Leprae Organisms Research Articles

Leprosy reactions: the effect of gender and household contacts

Various host-related factors have been reported as relevant risk factors for leprosy reactions. To support a new hypothesis that an antigenic load in local tissues that is sufficient to trigger the immune response may come from an external supply of Mycobacterium leprae organisms, the prevalence of reactional leprosy was assessed against the number of household contacts. The number of contacts was ascertained at diagnosis in leprosy patients coming from an endemic area of Brazil. The prevalence of reactions (patients with reactions/total patients) was fitted by binomial regression and the risk difference (RD) was estimated with a semi-robust estimation of variance as a measure of effect. Five regression models were fitted. Model 1 included only the main exposure variable "number of household contacts"; model 2 included all four explanatory variables ("contacts", "fertile age", "number of skin lesions" and "bacillary index") that were found to be associated with the outcome upon univariate analysis; models 3-5 contained various combinations of three predictors. Male and female patients were analyzed separately. In females, household contacts were a significant predictor for leprosy reactions in model 1 [crude RD = 0.06; 95% confidence interval (CI) = 0.01; 0.12] and model 5 (RD = 0.05; CI = 0.02; 0.09), which included contacts, bacillary index and skin lesions as predictors. Other models were unsatisfactory because the joint presence of fertile age and bacillary index was a likely source of multicollinearity. No significant results were obtained for males. The likely interpretation of our findings might suggest that in female patients, leprosy reactions may be triggered by an external spreading of M. leprae by healthy carrier family members. The small number of observations is an obvious limitation of our study which requires larger confirmatory studies.

How to prevent immunological reactions in leprosy patients and interrupt transmission of Mycobacterium leprae to healthy subjects: Two hypotheses

The basis of World Health Organization strategy for leprosy elimination is that the only source and reservoir for infection are patients with the disease. It was assumed that multi drug therapy (MDT) would reduce transmission of Mycobacterium leprae, but there is no convincing evidence for this. Furthermore, even if MDT has been proved to be extremely effective against the infectious disease, a noticeable proportion of leprosy patients can suffer from immunologic hypersensitivity reactions which are now the most significant issue in the managements of the disease. In endemic areas it was found that: M. leprae survives outside human body; healthy individuals harbor M. leprae bacilli in nasal cavity and shed micro-organisms in environment; there is widespread subclinical transmission of M. leprae with transient infection of the nose resulting in the development of a mucosal immune response. This disparate clinical, epidemiologic, and microbiologic evidence leads to the first hypothesis: that antigenic load in local tissues, sufficient to trigger the immune response, comes from external supply of M. leprae organisms. The hypothiocyanite anion (OSCN-) is generated in vivo by the reaction of thiocyanate with hydrogen peroxide catalyzed by lactoperoxidase. OSCN- is an antimicrobial oxidizing agent that prevents growth of bacteria, fungi, and viruses. OSNC- exists in lower airway secretions and lung has never been reported to be affected by leprosy. There is a sufficient concentration of OSCN- in the saliva, and accordingly mouth is rarely affected by leprosy. By contrast, the concentration of this compound is low or nil in nasal and lacrimal secretions and leprosy very often affects nose and eyes. The second hypothesis is that OSCN- may also protect from leprosy. Recently a method of OSCN- production, not involving enzymatic steps or use of toxic heavy-metal salts, has been patented. Studies on the susceptibility of M. leprae to hypothiocyanite could be carried out and, in case of positive results, the substance might be used in order to sterilize the nasal cavity of healthy carriers and prevent transmission of M. leprae to healthy subjects and to leprosy patients in whom it may trigger an immune response.

Experimental evaluation of possible new short-term drug regimens for treatment of multibacillary leprosy.

Groups of nude mice, with both hind footpads infected with 10(8) Mycobacterium leprae organisms, were treated with 4-week courses of different drug combinations. The effect treatment on each group was evaluated by subinoculating footpad homogenates from the treated mice into groups of normal and nude mice for subsequent regrowth, assessed 1 year later. A combination of rifampin (RMP) with clarithromycin (CLARI), minocycline (MINO), and ofloxacin (OFLO) resulted in the complete killing of M. leprae after 3 weeks of treatment. A combination of sparfloxacin (SPAR) and RMP also resulted in a similar bactericidal effect after 3 weeks of treatment. Other drug combinations showed variable effects. Very little or no effect was observed with any regimen if the treatment was given for less than 2 weeks. World Health Organization (WHO) multidrug therapy (MDT) given for 8 weeks was as effective as the two combinations described above. The results suggest that multidrug combinations consisting of RMP-OFLO (or SPAR)-CLARI (and/or MINO) are as effective as the WHO MDT for the treatment of experimental leprosy. Moreover, they imply that these combinations, which were found to be active in a 4-week experimental treatment protocol, could be administered as treatment to patients for a period of time shorter than the present 2-year regimen without a loss of effectiveness.

Cutaneous Malignancy and Leprosy

Leprosy is a chronic systemic infection caused by the bacillus Mycobacterium leprae. Cutaneous neoplasms have been observed in patients with leprosy. Also, albeit less commonly, M. leprae have been documented in the lesions of skin cancer. To describe a 62-year-old man with chronic sun exposure and exposure to armadillos who subsequently developed lepromatous leprosy, to discuss the cutaneous malignancies that have occurred in patients with leprosy, and to review the literature concerning the concurrent presence of an infectious pathogen and a cutaneous neoplasm in the same lesion. Our patient's basal cell carcinomas were excised, his abdominal plaques were biopsied, and his leprosy infection was treated with dapsone and rifampin. The types of cutaneous malignancies in leprosy patients and infectious pathogens concurrently found in lesions of skin tumors were summarized after evaluating previously published reports. Skin biopsies from our patient demonstrated M. leprae bacilli not only in his abdominal plaques, but also in all of his basal cell carcinoma lesions. Fungal, mycobacterial, and viral pathogens have concurrently been observed in skin lesions of basal cell carcinomas, Kaposi's sarcoma, melanoma, mycosis fungoides, and squamous cell carcinoma. Patients with leprosy can develop skin cancers and the histologic interpretation of those skin cancers can show evidence of leprosy. It is uncertain to what degree the decreased cell-mediated immunity in patients with lepromatous leprosy either enhances their susceptibility to and/or influences the course of their cutaneous neoplasms; also, in these patients, the coexistence of M. leprae organisms and cutaneous malignancy in the same lesion is likely to be secondary to the high bacillary load that is present.

A morphological and functional assessment of Mycobacterium leprae‐induced nerve damage in a guinea‐pig model of leprous neuritis

Nerve damage, resembling that caused by Mycobacterium leprae in man, was created by the injection of cobalt-irradiated M. leprae organisms into the tibial nerve of guinea-pigs. Assessment of nerve damage was made by clinical, electrophysiological and morphometric means at intervals up to 13 weeks after injection. Quantitative immunohistochemical analysis of neuropeptide-containing fibres in the skin of the foot was also carried out. Significant nerve damage occurred 3 weeks after injection of M. leprae organisms. Motor and sensory functional loss peaked at 5 weeks after injection, and there was a significant decrease of peptide-immunoreactive nerves in all skin compartments. The nerve damage was self-limiting and functional recovery had occurred by 13 weeks. The model shows many of the features found in the nerve damage of treated leprosy patients.

Vaccination of mice with a soluble protein fraction of Mycobacterium leprae provides consistent and long-term protection against M. leprae infection.

Groups of BALB/c mice were vaccinated intradermally with either Freund's incomplete adjuvant (FIA) alone, 10(7) heat-killed Mycobacterium leprae organisms in FIA, or a number of fractions of M. leprae containing soluble and/or cell wall components. At 1, 3, 6, 9, and 12 months later, vaccinated mice were challenged in the right hind footpad with 5,000 live M. leprae organisms, and vaccine protection was assessed 6 to 8 months later, at the peak of M. leprae multiplication in the negative control (FIA alone), by the two-sample rank-sum test. In these studies, a cell wall fraction rich in peptidoglycan was consistently ineffective. Both heat-killed M. leprae and a fraction containing cell wall and fixed proteins generally protected when the interval between vaccination and challenge was 1 or 3 months but not subsequently. On the other hand, soluble proteins of M. leprae alone or in combination (with cell wall fractions) consistently (14 of 14 instances) afforded highly significant protection (P less than or equal to 0.01) at all challenge intervals up to 1 year after vaccination. These results suggest that the soluble protein fraction of M. leprae offers promise for a vaccine against leprosy.

Phospholipase activity of Mycobacterium leprae harvested from experimentally infected armadillo tissue

Three types of phospholipase activity--phospholipase A1, A2, and lysophospholipase--were detected in Mycobacterium leprae harvested from armadillo tissue at about 25% of the specific activity found in a slowly growing mycobacterium, Mycobacterium microti, which was grown in medium to optimize its phospholipase activity. The highest activity found was lysophospholipase, which released fatty acid from 2-lyso-phosphatidylcholine. Phospholipase activity was detected by using phosphatidylcholine and phosphatidylethanolamine. Differences in relative activities with these three types of substrate distinguished phospholipase activity in M. leprae extracts from armadillo liver extracts. Furthermore, retention of activity in M. leprae after NaOH treatment showed that the activity associated with M. leprae was not host derived. The specific activity of phospholipase was 20 times higher in extracts of M. leprae than in intact M. leprae organisms. Diazotization, a treatment which abolishes activities of surface enzymes exposed to the environment by the formation of covalent azide bonds with exposed amino groups, did not affect M. leprae's phospholipase activity, with one exception: release of arachidonic acid from phosphatidylcholine, which was partially inhibited. Phenolic glycolipid I, the major excreted amphipathic lipid of M. leprae, inhibited phospholipase activity, including release of arachidonic acid, for both M. leprae- and armadillo-derived activity.

Enzymes for biosynthesis de novo and elongation of fatty acids in mycobacteria grown in host cells: is Mycobacterium leprae competent in fatty acid biosynthesis?

Fatty acid synthetase activity in extracts of Mycobacterium leprae was equivalent to 1.7 pmol malonyl-CoA incorporated into fatty acid min-1 (mg protein)-1. This activity--if representative of living M. leprae organisms--is insufficient to enable them to synthesize their lipid requirements rapidly enough to support growth. The major activity for scavenging fatty acids in extracts of Mycobacterium microti and Mycobacterium avium, as well as in extracts of M. leprae, was acetyl-CoA-dependent fatty acyl-CoA 'elongase'. This activity was about four times higher in M. avium and M. microti grown in a medium which contained lipids, or when grown in mice, than in medium without added lipids. In contrast, the de novo fatty acid synthetase activity was repressed in M. avium and M. microti when grown in medium that contained lipids, or when grown in mice. These results are consistent with the hypothesis that mycobacteria grown in vivo preferentially scavenge lipids from the host cells, and suggest that a source of lipid should be included in media for attempted axenic isolation of M. leprae.

Intracellular fate of Mycobacterium leprae in normal and activated mouse macrophages

Mycobacterium leprae replicates within mononuclear phagocytes, reaching enormous numbers in the macrophage-rich granulomas of lepromatous leprosy. To examine the capability of macrophages to digest M. leprae, we studied the intracellular fate of M. leprae organisms in normal and activated mouse macrophages by using the electron-dense secondary lysosome tracer Thoria Sol. Intracellular M. leprae organisms, surrounded by a characteristic electron-transparent zone, were contained within phagosomal vacuoles of macrophages cultured in vitro for 1 to 6 days. In normal macrophages, a majority of phagosomes containing freshly isolated live M. leprae cells resisted fusion with Thoria Sol-labeled lysosomes. The extent of fusion was not significantly affected by pretreatment of M. leprae with human patient serum high in specific immunoglobulin G and M antibodies. In contrast, a majority of phagosomes containing gamma-irradiated M. leprae cells underwent lysosome fusion in normal macrophages. In addition, increased phagolysosome fusion was observed with live M. leprae-containing phagosomes in macrophages activated with gamma interferon. Increased fusion was associated with an increase in the number of fragmented and damaged bacilli, suggesting that increased digestion followed fusion. This study indicates that activated macrophages may have an increased capacity for clearance of normally resistant M. leprae.

Mycobacterium leprae-specific Lyt-2+ T lymphocytes with cytolytic activity.

Mice were immunized intradermally with 10(7) irradiated Mycobacterium leprae organisms, and draining lymph nodes were collected after 4 weeks. Lymph node cells were restimulated in vitro with soluble M. leprae antigen and accessory cells. The resulting T-cell line was propagated in vitro in the presence of M. leprae antigen, accessory cells, and interleukin-2-containing supernatants from concanavalin A-stimulated rat spleen cells. Long-term cultured T cells were Thy-1+ L3T4- Lyt-2+ as revealed by analysis with the fluorescence-activated cell sorter. From this line, T-cell clones with the same phenotype were established. The T-cell clone A4 failed to secret interleukin-2 after stimulation with antigen and accessory cells, and its growth depended on exogeneous interleukin-2. A4 T cells produced gamma interferon in an antigen-specific, H-2-restricted, and interleukin-2-dependent way. Importantly, this T-cell clone was capable of lysing bone marrow macrophages presenting M. leprae antigen. Other T-cell clones as well as native Lyt-2+ T cells from M. leprae-immunized mice were also capable of lysing bone marrow macrophages expressing M. leprae antigens. These findings suggest that specific Lyt-2+ T cells participate in the immune response to M. leprae. It is postulated that cytolysis of M. leprae-infected macrophages or Schwann cells contributes to protection against and pathogenesis of leprosy.

Strain variations in the murine cellular immune response to the phenolic glycolipid I antigen of Mycobacterium leprae.

The cellular immune response to the Mycobacterium leprae-specific phenolic glycolipid I was examined in inbred mice immunized with M. leprae by in vivo delayed cutaneous hypersensitivity and in vitro lymphocyte proliferation. Whereas all mouse strains responded to M.leprae-induced delayed-type hypersensitivity and lymphocyte proliferation, only BALB.K was responsive in both assays to the glycolipid. Responsiveness was determined in part by non-H-2 genes, while the influence of H-2 genes was not apparent. Among congenic BALB/c mice differing only at Igh-C allotype loci, variations in responsiveness were found in both delayed-type hypersensitivity and lymphocytes proliferation assays, indicating a possible role for Igh-C loci-linked genes. Unresponsiveness in the lymphocyte proliferation assay to the glycolipid was inherited as a dominant trait in one set of responder X nonresponder F1 progeny. We conclude that after immunization with M. leprae organisms, the cell-mediated responses to the glycolipid, endowed with a single carbohydrate epitope, are under polygenic control, predominantly non-H-2-linked genes.

Oxidation of Carbon Sources through the Tricarboxylic Acid Cycle in Mycobacterium leprae Grown in Armadillo Liver

All the enzymes of the tricarboxylic acid cycle have now been demonstrated in extracts of Mycobacterium leprae grown in armadillo liver. Many were also present in homogenates of host-tissue, but biochemical evidence is presented which indicates that all enzymes detected in extracts from M. leprae were authentic bacterial enzymes. Further evidence for a complete tricarboxylic acid cycle in M. leprae was obtained by first establishing that citrate could be taken up and catabolized by whole M. leprae organisms, then showing that oxidation of radioisotopically labelled pyruvate to CO2 by suspensions of M. leprae was stimulated by adding unlabelled citrate. Control of tricarboxylic acid cycle activity in M. leprae by the inactivation of fumarase by a protease is speculated upon.

Cell envelope architectures of leprosy-derived corynebacteria,Mycobacterium leprae, and related organisms: a comparative study

Cell-envelope architectures of three strains of leprosy-derived corynebacteria (LDC) named Kim, FPSA, and 43LL were compared withMycobacterium leprae andM. avium as well as with related organisms (Nocardia asteroides andCorynebacterium psuedotuberculosis). Cytochemical studies were performed at the ultrastructural level after the lead citrate, silver proteinate, acidic phosphotungstic, and ruthenium red colorations. This study showed that, while the organisms belonging to theCorynebacterium-Mycobacterium-Nocardia (CMN) group had only the cytoplasmic membrane but not the cell wall reacting with the silver proteinate coloration, the LDC organisms had both the cell wall and the cytoplasmic membrane reacting with this coloration method. Moreover, the three strains of the LDC organisms differed from one another at the level of their exopolymer content.Mycobacterium leprae, on the other hand, gave a cytochemical response common to other mycobacteria and the members of the CMN group studied. Consequently, the envelopes of the LDC organisms were not identical toM. leprae, neither morphologically nor cytochemically.

Purine metabolism in Mycobacterium leprae grown in armadillo liver

Mycobacterium leprae organisms generally incorporated purines more rapidly than pyrimidines into nucleic acids from the incubation medium. Purine synthesis de novo took place at a very slow rate suggesting a preference of the organism for preformed purines. In cell-free extracts of leprosy bacilli, enzyme for scavenging and interconversion of purines were detected. The results are discussed in the light of the failure to cultivate M. leprae in vitro, and the use of labelled substrates to determine the viability of suspensions of leprosy bacilli and their sensitivity to anti-leprosy drugs.

Leprosy and Cancer: A Retrospective Cohort Study in Hawaii 2

We used data collected on a retrospective cohort of 1,123 leprosy patients living in Hawaii between 1940 and 1970, to test the hypotheses that patients with lepromatous leprosy, who have an impairment in their cellular immune response, would have an increased risk for cancer and that patients with tuberculoid leprosy, who are immunologically competent, would have a normal or even a reduced cancer risk from beneficial stimulation of their cellular immune system by exposure to the Mycobacterium leprae organisms. Based on the survival analysis method, the results of the study supported the predicted increase in cancer cases among the lepromatous leprosy patients (19 observed, 12.7 expected; risk ratio = 1.5) and the predicted decrease among the tuberculoid leprosy patients (14 observed, 17.8 expected; risk ratio = 0.8); in both groups, the findings were consistent across the five racial categories of the study. However, none of these differences between observed and expected cases was statistically significant at the 5% level. The study provided no support for the alternate hypothesis that chronic antigenic stimulation by the M. leprae organisms might lead to an increase in tumors of the lymphoreticular system.