O-diphenoloxidase concentrations in leprosy.

O-diphenoloxidase activity was studied in 15 patients with lepromatous leprosy, 15 with tuberculois leprosy, and 15 controls. O-diphenoloxidase isolated from skin and serum samples of patients with lepromatous leprosy had the specificity of a bacterially derived enzyme and not that of a mammalian-derived enzyme. Only the patients who had lepromatous leprosy for over two years showed enzyme activity in serum, though all showed it in skin tissue. O-diphenoloxidase activity in serum may be a useful diagnostic marker of lepromatous leprosy.     

Numerical Changes in T Cell Subsets (Tγ and Tμ) in Leprosy Patients

Eighty-six leprosy patients (49 active lepromatous, 24 inactive lepromatous, 7 borderline, and 6 tuberculoid) and nine healthy controls were examined for numerical changes in T cell subsets (Tγ and Tμ), and complement levels in peripheral blood to determine the roles of T cell subsets and complement in the etiology of leprosy. The percentage and number of Tγ and Tμ cells showed no significant differences among the different clinical groups, but 4 out of 49 active lepromatous, 3 out of 24 inactive lepromatous and 3 out of 7 borderline cases showed a high percentage of Tγ cells. Serum concentrations of C4, C3c, and C3 activator, an important factor in the alternative pathway of complement activation, were not significantly different among the groups. However, C3 activator and C3c concentrations were significantly high in active lepromatous patients complicated by an immune complex disease called “erythema nodosum leprosum” (ENL) compared with ENL-free active lepromatous leprosy.     

The HLA system and leprosy in Thailand

Summary To investigate immunogenetics of leprosy, 205 leprosy patients (26 with tuberculoid, 57 with borderline-tuberculoid, 21 with borderline, 31 with borderline-lepromatous, and 70 with lepromatous leprosy) have been typed for HLA antigens, and compared with 183 healthy controls from the same region (Nothern Thailand). There was no significant difference between the overal group of leprosy patients or the three borderline classes and the controls. The two polar forms, tuberculoid and lepromatous leprosy, however, showed significant associations: HLA-A2 is decreased and HLA-Bw17 is increased in tuberculoid leprosy; HLA-B7 is increased in lepromatous leprosy. When both polar forms are compared with each other, HLA-A2 is significantly higher, HLA-Bw40 lower in patients with lepromatous than in those with tuberculoid leprosy. The results are discussed with respect to the different immune responsiveness in the two polar forms of leprosy.     

Surface membrane changes in lepromatous macrophages affecting the adherence ofMycobacterium leprae

Macrophages from lepromatous leprosy patients showed poor adherence toMycobacterium leprae. The phagocytic activity of the macrophages was not correlated to the influence on the adherence ability. Based on the phagocytic behaviour of macrophages from normal individuals and from lepromatous leprosy, patients as well as the action of neuraminidase in reversing the extent of adherence, it is suggested that macrophages from lepromatous leprosy patients differ from those from normal individuals in regard to their surface properties. There was no relationship between the degree of adherence and the concentration of Fc receptors of the macrophages. It was also shown that an extract of lysed macrophages from lepromatous leprosy patient was able to reduce the adherence ofMycobacterium leprae to normal macrophages. This study shows that adherence is a good indicator of the surface property of macrophages which in turn could play an important role in the cell mediated immunity of the patient. The observations suggest altered macrophage membrane structure in the long term-treated, otherwise normal, lepromatous leprosy patients.     

Factors contributing to impairment of the mixed lymphocyte reaction in leprosy.

Whereas the mixed lymphocyte reaction was essentially normal in inactive lepromatous leprosy and tuberculoid leprosy, it was severely impaired in active lepromatous leprosy. The impairment was found to be contributed by certain unknown factors in their plasma and subnormal reactivity of their T lymphocytes. The plasma derived from active lepromatous leprosy patients depressed the reaction of normal cells and normal plasma enhanced the reaction of active lepromatous lymphocytes. The cellular factor was studied by using a one-way reaction in which one of the two lymphocyte preparations was inactivated with mitomycin C. The impairment of blastogenesis of active lepromatous lymphocytes was partially reversed by substituting inactivated normal cells for similarly treated leprous cells, and conversely the response of normal allogeneic lymphocytes was depressed by substituting inactivated leprous lymphocytes as the stimulator cells.     

Suppressor T lymphocytes from lepromatous leprosy skin lesions

The immune response in leprosy forms a spectrum with lepromatous leprosy patients exhibiting specific unresponsiveness to antigens of Mycobacterium leprae. This unresponsiveness is thought to be related to the prevalence of T8-positive lymphocyte in these lepromatous lesions. To analyze the immunoregulatory function of these T8 cells, we developed simple procedures to extract lymphocytes from skin biopsy specimens of patients with leprosy. These lymphocytes were sorted for T8 and T4 positive cells, and cell lines were established by expansion with interleukin 2 (IL 2) and irradiated feeder cells. All T8 positive lines tested were positive for IL 2 receptors and HLA-DR determinants. These lines were additionally assayed for lepromin-induced suppression of the normal peripheral blood lymphocyte Con A proliferative response. Thirteen of 32 lines from six lepromatous patients showed significant suppressor activity, whereas nine lines from six tuberculoid patients and one line from normal peripheral blood failed to show suppression (p less than 0.001). Taken together, the finding of M. leprae-triggered suppressor cells within lepromatous skin lesions may in part explain the M. leprae unresponsiveness of lepromatous leprosy patients.     

Mycobacterium leprae mediated stimulation of macrophages from leprosy patients and hydrogen peroxide production

Macrophages cultured from the peripheral blood of normal individuals, tuberculoid leprosy patients and long-term-treated, bacteriologically negative lepromatous leprosy patients are able to release hydrogen peroxide on stimulation withMycobacterium leprae. Macrophages from lepromatous leprosy patients who are bacteriologically positive produce considerably lower levels of hydrogen peroxide, even though stimulation of these cells withMycobacterium leprae is definitely demonstrable. This differential stimulation of macrophages appears to be largely specific toMycobacterium leprae. There is also a good indication that decreased stimulation of macrophages from positive patients could be due to an after-effect of infection. It is possible that while other factors aid survival ofMycobacterium leprae in the macrophages, hydrogen peroxide may not be as effective in the killing of the bacteria in infected patients as it would be, perhaps, in other infections.     

Lepromin-induced suppressor cells in patients with leprosy.

The possibility of an active mechanism of immunologic suppression in leprosy was explored by assessing the in vitro lymphocyte responses of 61 leprosy patients and 30 normal individuals to the mitogen Con A in the presence or absence of Dharmendra lepromin. Lepromin-induced suppression of Con A stimulation was found in 32 of 35 lepromatous patients and 15 of 15 borderline patients, but only 2 of 15 tuberculoid patients and 2 of 30 normal controls. Cell fractionation studies indicated at least two cell populations involved in the in vitro lepromin-induced suppressor activity, adherent cells and T gamma-cells.     

An indomethacin sensitive suppressor factor released by macrophages of leprosy patients

Reduction inF _c receptor expression as assayed by ‘erythrocyte’ rosetting of macrophage cultures from long term treated lepromatous leprosy patients (bactereologically negative) was seen in the presence of viableMycobacterium leprae. Macrophages with and without intracellular bacilli demonstrated this reduction. On the basis of this observation the conditioned medium ofMycobacterium leprae infected macrophage cultures of lepromatous patients, were tested on macrophages from normal individuals for [³H]-leucine incorporation and antigen specific physical interaction with lymphocytes. Both these parameters showed decreased values as compared to the controls which were not exposed to this conditioned medium. Lymphocyte transformation toMycobacterium leprae in leucocyte cultures of normal individuals was also reduced in the presence of the conditioned medium from lepromatous patients’ macrophages. The indication that this factor may be a prostaglandin was suggested by the observation that its synthesis was inhibited by indomethacin. Its importance in the non-specific depression in cell-mediated immunity seen in lepromatous patients is discussed.     

Impairment of alternate pathway (CD2) of T cell activation in leprosy

Recent studies in basic immunology have been directed towards the understanding of the mechanism of T cell activation. T cells can be activated to proliferatevia the classical pathway through the antigen receptor (CD3-Ti) orvia the alternate pathway through the CD2 receptor. Since immunologic unresponsiveness in lepromatous leprosy is considered to be due to the inability of T cells to proliferate upon stimulation, we have been interested in the nature of these receptors and the activation pathways in lymphocytes of leprosy patients. In the present investigation we demonstrate: (i) CD2 receptor (E-receptor) is downregulated in bacterial index positive lepromatous leprosy patients. (ii) The alternate pathway of T cell activation is impaired in lepromatous patients as revealed by the inability of their lymphocytes to proliferate in response to a pair of mitogenic anti-CD2 monoclonals. (iii) The addition of recombinant interleukin 2in vitro restores the ability of lymphocytes from lepromatous patients to proliferate in response to anti-CD2 antibodies. (iv) Interestingly, CD2 modulation and the associated functional impairment could be brought about in peripheral blood lymphocytes from normal subjects by prior treatment withMycobacterium leprae in vitro. This approach would be useful in understanding the molecular events leading to the defective T cell functions in leprosy.     

Image analysis to quantify S100-positive dendritic cells in leprosy-affected skin

A morphometric analysis of skin dendritic cells was done on biopsies of patients with different forms of leprosy. An anti S100 antibody was used to determine dendritic cell quantity and extension. Patients with a better immune response to the bacillus showed a greater number of dendritic cells in the cases of dimorphic tuberculoid leprosy and tuberculoid leprosy. This result contrasted with that from patients with dimorphic lepromatous leprosy and lepromatous leprosy.     

Signaling lymphocytic activation molecule expression and regulation in human intracellular infection correlate with Th1 cytokine patterns.

Induction of Th1 cytokines, those associated with cell-mediated immunity, is critical for host defense against infection by intracellular pathogens, including mycobacteria. Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein expressed on lymphocytes that promotes T cell proliferation and IFN-gamma production. The expression and role of SLAM in human infectious disease were investigated using leprosy as a model. We found that SLAM mRNA and protein were more strongly expressed in skin lesions of tuberculoid patients, those with measurable CMI to the pathogen, Mycobacterium leprae, compared with lepromatous patients, who have weak CMI against M. leprae. Peripheral blood T cells from tuberculoid patients showed a striking increase in the level of SLAM expression after stimulation with M. leprae, whereas the expression of SLAM on T cells from lepromatous patients show little change by M. leprae stimulation. Engagement of SLAM by an agonistic mAb up-regulated IFN-gamma production from tuberculoid patients and slightly increased the levels of IFN-gamma in lepromatous patients. In addition, IFN-gamma augmented SLAM expression on M. leprae-stimulated peripheral blood T cells from leprosy patients. Signaling through SLAM after IFN-gamma treatment of Ag-stimulated cells enhanced IFN-gamma production in lepromatous patients to the levels of tuberculoid patients. Our data suggest that the local release of IFN-gamma by M. leprae-activated T cells in tuberculoid leprosy lesions leads to up-regulation of SLAM expression. Ligation of SLAM augments IFN-gamma production in the local microenvironment, creating a positive feedback loop. Failure of T cells from lepromatous leprosy patients to produce IFN-gamma in response to M. leprae contributes to reduced expression of SLAM. Therefore, the activation of SLAM may promote the cell-mediated immune response to intracellular bacterial pathogens.     

Hansen's disease (leprosy). Diagnosis by aspiration biopsy of lymph nodes

A 61-year-old male native of Mexico presented with generalized enlargement of lymph nodes. Fine needle aspiration (FNA) biopsy established lepromatous leprosy as the cause of the lymphadenopathy. The cytologic findings included abundant, frequently multinucleated histiocytes (globus cells), the cytoplasm of which showed multiple vacuoles; cytoplasmic membrane-bound vacuoles were seen free in the background. The vacuoles contained large numbers of acid-fast bacilli. Globus cells, while characteristic, are not specific for Mycobacterium leprae infection and are seen in certain atypical mycobacterioses in immunodeficient patients. This appears to be the first report of lymphadenopathy due to lepromatous leprosy in which the diagnosis was made by FNA biopsy. The immunologic spectrum of leprosy is correlated with clinical and pathologic findings, and the need to remember infectious processes in evaluating lymphadenopathy and the value of reserving air-dried and alcohol-fixed smears for special stains are emphasized.     

Effect of recombinant interferon-gamma on hydrogen peroxide-releasing capacity of monocyte-derived macrophages from patients with lepromatous leprosy

Monocyte-derived macrophages from 14 patients with lepromatous leprosy respond to rIFN-gamma with an enhanced secretion of H2O2 in a fashion similar to that of cells obtained from normal donors. The activation is not dependent on the cutaneous bacterial index, the length of treatment, or the stage and activity of the disease. H2O2 release can be triggered in these cells both by phorbol myristate acetate and by intact irradiated Mycobacterium leprae. Uptake of M. leprae by both normal donors' and patients' macrophages is proportional to the number of bacilli added. Prior ingestion of M. leprae does not interfere with the ability of macrophages to respond to IFN-gamma by the production of oxygen intermediates. We conclude that the immune defect in lepromatous leprosy probably results from a lack of response to M. leprae by the patients' T cells rather than an inability of mononuclear phagocytes to respond to IFN-gamma.     

Immunoglobulin G response against 10-kDa and 65-kDa heat-shock proteins in leprosy patients and their household contacts

Abstract We measured antibody responses to recombinant Mycobacterium leprae 65-kDa (rML65) and 10-kDa (rML10) by indirect ELISA in sera from leprosy patients, household contacts and healthy controls in a leprosy-endemic area in the north east of Argentina. Serum antibody levels to those antigens were correlated with IgM anti-phenolic glycolipid I (PGL-I) levels, with bacterial index and the period of time under chemotherapy. Bacterial index positive (BI⁺) patients showed higher mean values when compared with BI negatives (BI⁻). Among lepromatous patients a positive correlation was observed between IgG antibody responses to both recombinant antigens and IgM antibody response to PGL-I. Anti-rML10 test detected a higher percentage of positive/total than anti-rML65 in all leprosy groups and healthy contacts. Bacterial load, leprosy clinical form and the time under chemotherapy were factors which could influence levels of the antibody response. The contribution of these antibody studies for a precise and early diagnosis in leprosy is discussed.     

Rifampicin for lepromatous leprosy: nine years' experience.

Over 100 patients with lepromatous leprosy were treated with rifampicin in a series of pilot, uncontrolled, and controlled trials in 1968-77. The rapid bactericidal effect of rifampicin on Mycobacterium leprae was confirmed. Clinical improvement became apparent sometimes as early as 14 days after the start of treatment. Nevertheless, a few persisting viable M leprae were detected as long as five years after the start of treatment with rifampicin either by itself or in combination with the bacteriostatic drug thiambutosine. Treatment with rifampicin and dapsone for six months reduced the number of persisting leprosy bacteria more than treatment with dapsone alone. Although rifampicin proved more effective than dapsone, it is unlikely that used by itself if can significantly shorten the length of treatment in lepromatous leprosy. Therefore initial intensive combined treatment with two or more bactericidal drugs (including rifampicin) warrants further investigation in both untreated leprosy and lepromatous leprosy resistant to dapsone.     

Increase in TGF-β Secreting CD4+CD25+ FOXP3+ T Regulatory Cells in Anergic Lepromatous Leprosy Patients

Background

Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25⁺FOXP3⁺ regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples.

Methodology/Principle Findings

Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3⁺, TGF-β in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4⁺CD25⁺FOXP3⁺ T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-β which was further confirmed by increase in TGF-β in culture supernatants (p<0.003). Furthermore, TGF-β in iTreg cells was associated with phosphorylation of STAT5A. TGF-β was seen in CD25⁺ cells of the CD4⁺ but not that of CD8⁺ T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients.

Conclusions/Significance

Our results indicate that FOXP3⁺ iTregs with TGF-β may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.     

Serum proteome of leprosy patients undergoing erythema nodosum leprosum reaction: regulation of expression of the isoforms of haptoglobin

Validated proteome profile allows better understanding of disease progression, subtype classification, susceptibility patterns, and disease prognosis. Leprosy is a spectral disease, with clinically, histologically, immunologically, and bacteriologically distinguishable subtypes. In addition, a significant fraction of patients undergo immune mediated reactions even after multidrug therapy (MDT). Erythema nodosum leprosum (ENL) is an immune complex mediated reactional condition in leprosy, characterized by a systemic inflammatory condition afflicting borderline lepromatous (BL) and lepromatous leprosy patients (LL). In this study, we have analyzed serum proteome of leprosy patients undergoing ENL reactions and compared it with that of healthy noncontact controls. Depletion of albumin and immunoglobulin G (IgG) was optimized using Aurum serum protein mini kit (Bio-Rad), and then two-dimensional gel electrophoresis (2-DE) of these serum samples was performed. Differentially expressed proteins were identified by MALDI-TOF and MALDI-TOF MS/MS mass spectrometry. Significant increase in one of the isoforms of alpha2 chain of haptoglobin was observed in ENL condition. In addition, haptoglobin phenotype was determined for healthy controls and leprosy patients. Hp 0-0 phenotype was detected in 21.4% of the ENL patients undergoing treatment, which on follow up examination showed typable phenotype, thus showing a condition of acquired anhaptoglobinemia. Since ENL still remains a threat to leprosy disease management, the above findings may provide new insights in understanding the development and progression of this inflammatory condition.     

Antibodies against BCG antigen 60 in mycobacterial infection.

A sensitive specific radioimmunoassay was developed to measure antibodies against BCG antigen 60, a prominent antigenic component of BCG bacilli which cross-reacts with similar components in many mycobacterial species including Mycobacterium leprae and M tuberculosis. A lepromatous serum pool had anti-BCG-60 activity with a titre of 10(5) and the tuberculoid pool a titre of 10(4). Testing of individual sera showed striking variations within groups of patients with lepromatous and tuberculoid leprosy. In five of the 20 tuberculoid leprosy sera the anti-BCG-60 activity was above the median for the lepromatous group. The current view that antibody formation against mycobacterial antigens is very low in tuberculoid leprosy thus no longer appears to be tenable. Sera from eight patients with active pulmonary tuberculosis also showed a striking variation in anti-BCG-60 content, and the median value of this group was even higher than in those with lepromatous leprosy.     

Analysis of apoptosis and Bcl-2 expression in polar forms of leprosy

Apoptosis eliminates pathogen-infected cells. Its modulation can influence the course of infections, permitting the survival of intracellular pathogens. In leprosy, which presents several clinical manifestations related to bacillary burden and host immune status, the mechanisms responsible for the persistence of the bacillus are unknown. Few studies have focused on apoptosis over the disease spectrum and as a defense mechanism against Mycobacterium leprae. We evaluated apoptosis using terminal transferase dUTP nick end labeling and the expression of Bcl-2 by immunohistochemistry in skin lesions from 11 tuberculoid and 15 lepromatous leprosy patients. Each specimen was evaluated by determining the number of positive cells in 10 fields at × 400 magnification. We observed a higher number of apoptotic cells in tuberculoid lesions in comparison with lepromatous leprosy (42.5 cells per 10 fields vs. 11.5 cells per 10 fields, P<0.0001). Expression of Bcl-2, conversely, was larger in lepromatous than in tuberculoid samples (172.0 cells per 10 fields vs. 17.7 cells per 10 fields, P<0.0001). These observations suggest modulation of apoptosis in leprosy, primarily in lepromatous patients, for which the decrease in cell death could support M. leprae survival and contribute to the success of infection. Conversely, in tuberculoid patients, apoptosis could contribute to reducing propagation of the bacillus.