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Background: The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues., Methodology/principle Findings: Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations., Conclusions: hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.

Early diagnosis of leprosy and a multi-drug therapy (MDT) regimen will block the trajectory of nerve damage, disability and deformity that are the hallmarks of this chronic disease. However, the diagnosis of leprosy is made solely by recognition of clinical signs and symptoms, requiring special expertise. These limitations also result in the under reporting of worldwide prevalence and incidence rates for leprosy. Sorely needed is an objective laboratory test for detecting early leprosy. As the antigenic burden of M. leprae can be virtually undetectable in early clinical leprosy, cell mediated immunity and antibody responses will likely be weak. So the sensitivity of new diagnostic tests is as important as specificity. Major efforts are underway employing recombinant M. leprae antigens and synthetic peptides, to develop diagnostic assays for early leprosy infection, using in vitro T cell reactivity or serological tests. We have used the initial phase of the mouse foot pad model as an 'early' model of leprosy infection to screen T cell responses against M. leprae specific antigens and synthetic peptides. Unlike human disease in animal models we can control infection progress and monitor bacillary growth relative to time course of development of T cell response to specific M. leprae antigens. The study employed splenic T cells instead of draining lymph node T cells to model the systemic response as opposed to a local one. We found that 10(5) live M. leprae is the minimum dose required for any meaningful and consistent in vitro splenic IFN-gamma response against M. leprae antigens 3 months after foot pad inoculation. Using this model we found that several M. leprae recombinant proteins, ML0840, ML2028, ML2307, ML2346, ML2478, and ML2532, induced significant levels of IFN-gamma secretion. By controlling for variables that can be confounding factors in the sensitivity of human testing, this mouse model provides an interface between M. leprae diagnostic antigen development and the screening of these antigens in humans under field conditions.

The role played by apoptosis in host response to Mycobacterium leprae is unclear. Here, we studied in vitro induction of apoptosis in mouse bone marrow-derived macrophages infected with live and irradiated M. leprae, as a function of multiplicity of infection under permissive (33 degrees C) and nonpermissive (37 degrees C) temperatures. The infected macrophages were scored for apoptosis by using DAPI (4',6-diamindino-2-phenylindole dihydrochloride) and Annexin V staining, along with activated Caspases 3 and 9 and TUNEL (terminal dUTP nick end labeling) assay. Our results show that, in contrast to uninfected cells, murine macrophages infected with live M. leprae demonstrated little, if any, apoptosis, even when macrophages had a heavy burden of live leprosy bacilli. In contrast, elevated levels of apoptosis were observed when macrophages were infected with irradiated M. leprae. The results strongly suggest that the viability and purity of the leprosy bacilli used for in vitro studies determines the extent of apoptosis observed in infected host cells.

Recent studies identified an association between genetic variants in the lymphotoxin-alpha (LTalpha) gene and leprosy. To study the influence of LTalpha on the control of experimental leprosy, both low- and high-dose Mycobacterium leprae foot pad (FP) infections were evaluated in LTalpha-deficient chimeric (cLTalpha(-/-)) and control chimeric (cB6) mice. Cellular responses to low-dose infection in cLTalpha(-/-) mice were dramatically different, with reduced accumulation of CD4(+) and CD8(+) lymphocytes and macrophages and failure to form granulomas. Growth of M. leprae was contained for 6 months, but augmented late in infection. In contrast, tumor necrosis factor knockout and tumor necrosis factor receptor 1 knockout FPs exhibited extensive inflammatory infiltration with an increase in M. leprae growth throughout infection. Following high-dose infection, cB6 FP induration peaked at 4 weeks and was maintained for 12 weeks. Induration was not sustained in cLTalpha(-/-) FPs that contained few lymphocytes and no granulomas. There was a reduction in the expression levels of inflammatory cytokines, chemokines, and chemokine receptors, including nitric oxide synthase 2, vascular cell adhesion molecule, and intercellular cell adhesion molecule. Furthermore, cLTalpha(-/-) popliteal lymph nodes contained a higher proportion of naïve CD44(lo)CD62L(hi) T cells than cB6 mice, suggestive of reduced T cell activation. Therefore, both LTalpha and tumor necrosis factor are essential for the regulation of the granuloma, but they have distinctive roles in the recruitment of lymphocytes and maintenance of the granulomatous response during chronic M. leprae infection.

Objectives: Leprosy transmission remains poorly understood, though, prolonged skin contact and/or infection via nasal mucosa, are considered likely. Problematic in any transmission hypothesis is the fastidious nature of Mycobacterium leprae outside its host cell and the requirement for temporary survival in the environment, soil or water. Experiments were carried out to test the hypothesis that free living pathogenic amoeba might serve as host cells for M. leprae, protecting them from adverse environmental conditions., Design: In this study we employed cultures of Acanthamoeba castellanii, a free-living pathogenic soil amoeba, to determine whether these protozoa can ingest M. leprae and whether the intracellular bacilli remain viable., Results: More than 90% of cultured amoeba ingested M. leprae at a 20:1 multiplicity of infection while the infected amoebae thrived and multiplied normally. The ingested M. leprae were not degraded and remained viable for at least 72 hours as determined by their metabolic activity (radiorespirometry) and cell wall integrity (viability staining). M. leprae isolated from infected amoebae multiplied at the same rate as freshly harvested bacilli in the foot pads of nu/nu mice., Conclusions: These findings provide proof of principle that free-living pathogenic amoebae are capable of ingesting and supporting the viability of M. leprae expelled into the environment. Studies are underway to determine whether M. leprae-infected A. castellanii and other pathogenic amoebae may also play a role in transporting leprosy bacilli through broken skin or the nasal mucosa.

Objective: The immune-mediated events that precipitate erythema nodosum leprosum (ENL) are not well understood. One component may be the complexing of antibody with antigens released from infected macrophages, the activation of complement and the subsequent local inflammation. We assess here the ability of highly-purified, disrupted M. leprae, to activate complement., Results: Intact and sonically-disrupted crude and alkali-purified nu/nu mouse-derived M. leprae suspensions were incubated with normal serum and a hemolytic titer (CH50) was determined as a measure of complement fixation. Crude M. leprae consumed complement, and disrupted preparations more than the intact. Purified M. leprae preparations did not consume complement unless disrupted., Conclusion: M. leprae, if disrupted, can activate complement. This supports a hypothesis that links released antigens with ENL, and may explain the increased probability of an occurrence of ENL following chemotherapy.

Armadillos (Dasypus novemcinctus) manifest the full histopathological spectrum of leprosy, and are hosts of choice for in vivo propagation of Mycobacterium leprae. Though potentially useful as a model of leprosy pathogenesis, few armadillo-specific reagents exist. We have identified a region of high homology to the interferon gamma (IFN-gamma) of other mammals within the recently published armadillo whole genomic sequence. cDNA was made from ConA-stimulated armadillo peripheral blood mononuclear cells (PBMC), amplified, and cloned into a pET expression vector for transformation and over-expression in Escherichia coli. The recombinant protein (rDnIFN-gamma) was characterized by western blot and its biological function confirmed with bioassays including intracellular killing of Toxoplasma gondii and induction of indoleamine 2, 3-dioxygenase activity. In using rIFN-gamma to activate macrophages from mice, humans or armadillos, similar to humans, rIFN-gamma-activated armadillo MPhi did not produce nitrite and or inhibit the viability of M. leprae in vitro. Conversely, murine rIFN-gamma-activated mouse MPhi produced high levels of nitrite and killed intracellular M. leprae in vitro. These data indicate that the response of armadillo MPhi to rDnIFN-gamma is similar to that which occurs in humans, and demonstrates a potentially important value of the armadillo as a model in leprosy research.

Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae-infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.

Leprosy affects skin and peripheral nerves, and acute inflammatory type 1 reactions (reversal reaction) can cause neurologic impairment and disabilities. Single skin lesion paucibacillary leprosy volunteers (N = 135) recruited in three Brazilian endemic regions, treated with single-dose rifampin, ofloxacin, and minocycline (ROM), were monitored for 3 years. Poor outcome was defined as type 1 reactions with or without neuritis. IgM anti-phenolic glycolipid I, histopathology, Mitsuda test, and Mycobacterium leprae DNA polymerase chain reaction (ML-PCR) were performed at baseline. chi(2) test, Kaplan-Meir curves, and Cox proportional hazards were applied. The majority of volunteers were adults with a mean age of 30.5 +/- 15.4 years; 44.4% were ML-PCR positive. During follow-up, 14.8% of the patients had a poor clinical outcome, classified as a type 1 reaction. Older age (> or = 40 years), ML-PCR positivity, and lesion size > 5 cm were associated with increased risk. In multivariate analysis, age (> or = 40 years) and ML-PCR positivity remained baseline predictors of type 1 reaction among monolesion leprosy patients.

Leprosy is caused by infection with Mycobacterium leprae. The immune response of leprosy patients can be highly diverse, ranging from strong cellular responses accompanied by an apparent deficit of M. leprae-specific antibodies to strong humoral responses with a deficit of cell-mediated responses. Leprosy takes many years to manifest, and this has precluded analyses of disease and immune response development in infected humans. In an attempt to better define development of the immune response during leprosy we have developed an M. leprae ear infection model. Intradermal inoculation of M. leprae into the ear supported not only infection but also the development of a chronic inflammatory response. The inflammatory response was localized, comprising a T-cell infiltration into the ear and congestion of cells in the draining lymph nodes. The development of local chronic inflammation was prevented by rifampin treatment. Importantly, and in contrast to subcutaneous M. leprae footpad infection, systemic M. leprae-specific gamma interferon and antibody responses were detected following intradermal ear infection. These results indicate the utility of intradermal ear infection for both induction and understanding of the immune response during M. leprae infection and the identification or testing of new leprosy treatments.

Cytokine-activated macrophages (MPhi) employ reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to combat pathogens. The requirement for ROI for an effective host response to experimental leprosy using mice which have a disruption in the 91-kD subunit of the NAPDH oxidase cytochrome b (phox91-/-) was examined. Mycobacterium leprae multiplication in phox91-/- foot pads (FP) was elevated early in infection but subsequently arrested similarly to control mice within a noninvasive granuloma. Using a modified lepromin test model, a similar cellular composition in the M. leprae-induced FP granuloma in both strains with lymphocyte infiltration consisting primarily of CD4+CD44(hi)CD62L(lo) effector cells was found. Of great interest was the disparity in the T cell population between the granuloma and the draining lymph node which contained predominantly naïve CD4+CD44(lo)CD62L(hi) cells and was, therefore, not representative of the infection site. TH1 cytokines, chemokines and inducible nitric oxide synthase were comparably expressed in the FP of both strains. When infected in vitro, normal MPhi from B6 and phox91-/- mice supported bacterial viability, whereas IFNgamma-activated MPhi killed M. leprae in a RNI-dependent manner, emphasizing that ROI was dispensable. These data show that phox91-/- mice generate a strong adaptive immune response and control long-term infection with M. leprae.

Leprosy responds very slowly to the current multidrug therapy, and hence there is a need for novel drugs with potent bactericidal activity. PA-824 is a 4-nitroimidazo-oxazine that is currently undergoing phase I clinical trials for the treatment of tuberculosis. The activity of PA-824 against Mycobacterium leprae was tested and compared with that of rifampin in axenic cultures, macrophages, and two different animal models. Our results conclusively demonstrate that PA-824 has no effect on the viability of M. leprae in all three models, consistent with the lack of the nitroimidazo-oxazine-specific nitroreductase, encoded by Rv3547 in the M. leprae genome, which is essential for activation of this molecule.

Leprosy is best understood as two conjoined diseases. The first is a chronic mycobacterial infection that elicits an extraordinary range of cellular immune responses in humans. The second is a peripheral neuropathy that is initiated by the infection and the accompanying immunological events. The infection is curable but not preventable, and leprosy remains a major global health problem, especially in the developing world, publicity to the contrary notwithstanding. Mycobacterium leprae remains noncultivable, and for over a century leprosy has presented major challenges in the fields of microbiology, pathology, immunology, and genetics; it continues to do so today. This review focuses on recent advances in our understanding of M. leprae and the host response to it, especially concerning molecular identification of M. leprae, knowledge of its genome, transcriptome, and proteome, its mechanisms of microbial resistance, and recognition of strains by variable-number tandem repeat analysis. Advances in experimental models include studies in gene knockout mice and the development of molecular techniques to explore the armadillo model. In clinical studies, notable progress has been made concerning the immunology and immunopathology of leprosy, the genetics of human resistance, mechanisms of nerve injury, and chemotherapy. In nearly all of these areas, however, leprosy remains poorly understood compared to other major bacterial diseases.

Over the years, researchers have carried out experiments with Mycobacterium leprae obtained from either human multibacillary lesions, or infected armadillo tissues, or infected footpad tissues of conventional mice as well as athymic nu/nu mice. In general, these sources of leprosy bacilli are satisfactory for most biochemical and mouse footpad studies, but less than satisfactory for studies in cell biology and immunology where contaminating host tissues pose a serious problem. We examined the utility of a procedure for eliminating mouse footpad tissue from M. leprae suspension using sodium hydroxide solution and its subsequent effect on the viability of the organism by determining the rate of palmitic acid oxidation, bacterial membrane integrity, and growth in the mouse footpad. We found that treating M. leprae suspension, obtained from infected nu/nu mouse footpad, with 0.1N NaOH for 3 min was sufficient to remove the majority of mouse tissue without adversely affecting the viability of the organism. This is a simple and rapid method to get suspensions of nu/nu footpad-derived viable M. leprae essentially free of host tissues, which can be a research reagent for studying the host-pathogen relationship in leprosy. We also report here a method for labeling M. leprae with the fluorescent dye PKH26, without compromising on the viability of the organism. This method may be useful in intracellular trafficking studies of M. leprae or in other cell biology studies that require tracking of the bacteria using fluorescent tag. We observed the staining to be stable in vitro over considerable lengths of time and did not affect the viability of the bacteria.

The lepromatous leprosy granuloma is a dynamic entity requiring a steady influx of macrophages (Mphi) for its maintenance. We have developed an in vitro model to study the fate of Mycobacterium leprae in a LL lesion, with and without immunotherapeutic intervention. Target cells, consisting of granuloma Mphi harvested from the footpads of M. leprae-infected athymic nu/nu mice, were cocultured with normal or IFN-gamma-activated (ACT) effector Mphi. The bacilli were recovered and assessed for viability by radiorespirometry. M. leprae recovered from target Mphi possessed high metabolic activity, indicating a viable state in this uncultivable organism. M. leprae recovered from target Mphi incubated with normal effector Mphi exhibited significantly higher metabolism. In contrast, bacilli recovered from target Mphi cocultured with ACT effector Mphi displayed a markedly decreased metabolic activity. Inhibition by ACT Mphi required an E:T ratio of at least 5:1, a coculture incubation period of 3-5 days, and the production of reactive nitrogen intermediates, but not reactive oxygen intermediates. Neither IFN-gamma nor TNF-alpha were required during the cocultivation period. However, cell-to-cell contact between the target and effector Mphi was necessary for augmentation of M. leprae metabolism by normal effector Mphi as well as for inhibition of M. leprae by ACT effector Mphi. Conventional fluorescence microscopy and confocal fluorescence microscopy revealed that the bacilli from the target Mphi were acquired by the effector Mphi. Thus, the state of Mphi infiltrating the granuloma may markedly affect the viability of M. leprae residing in Mphi in the lepromatous lesion.

We explored the prognostic value of in situ cytokine patterns in 39 patients with single-skin-lesion paucibacillary leprosy before single-dose therapy, with 3 years of follow-up. Interferon (IFN)-gamma, interleukin (IL)-12, IL-10, IL-4, tumor necrosis factor (TNF)-alpha, and macrophage inflammatory protein (MIP)-1alpha mRNA was quantified in skin biopsy samples at diagnosis, and Mycobacterium leprae DNA was detected in 51.4% of cases. Type 1 immunity predominance with measurable IFN-gamma and undetectable IL-4, which is indicative of effective cell-mediated immunity, is compatible with both the reversal reactions (33.3%) and the resolution of lesions (64.1%) observed. A positive correlation between IL-12 and IFN-gamma indicated type 1 polarization via IL-12. The TNF-alpha/MIP-1alpha correlation implied the TNF-alpha induction of chemokines, which is important for granuloma formation. Positive correlations between key regulatory cytokines-IL-10 and IFN-gamma, IL-10 and IL-12, and IL-10 and TNF-alpha-suggests that there may be some level of an intralesional pro- or anti-inflammatory mechanism essential in avoiding immunopathology.

Mycobacterium leprae infection was evaluated in interferon-gamma knockout (GKO) mice. At 4 months, growth of the bacilli in the footpads of GKO mice plateaued a log(10) higher than that in control mice. Control mice exhibited mild lymphocytic and histiocytic infiltrates, whereas GKO mice developed large, unorganized infiltrates of epithelioid macrophages and scattered CD4 and CD8 T cells. Flow cytometric analysis of popliteal lymph node cells demonstrated similar profiles of T cells; however, GKO cells exhibited an elevated proliferative response to M. leprae antigen. Expression of inducible nitric oxide synthase mRNA was decreased in GKO mice, whereas macrophage inflammatory protein-1alpha and interleukin-4 and -10 mRNA expression were augmented. Control and GKO activated macrophages inhibited bacterial metabolism and produced nitrite. Thus, although deficient in an important Th1 cytokine, GKO mice possess compensatory mechanisms to control M. leprae growth and feature elements resembling mid-borderline leprosy in humans.

Mycobacterium leprae remain a rare research resource. They cannot be cultivated on artificial media, and the only established means to quantify viability of M. leprae has been by its relative growth in the foot pads of conventional mice (MFP). The MFP method is technically difficult and requires several months to yield results. More effective methods are needed. We examined the association between M. leprae's ability to oxidize 14C-palmitate in axenic culture and the MFP growth results of a large number of suspensions. Oxidative activity was assessed by radiorespirometry (RR) using the Buddemeyer-type biphasic culture vessels containing 7H12 liquid medium and 14C-palmitate, or with commercially prepared BACTEC 12B vessels containing the same medium. The RR results were highly correlated (r = 0.71) with the growth level that each M. leprae suspension achieved by the MFP technique. In using this technique to examine the effects that many common laboratory practices have on M. leprae viability, we found that viability varies markedly between bacillary suspensions derived from different hosts and tissues. The highest viabilities were obtained with bacilli from moderately enlarged nude MFP (< 1 g). Viability tended to be lower among very large nude MFP or long-duration infections and from armadillo tissues. After their harvest from host tissues, leprosy bacilli lost viability quickly. Suspensions stored in 7H12 liquid medium retained < 1% of their viability within 3 weeks of harvest, and freezing bacillary preparations or incubating them at 37 degrees C resulted in nearly an immediate equivalent loss in metabolic activity and viability. M. leprae viability is maintained best when bacilli are stored for only short periods of time at 4 degrees C-33 degrees C. Palmitate oxidation is a rapid, reliable and objective means by which to estimate the viability of M. leprae and can be used effectively as a surrogate for the conventional MFP technique in many studies.

In Brazil, there is little information about the clinical and epidemiological characteristics of paucibacillary, single skin lesion leprosy patients (SSL-PB). Only recently has the official notification system distinguished leprosy patients with a single lesion as a clinical entity, for whom the single-dose ROM (rifampin, ofloxacin and minocycline) regimen has been recommended. In this paper, we describe the baseline clinical features and the immunological background of a multicenter cohort of SSL-PB leprosy cases enrolled between December 1997-1998. Patients were recruited at health centers located in the following regions: Southeast = Rio de Janeiro; North = Amazon and Rondônia states and Center-West = Goiás state. Eligible cases were newly detected, untreated single-lesion leprosy patients without thickened nerve involvement, and were assessed by clinical, bacilloscopic and histopathological exams. The Mitsuda skin test and anti-PGL-I serology (ELISA) were also performed. Of the 299 SSL-PB leprosy patients, 259 (86.6%) fulfilled the criteria for single-dose ROM intervention. Our results showed that patients recruited from different sites had similar features, considering the clinical and immunological profiles. There was a predominance of adults (mean age 32.4; S.D. = 16.0), and a BCG scar was detected in 76.7% of the children (< or = 15 years old). Only 7 cases were diagnosed as the multibacillary type, representing less than 3% of the patients being misclassified. Our data indicate that in Brazil SSL-PB case ascertainment based on clinical and bacilloscopic criteria can be accurately defined under a routine control program; 75.0% of SSL-PB cases were Mitsuda positive (> or = 5 mm) and seropositivity for anti-PGL-I was detected in 17.3% of the patients. These data are compatible with effective cell-mediated immunity and low bacillary load, suggesting favorable clinical outcomes for most SSL-PB participants of this cohort.

The manifestation of leprosy in humans is largely determined by host immunity to Mycobacterium leprae and is a model for immunoregulation in a human disease. However, animal models available for exploration of the leprosy spectrum are inadequate. This study explored M. leprae infection in mice deficient in inducible nitric oxide synthase, and this report describes elements resembling borderline tuberculoid leprosy in humans.

Mycobacterium leprae is uncultivable on artificial medium, but viability can be maintained without multiplication for a limited time in vitro. In this study, we evaluated gamma-irradiation (gamma-irr) as a means to kill this slowly growing organism. Freshly harvested, viable, athymic, nu/nu mouse-derived M. leprae were exposed to varying doses of gamma-irr from a 60Co source. Two indicators of bacterial viability were determined: metabolism, measured by oxidation of 14C-palmitic acid to 14CO2 in the BACTEC 460 system, and multiplication, measured by titration in the mouse foot pad. gamma-Irr of both M. leprae and M. lufu, a cultivable control mycobacterium, resulted in a dose-dependent inhibition of viability. gamma-Irr of up to 10(3) rad had little effect on the metabolic activity of either organism. For M. leprae, 10(4)-10(5) rad caused an intermediate inhibitory effect; whereas 10(6) rad yielded almost total inhibition. In the mouse foot pad assay, up to 10(4) rad had little effect on M. leprae growth; however, 10(5) rad resulted in at least a 2-log reduction in the number of bacilli recovered and no M. leprae growth was measurable after exposure to 10(6) rad. With M. lufu, 10(5) rad inhibited metabolic activity by 99% and caused > or = 2-log reduction in the number of colony forming units (CFU). No CFU of M. lufu were recovered after exposure to 10(6) rad. Scanning electron microscopy revealed the presence of some aberrant protrusions on the cell surface of lethally irradiated M. leprae; whereas boiling and autoclaving caused obvious morphological denaturation. These data suggest that gamma-irr is an effective way to kill M. leprae without causing extensive damage to the cell architecture. Killing M. leprae by gamma-irr may be preferable when comparing cellular responses to live versus dead bacilli in vitro and in vivo.

The therapeutic efficacy of liposomal clofazimine (L-CLF) was studied in mice infected with Mycobacterium tuberculosis Erdman. Groups of mice were treated with either free clofazimine (F-CLF), L-CLF, or empty liposomes twice a week for five treatments beginning on day 1 (acute), day 21 (established), or day 90 (chronic) postinfection. One day after the last treatment, the numbers of CFU of M. tuberculosis in the spleen, liver, and lungs were determined. F-CLF at the maximum tolerated dose of 5 mg/kg of body weight was ineffective; however, 10-fold-higher doses of L-CLF demonstrated a dose response with significant CFU reduction in all tissues without any toxic effects. In acutely infected mice, 50 mg of L-CLF/kg reduced CFU 2 to 3 log units in all three organs. In established or chronic infection, treated mice showed no detectable CFU in the spleen or liver and 1- to 2-log-unit reduction in the lungs. A second series of L-CLF treatments cleared M. tuberculosis in all three tissues. L-CLF appears to be bactericidal in the liver and spleen, which remained negative for M. tuberculosis growth for 2 months. Thus, L-CLF could be useful in the treatment of tuberculosis.

Leprosy is an ancient disease which is still poorly understood and often feared by the general public and even by some healthcare professionals. Fortunately, improvements in the management of leprosy over the past three decades have diminished the stigma and greatly altered the outlook for patients. Public understanding of the disease has benefited from WHO's goal of eliminating leprosy as a public health problem by the year 2000. Unfortunately that goal has also led many to believe that leprosy has been or will soon be eradicated. This will not happen in the near future because, despite a fall in registered cases, the incidence of the disease has changed very little, and eradication of a bacterial infectious disease such as this is unlikely with chemotherapy alone. Nevertheless, as a result of the WHO's efforts, patients nearly everywhere should have access to care, and the incidence may begin to diminish if adequate control efforts are maintained beyond the year 2000. Given the mobility of patients today a physician anywhere may occasionally see a case or be asked about the disease so a basic understanding of leprosy and its management should prove useful.

The macrophage is a major component of the inflammatory response induced by lymphatic tissue-dwelling filariae. Intraperitoneal (i.p.) infections with Brugia pahangi in Mongolian gerbils, or jirds (Meriones unguiculatus), induce a peritoneal inflammatory response characterized by accumulation of numerous macrophages and fewer eosinophils. This inflammatory response is associated with the release of microfilariae by female worms. The aim of this study was to investigate the activation state of the peritoneal macrophages during the course of i.p. infections with either male or female worms. Activation was determined by a toxoplasmacidal assay and assays which measured the production of tumor necrosis factor (TNF)-like activity and nitric oxide (NO) production. The development of these assays with jirds was initially conducted in parallel with the mouse system, which served as a positive control. Jird macrophages became activated to kill Toxoplasma gondii by in vivo immunization with Mycobacterium bovis BCG in a pattern similar to that of mouse macrophages. However, unlike the mouse system, supernatants from purified protein derivative- or concanavalin A-stimulated jird splenocytes plus lipopolysaccharide failed to activate jird macrophages in vitro or induce NO production. These results indicate that factors involved in jird macrophage activation may differ from those demonstrated in the mouse system and other systems. i.p. infections of 15 days in duration with either male or female worms induced macrophage activation as measured by Toxoplasma killing and TNF production. These responses decreased as the infection progressed to the chronic period on a time course that parallels the down regulation of experimental B. pahangi granulomas. There was no evidence of NO production by activated jird macrophages. These data indicate that macrophage function is down modulated during filarial infection and suggest that mechanisms involved in macrophage deactivation are related to those that induce down modulation of the systemic granulomatous inflammatory response in the jird. This response is not dependent on the microfilarial stage of the parasite and is also independent of mechanisms which induce peritoneal accumulations of macrophages.

Thalidomide is effective in the treatment of inflammatory conditions like erythema nodosum leprosum in leprosy patients, and aphthous ulcers in AIDS patients. Its mechanism of action is uncertain and reports of its effect on the synthesis of inflammatory cytokines such as IL-2 and TNF-alpha are contradictory. As thalidomide is labile to spontaneous hydrolysis at pH 7.4, studies were carried out to explore the effects of deliberate hydrolysis or the ability of thalidomide to modulated cytokine production by human mononuclear cells stimulated in vitro with Staphylococcal enterotoxin A (SEA)(IL-2) or lipopolysaccharide from Salmonella minnesota (LPS)(TNF-alpha). Unhydrolyzed thalidomide at 4.0 micrograms/ml consistently enhanced the synthesis of IL-2 in SEA-stimulated cells, and suppressed the synthesis of TNF-alpha in LPS-stimulated cells; whereas, hydrolyzed thalidomide had no enhancing effect on SEA stimulated-cell synthesis of IL-2 or suppressive effect on LPS stimulated-cell synthesis of TNF-alpha. These findings demonstrate that thalidomide's ability in vitro to enhance IL-2 and to suppress TNF-alpha in stimulated cells is dependent on the intact molecule and underscore the necessity to employ thalidomide under appropriate physicochemical conditions.

Results from animal and in vitro studies suggest that essential fatty acid (EFA) deficiency enhances cell-mediated immunity by reducing production of prostaglandins with immunosuppressive actions. However, direct experimental evidence that EFA deficiency enhances T-lymphocyte function in vivo has not been obtained. In this study, athymic (nu/nu) mice were infected in the footpads with Mycobacterium leprae and fed a linoleic acid-free diet. These mice, and infected nu/nu mice on control diets, were given an adoptive transfer of M. leprae-primed, T-cell-enriched lymphocytes. After 2 weeks, M. leprae bacilli were harvested from the recipient mice and bacterial viability was determined by the BACTEC system. M. leprae recovered from recipient mice fed control diets displayed little reduction in metabolic activity. In contrast, M. leprae from recipient mice fed the EFA-deficient (EFAD) diet exhibited markedly reduced viability. In vitro, donor cells from M. leprae-primed mice secreted elevated levels of gamma interferon upon exposure to the bacilli. These cells also exhibited an enhanced proliferative response, which was reduced by exogenous prostaglandin E2 (PGE2). In addition, M. leprae-infected granuloma macrophages (Mphi) from EFAD recipient nu/nu mice secreted significantly less PGE2 than granuloma Mphi from mice on control diets. These data suggest that enhanced levels of Mphi-generated PGE2, induced by M. leprae or its constituents, could act as an endogenous negative modulator of the immune response occurring in the microenvironment of the lepromatous granuloma.

Objective: To study the role of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) in host response to Mycobacterium tuberculosis., Design: M. tuberculosis infection (i.v.) was compared in B6 control and two strains of knockout (KO) mice. X-CGD mice with a nonfunctional allele for the gp91phox subunit of the phagocyte oxidase cytochrome b are unable to produce ROI whereas iNOS KO mice lack a functional inducible nitric oxide synthase (iNOS) gene and fail to make RNI., Results: M. tuberculosis growth was markedly enhanced in the lungs of X-CGD mice compared to B6 mice, but was controlled in the spleen and liver. In iNOS KO mice, M. tuberculosis growth was exacerbated in the spleen, but was unremarkable in the lungs compared to B6 mice until later (Day 60) in the infection. In vitro, X-CGD alveolar and peritoneal macrophages (M phi) produced no ROI, but did produce RNI and inhibited growth of M. tuberculosis when activated with interferon gamma. iNOS KO M phi produced ROI, but failed to produce RNI and could not cope with M. tuberculosis in vitro when activated. The inhibition of M. tuberculosis growth observed in activated B6 and X-CGD M phi) was reversed in the presence of aminoguanidine., Conclusion: These KO mouse strains demonstrate the relative potent effects of ROI and RNI in resistance to M. tuberculosis and should prove useful for the study of regulatory and compensatory mechanisms of immunity.

The accumulation of various T cell subsets in Bcg-susceptible (C57BL/6) and- resistant (C3H/HeN) strains of mice were compared following an intraperitoneal infection with Mycobacterium paratuberculosis. Groups of mice from both strains were killed at 3, 5, 10, 15, 30, and 150 days after infection and lymphocytes were harvested from the peritoneal exudate cells (PEC), spleen, intestinal epithelial lymphocytes (IEL), lamina propria lymphocytes (LPL), Peyer's patches, and mesenteric lymph node (MLN) and labelled with monoclonal antibodies to CD3, CD4, CD8, gamma delta TCR, CD25, and CD44 for flow cytometric analysis. Uninfected C3H/HeN mice had higher proportions of CD4+ cells in the spleen, MLN, LPL, IEL, and Peyer's patches, while uninfected C57BL/6 mice had higher proportions of CD8+ and/or gamma delta T cells. Significant increases in accumulation of CD8+ and gamma delta T cells were detected in the peritoneum and other tissues in both strains of mice after infection. Higher CD4/CD8 ratios were observed in most lymphoid tissues of C3H/HeN mice, while increased proportions of CD8+ and/or gamma delta T cells were present in C57BL/6 mice. These results indicate that significant differences in T cell profiles exist between these two strains of mice, both inherently and in response to infection with M. paratuberculosis. Innately lower levels of CD4+ cells and/or higher percentages of CD8+ and gamma delta T cells may play a role in the increased suspectibility of C57BL/6 mice to infection with M. paratuberculosis.

Leprosy, a chronic infectious disease caused by Mycobacterium leprae, affects primarily the skin, mucous membranes, and peripheral nerves. It is characterized by a spectrum of symptoms, largely a result of the immunological response of the host to the antigens of M. leprae. At one pole [lepromatous leprosy (LL)] there is an enormous growth of the bacilli in the tissues resulting from a specific and profound lack of cell-mediated immunity (CMI) of the host to M. leprae. At the opposite pole [tuberculoid leprosy (TT)], patients are responsive to M. leprae antigens, and there are few bacilli present in the lesions. A variety of techniques have been applied in the analyses of leprosy granulomas, from standard histological examination to molecular immunological probes. Each technique provides a limited, yet distinctive, look into the constitution of the granuloma. Together, they offer a comprehensive view into the regulation of leprosy granuloma development.

Susceptibility of C57BL/6 (Bcgs) and C3H/HeN (Bcgr) mice to an intraperitoneal infection with Mycobacterium paratuberculosis strain 19698 was compared (by histopathology and the number of mycobacteria isolated from the spleen). Mycobacterial counts from the spleen of Bcgr mice progressively decreased over the course of infection but remained unchanged in Bcgs mice. Granulomatous lesions and acid-fast bacteria were consistently present in the liver and lymph nodes of Bcgs mice, whereas lesions were transient or absent in Bcgr mice. These results indicate that Bcgr mice are inherently resistant to M. paratuberculosis, whereas Bcgs mice are inherently susceptible. These differences may prove useful in elucidating the mechanisms of resistance and susceptibility to paratuberculosis and other mycobacterial infections.

The susceptibility of C57BL/6 mice to oral inoculation with Myobacterium paratuberculosis was evaluated histopathologically. Granulomatous lesions containing acid-fast bacteria developed in the mesenteric lymph nodes in over 50% of the mice by 11 months after inoculation. The results suggest that C57BL/6 mice may be useful for studying infection, pathogenesis, and other aspects of paratuberculosis.

Tumor necrosis factor (TNF) plays a pivotal role in inflammatory phenomena that culminate in either pathogenesis or resistance in mycobacterial disease. The regulatory role of TNF in murine tuberculosis was examined by administering a recombinant adenovirus encoding a fusion protein consisting of the human 55-kDa TNF receptor extracellular domain and the mouse IgG heavy chain domain (AdTNFR). During acute infections with Mycobacterium tuberculosis, AdTNFR pretreatment induced elevated mycobacterial burdens of 1 log10 in the tissues of H37Ra-infected mice and 2 log10 (spleen and liver) and 4 log10 (lungs) in H37Rv-infected mice. In mice infected chronically with H37Rv, AdTNFR treatment induced a 3-log10 increase of M. tuberculosis in the lungs, in which a tuberculous bronchopneumonia developed with numerous acid-fast bacilli visible in alveoli and bronchi. Administration of AdTNFR may serve as a useful model for studying the pathogenesis and chemotherapy of progressive primary tuberculosis.

Because of the similarities in causative agents of Hansen's disease and tuberculosis, Hansen's disease research is now being used in the identification, treatment, and prevention of tuberculosis. Numerous studies are under way to screen and develop new drugs to combat the threat of multiple drug-resistant tuberculosis. Additional studies focus on factors to reduce the transmission of tuberculosis and on the development of techniques for early diagnosis and identification of drug resistance. Advances in Hansen's disease research and treatment also are being applied to the prevention of ulcers and amputations in diabetics and others without protective sensation in their feet. The Lower Extremity Amputation Prevention Program, developed at the Gillis W. Long Hansen's Disease Center in Carville, LA, is a multidisciplinary approach that includes screening, risk assessment, and the development of a treatment plan with an emphasis on patient involvement. Expected to prevent up to 90 percent of diabetes-related amputations, the program is being implemented in Jackson, MS, in a community-based diabetic foot program and will be replicated throughout the United States.

Because of the limited chemotherapeutic approaches available to treat reactivated latent Toxoplasma gondii infection manifested as toxoplasmic encephalitis in AIDS patients, investigation of novel chemotherapeutic agents is warranted. Several poloxamers (nonionic block copolymers composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene) were tested for their abilities to alter the course of acute infection with a highly virulent T. gondii in mice. The effect varied markedly with the length of the constituent chains of the copolymers. The most effective preparations were highly effective when administered after infection and afforded remarkable protection against 10 to 1,000 100% lethal doses of T. gondii. Protection was dose dependent, and multiple treatments were more effective than single treatment. These preliminary findings warrant additional studies to determine whether this novel form of antitoxoplasma chemotherapy may prove promising in the treatment or prevention of acute toxoplasmic encephalitis in humans.

Lipoarabinomannan (LAM) is the major arabinose- and mannose-containing phosphorylated lipopolysaccharide (LPS) in mycobacterial cell walls. LAM preparations from a virulent strain (Erdman) (LAM(Erdman)) and an attenuated strain (H37Ra) (LAMH37Ra) of Mycobacterium tuberculosis, as well as from M. leprae (a virulent mycobacterium), were analyzed for their effects on various macrophage (M phi) effector functions. LAMH37Ra, like gram-negative LPS, exhibited a dose-dependent ability to induce tumor necrosis factor alpha (TNF-alpha) production in normal M phi, and gamma interferon (IFN-gamma) priming of the M phi greatly augmented the levels of TNF-alpha. However, the effects of LAMH37Ra were unaffected by polymyxin B, which totally abrogated the effects of LPS. LAM(Erdman) and LAM from M. leprae, on the other hand, induced virtually no TNF-alpha production. Analysis of M phi mRNA by reverse transcription-polymerase chain reaction revealed that the levels of production. Analysis of M phi mRNA by reverse transcription-polymerase chain reaction revealed that the levels of TNF-alpha mRNA induced by the various preparations correlated with the levels of TNF-alpha protein detected. Interestingly, both LAMH37Ra and LAM(Erdman) could block subsequent IFN-gamma- and LPS-induced M phi activation, a previously reported measure of the potent ability of LAM to down-regulate M phi effector functions. Two lines of evidence suggested, however, that M phi cyclooxygenase products did not play a role in this down-regulation. LAMH37Ra and LPS could induce the production of NO2- in both normal and IFN-gamma-primed M phi, whereas LAM(Erdman) could stimulate NO2- production only in primed M phi. Both LAMH37Ra and LAM(Erdman) could substitute for LPS as a triggering signal for IFN-gamma-primed M phi in a toxoplasma killing assay. The triggering ability of LAM(Erdman), however, was abrogated by an anti-TNF-alpha antibody, suggesting that sufficient TNF-alpha production was stimulated by LAM(Erdman) to drive a M phi function relevant in host resistance. Thus, mycobacterial LAM is a potent regulator of M phi functions, a fact that may have important consequences in mycobacterial disease.

IFN-gamma is an important mediator of cellular resistance against microbial pathogens and tumor cells due in part to its potent capacity to activate macrophages for enhanced cytotoxicity. The present study demonstrates that TNF-alpha regulates the expression of enhanced antimicrobial activity by triggering IFN-gamma primed macrophages to kill or inhibit intracellular Toxoplasma gondii. Resident mouse macrophages stimulated with rIFN-gamma at levels up to 2500 U/ml failed to display enhanced antitoxoplasmal activity when cultured in vitro under low endotoxin conditions (less than 10 pg/ml), but were triggered by addition of small amounts of LPS (0.1 ng/ml). A similar requirement for LPS as a second signal necessary to trigger antitoxoplasmal activity was observed when IFN-gamma was administered to mice in vivo. The essential nature of this triggering step allowed us to explore the role of cytokines that act as endogenous regulators of macrophage activation. rTNF-alpha, although unable to confer antitoxoplasmal activity when used alone to treat macrophages, was capable of triggering IFN-gamma-primed macrophages cultured under low endotoxin conditions. The ability of TNF-alpha to trigger IFN-gamma-primed macrophages was blocked by rabbit anti-TNF-alpha polyclonal antisera but was not affected by polymyxin B indicating that TNF-alpha triggering was not due to contamination with LPS. Collectively, these findings demonstrate that TNF-alpha performs an important regulatory role in the expression of enhanced anti-microbial activity by IFN-gamma-primed macrophages.

Recently, L-arginine has been shown to be a necessary substrate for murine-activated macrophage-mediated tumor cytostasis and microbiostasis of certain fungi, bacteria, and intracellular protozoa. We report here the effects of the L-arginine-dependent pathway of activated mouse macrophages (MO) on the obligate intracellular prokaryote, Mycobacterium leprae. Due to the inability to culture M. leprae in vitro, a simple, quantitative assay was employed to measure the metabolism/viability of M. leprae released from MO: the metabolic capacity of M. leprae to oxidize 14C-palmitic acid to 14CO2. Murine normal MO or MO activated in vitro with IFN-gamma or in vivo by injection with Corynebacterium parvum were infected with viable M. leprae freshly harvested from the footpads of nu/nu mice. Activated MO strikingly inhibited the metabolism of M. leprae; however, in L-arginine-free medium or in medium containing L-arginase, the inhibitory effects of activated MO on M. leprae metabolism were abolished. The competitive inhibitor of L-arginine, NG-monomethyl-L-arginine, also blocked the inhibitory effects of activated MO for M. leprae, but the addition of supplemental L-arginine overcame the NG-monomethyl-L-arginine-induced block. Furthermore, in the culture supernatants, the levels of NO2-, an end product of L-arginine degradation, were directly proportional to the ability of the activated MO to inhibit M. leprae metabolism. These data present five lines of evidence that suggest that activated MO utilize the L-arginine-dependent pathway to cope with M. leprae.

Five alternative methods were used to explore in vitro the effects of normal and activated murine macrophages on the metabolic well-being of intracellular Mycobacterium leprae: fluorescein diacetate-ethidium bromide staining, ATP content, synthesis of phenolic glycolipid 1, and two techniques to quantitate oxidation of palmitic acid. In relatively short-term experiments (7 to 10 days), each of these procedures provided strong evidence that activated macrophages exerted a deleterious effect on the leprosy bacillus. These findings appear to confirm the contention that activated macrophages underlie host resistance to clinical leprosy and limitation of M. leprae growth in paucibacillary leprosy.

We have shown that preactivated MACs are able to cope with newly acquired leprosy bacilli, and a high intracellular burden of live M. leprae induces a refractory response of the host MAC to activation by IFN-gamma. Our studies underscore the fact that MAC function in LL is dependent on localized conditions, influenced by the high intracellular burden of leprosy bacilli and, in part, involving the production of prostanoids. These findings preclude inferences about the function of granuloma MAC s in leprosy based on the responses of MACs from other easily accessible anatomical compartments such as the peritoneal cavity or peripheral blood. We feel that the clearance of bacilli from the LL lesion as a consequence of local immunotherapeutic measures or chemotherapy likely depends on the influx of new competent MAC rather than the activation of resident lepromatous MACs.

Recent studies show the importance of a single amino acid, L-arginine, as a necessary substrate for activated macrophage-mediated cytotoxic activity for tumor target cells and microbiostatic function for Cryptococcus neoformans. The present studies were carried out to determine the role of the L-arginine-dependent macrophage effector function on the microbiostatic effects of activated macrophages on the obligate intracellular protozoan, Toxoplasma gondii. A guanidino methylated derivative of L-arginine, NGmonomethyl-L-arginine (NGMMA), a competitive inhibitor of the L-arginine-dependent effector pathway, virtually abolished the normally potent microbiostatic effect of macrophages for Toxoplasma gondii after activation of the macrophages in vitro by IFN-gamma and LPS or in vivo by i.p. injection of killed Corynebacterium parvum. Addition of supplemental L-arginine to the culture medium overcame the capacity of NGMMA to block activated macrophage-mediated microbiostasis of Toxoplasma. The ability of NGMMA to inhibit the microbiostatic capacity of activated macrophages for Toxoplasma gondii correlated with almost total inhibition of synthesis of nitrite, nitrate, and L-citrulline from L-arginine. Therefore, as is the case for tumor target cells and C. neoformans, the synthesis of inorganic nitrogen oxides from a terminal guanidino nitrogen atom of L-arginine appears to be essential for murine cytotoxic activated macrophage mediated microbiostatic capacity for T. gondii.

Lipoarabinomannan (LAM), purified from the cell walls of Mycobacterium leprae and M. tuberculosis, is a potent inhibitor of interferon-gamma (IFN-gamma) mediated activation of macrophages. The capability of LAM to inhibit IFN-gamma activation of macrophages in vitro was dose dependent and required a 24-h pre-exposure. Defective activation was evident as a block in IFN-gamma-induced cytocidal activity for tumour cell targets and microbicidal capacity for intracellular Toxoplasma gondii. Additionally, LAM treatment blocked the induction of surface Ia antigens on peritoneal macrophages by IFN-gamma. The requirement for pretreatment with LAM was further substantiated by the finding that peritoneal macrophages that were activated in vivo were not affected by LAM treatments and retained full microbicidal function. However, once inhibited by LAM treatment in vitro, macrophages remained fully refractory to IFN-gamma activation for up to 5 days in culture. Inhibition of IFN-gamma activation in macrophages treated with LAM was not overcome by 100-fold increases in the dose of IFN-gamma used or by a constant dose of IFN-gamma in combination with 100-fold increases in the level of endotoxin used to trigger cytotoxic activity. The defect in IFN-gamma unresponsiveness was not due to altered receptor function, as control and LAM-treated macrophages showed similar capacity to bind, internalize, and digest radiolabelled IFN-gamma. Based on the in vitro findings reported here, the inhibition of IFN-gamma-mediated macrophage activation by exposure to LAM may contribute to defective macrophage function observed in lepromatous granulomas and thus constitutes an important aspect of pathogenesis in mycobacterioses.

Mycobacterium leprae grows to enormous numbers in the nu/nu mouse footpad, producing granulomas resembling those of lepromatous leprosy in humans. Footpad granuloma cells gorged with M. leprae were established in primary cell culture to examine their functional capabilities. These cells were classified as macrophages by the following criteria: positive staining for nonspecific esterase, reduction of Nitro Blue Tetrazolium during phagocytosis of Candida albicans, possession of Fc receptors, and possession of Mac-1 antigen. Footpad macrophages also phagocytized and supported the intracellular growth of Toxoplasma gondii. However, unlike peritoneal macrophages, footpad macrophages could not be activated to kill or inhibit T. gondii by macrophage-activating factor produced by mitogen-stimulated spleen cells or by recombinant gamma interferon. Thus, although the lepromatous macrophages appeared to be normal in many of their functions, they were defective in response to macrophage-activating signals.

A highly sensitive assay for quantitating the presence of activated macrophages was employed to show that foot pad infection of mice with neither M. leprae nor M. marinum induced a population of activated peritoneal macrophages. In vitro attempts to demonstrate local activated macrophages in the foot pads of M. leprae infected mice failed, but, because of the technical problems encountered, do not preclude their presence. Additional studies are being carried out.

Mycobacterium leprae synthesizes large quantities of a specific phthiocerol-containing phenolic glycolipid in vivo. We have shown earlier that viable M. leprae readily incorporates radiolabeled palmitic acid into phenolic glycolipid I when residing in cultured macrophages in vitro and that this process is inhibited by the antileprosy drug rifampin. In the present paper we report that application of this observation to the rapid evaluation of over 25 antimicrobial agents for potential antileprosy activity in vitro. All the known antileprosy drugs rifampin, dapsone, clofazimine, and ethionamide inhibited phenolic glycolipid I synthesis. Rifabutin, a spiropiperidyl derivative of rifamycin, also reported to be active in the mouse model, was very effective. Interestingly, the macrolides erythromycin, clarithromycin, and roxithromycin were also found to be active in this system, while D-cycloserine and other cell wall synthesis inhibitors showed no effect. Many of the compounds found to be active in this system have been reported to be effective in vivo in mice. This correlation lends support to the feasibility of using phenolic glycolipid I synthesis for the rapid evaluation of new drugs against leprosy.

Studies were carried out to determine whether treatment of mice with the synthetic adjuvant muramyl dipeptide (MDP) afforded resistance to infection with Mycobacterium leprae or M. marinum in mice. Regardless of the timing, dose, or route of administration, there was no evidence that treatment with MDP or 3 of its analogs (desMDP, abuMDP, valMDP) enhanced resistance to food pad infection with M. leprae or M. marinum. In parallel studies, systemic treatment with Propionibacterium acnes failed to protect against either M. leprae or M. marinum. Administration of P. acnes locally into the foot pad afforded marginal resistance to M. marinum when injected prior to infection. Local treatment with P. acnes afforded marked resistance to growth of M. leprae, even when administered months after infection.

Toxoplasma gondii belongs to a group of highly virulent intracellular parasites that reside in host cell vacuoles which resist typical phagosome-lysosome fusion. Live Toxoplasma replicate prodigiously within modified phagocytic vacuoles formed during invagination of the host plasma membrane. In contrast, heat-killed Toxoplasma or specific antibody (heat-inactivated)-coated live Toxoplasma-containing vacuoles readily undergo lysosome fusion and digestion in normal macrophages. Of newly recognized significance to Toxoplasma survival is the microbicidal effect of phagosome acidification, which reportedly can occur independently of fusion with other acidic vesicles. We report here that modified live Toxoplasma-containing vacuoles fail to acidify in normal macrophages, as indicated by the sensitive pH probe fluorescein. In contrast, when live Toxoplasma are coated with specific antibody (heat-inactivated), they trigger phagosome acidification when entering normal macrophages. A similar acidification is observed when normal phagocytes ingest dead Toxoplasma. Extracellular Toxoplasma are highly susceptible to acidic pH conditions, indicating that the acidification block in the modified vacuoles may be important for intracellular survival.