Molecular assays for determining Mycobacterium leprae viability in tissues of experimentally infected mice.

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.

Development of a mouse food pad model for detection of sub clinical leprosy.

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.

Infection of mouse macrophages with viable Mycobacterium leprae does not induce apoptosis.

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.

Mycobacterium leprae actively modulates the cytokine response in naive human monocytes.

Leprosy is a chronic but treatable infectious disease caused by the intracellular pathogen Mycobacterium leprae. Host immunity to M. leprae determines the diversity of clinical manifestations seen in patients, from tuberculoid leprosy with robust production of Th1-type cytokines to lepromatous disease, characterized by elevated levels of Th2-type cytokines and a suboptimal proinflammatory response.

The role of free-living pathogenic amoeba in the transmission of leprosy: a proof of principle.

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.

Lymphotoxin-alpha and TNF have essential but independent roles in the evolution of the granulomatous response in experimental leprosy.

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.

Activation of complement by Mycobacterium leprae requires disruption of the bacilli.

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.

Enumeration of Mycobacterium leprae using real-time PCR.

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.

Mycobacterium leprae DNA associated with type 1 reactions in single lesion paucibacillary leprosy treated with single dose rifampin, ofloxacin, and minocycline.

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.

Antigen-specific cellular and humoral responses are induced by intradermal Mycobacterium leprae infection of the mouse ear.

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.

Emergence of an effective adaptive cell mediated immune response to Mycobacterium leprae is not impaired in reactive oxygen intermediate-deficient mice.

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.

Mycobacterium leprae inhibits dendritic cell activation and maturation.

Leprosy presents with a clinical spectrum of skin lesions that span from strong Th1-mediated cellular immunity and control of bacillary growth at one pole to poor Ag-specific T cell immunity with extensive bacillary load and Th2 cytokine-expressing lesions at the other. To understand how the immune response to Mycobacterium leprae is regulated, human dendritic cells (DC), potent inducers of adaptive immune responses, exposed to M. leprae, Mycobacterium tuberculosis (Mtb), and Mycobacterium bovis bacillus Calmette-Guerin (BCG) were studied for their ability to be activated and to prime T cell proliferation.

Mycobacterium leprae is naturally resistant to PA-824.

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.

Effects of purification and fluorescent staining on viability of Mycobacterium leprae.

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.

DC-SIGN interacts with Mycobacterium leprae but sequence variation in this lectin is not associated with leprosy in the Pakistani population.

The C-type lectin DC-SIGN is involved in early interactions between human innate immune cells and a variety of pathogens. Here we sought to evaluate whether DC-SIGN interacts with the leprosy bacillus, Mycobacterium leprae, and whether DC-SIGN genetic variation influences the susceptibility and/or pathogenesis of the disease. A case-control study conducted in a cohort of 272 individuals revealed no association between DC-SIGN variation and leprosy.

Application of a viability-staining method for Mycobacterium leprae derived from the athymic (nu/nu) mouse foot pad.

Mycobacterium leprae cannot be cultured, so ascertaining viability of the organism remains a major obstacle, impeding many avenues of investigation. This study tested a two-colour, Syto9 and propidium iodide, fluorescence assay, which scores for membrane damage in individual bacilli, to determine if a rapid direct-count viability-staining technique can be reliably applied to M. leprae.

IL-10 treatment of macrophages bolsters intracellular survival of Mycobacterium leprae.

In these studies, metabolically active Mycobacterium leprae were maintained for as long as 8 weeks in monolayer cultures of mouse peritoneal macrophages (MPhi). Supplemental IL-10, but not TGF-beta, bolstered, directly or indirectly, M. leprae metabolism in mouse MPhi.

An in vitro model for the lepromatous leprosy granuloma: fate of Mycobacterium leprae from target macrophages after interaction with normal and activated effector macrophages.

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.

In situ type 1 cytokine gene expression and mechanisms associated with early leprosy progression.

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.

The study of Mycobacterium leprae infection in interferon-gamma gene--disrupted mice as a model to explore the immunopathologic spectrum of leprosy.

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.