides from the glycopepti-
The Baton Rouge Area Foundation, and the Hansen's Disease dolipid antigens of serovars in the Mycobacterium avium-
Foundation. Mycobacterium intracellulare-Mycobacterium-scrofulaceum
We are deeply grateful to Rened Painter, Rosie Hauge, and Penne serocomplex. J. Biol. Chem. 256:6817-6822.
Cason for their help in the literature search and in preparation of the 20. Brennan, P. J., and W. W. Barrow. 1980. Evidence for
manuscript. species-specific lipid antigens in Mycobacterium leprae. Int. J.
Lepr. 48:382-387.
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