Glycoside-bearing liposomal delivery systems against macrophage-associated disorders involving Mycobacterium leprae and Mycobacterium tuberculosis.

Asiaticoside, a plant glycoside with rhamnose as end sugar and having microbicidal properties was tested against Mycobacterium leprae and Mycobacterium tuberculosis both in vivo and in vitro. As rhamnose is reported to have no tissue specificity, corchorusin D having glucose as end sugar was used for targeting with an equimolar proportion of asiaticoside in liposomal form for testing the drug value. Results showed that liposomal asiaticoside had better microbicidal property against M. leprae and M. tuberculosis when compared to that of free asiaticoside whereas liposomes containing asiaticoside and corchorusin D were found to be equally or more active in comparison to liposomal asiaticoside alone. It is inferred that appropriate glycosides, if used in liposomal form (incorporated or covalently grafted) have enhanced drug efficacy and such glycoside bearing liposomes as targeted delivery systems could be used for chemotherapeutic control of several other diseases.

T lymphocyte reactivity of leprosy patients and healthy contacts from a leprosy-endemic population to delipidified cell components of Mycobacterium leprae.

In this study, we measured in vitro proliferative responses of peripheral blood mononuclear cells from both leprosy patients across the clinical spectrum and also healthy contacts from a leprosy-endemic population to delipidified cell components of Mycobacterium leprae (DCC) and Dharmendra lepromin. Dharmendra lepromin was poor in inducing in vitro T cell proliferation in all the study groups, even though it elicited marked in vivo skin test reaction in tuberculoid leprosy patients and healthy contacts. In contrast, Dharmendra preparation of BCG induced marked T-cell response in tuberculoid as well as bacterial index negative lepromatous patients. DCC induced a significantly higher lymphoproliferative response than Dharmendra lepromin in all study groups. A significant positive correlation was observed between the lymphoproliferative responses to DCC and BCG. The present study, based on a large number of leprosy patients and healthy contacts, clearly demonstrates that DCC, depleted of glycolipids and lipopolysaccharides, is a good antigenic preparation for evaluating T-cell reactivity to M. leprae.

Nature of peritoneal macrophages from DCC immunized mice.

Peritoneal macrophages from mice immunized with the delipidified cell component (DCC) of Mycobacterium leprae showed changes in various parameters such as increased protein synthesis, levels of hydrolytic enzyme and augmented phagocytic ability indicating activation of the cells. Furthermore, the surface structure of the cells were quite different from that of the macrophages of normal mice. These observations indicate that the peritoneal macrophages have been activated to phagocytose and kill M. leprae better in the immunized mice. The ability to kill the pathogen by these cells was reported by us earlier.

In vivo effect of delipidified cell component of Mycobacterium leprae in relation to infection with leprosy bacteria in mice.

The delipidified cell component (DCC) of Mycobacterium leprae was used as an immunomodulatory agent in Swiss white mice. The peritoneal macrophages of these mice were activated to produce increased amount of reactive oxygen intermediates like hydrogen peroxide (H2O2) and superoxide. These macrophages also attained the ability to kill M. Leprae in vitro as shown by several assay systems including the conventional mouse foot-pad technique. The increased levels of superoxide seem to be responsible for the killing of M. leprae as addition of the enzyme superoxide dismutase, which breaks down O2, resulted in survival of these bacilli inside the macrophages. The increased production of H2O2 does not seem to be responsible for killing M. leprae. The results indicate that the DCC of M. leprae acts as an effective immunomodulator in mice leading to the activation of macrophages with increased production of H2O2 and superoxide as well as enabling them to kill M. leprae via the action of superoxide anions.

Antigenic similarity of a cultivable acid fast bacterium to Mycobacterium leprae.

A cultivable acid fast stainable bacterium obtained from leprosy nodule showed similarity to M. leprae in antigenicity to serum antibodies of lepromatous leprosy patients. The antigenic similarity has been seen more clearly in the delipidified cell components of both these bacteria. An antigen of 35-38 kDa has been seen as a common antigen between M. leprae and the cultivable bacilli with binding ability to sera from leprosy patients. This cultivable bacterial component could be used for serodiagnosis of lepromatous leprosy.

The use of an amino acid as a precursor of a bioactive metabolite in combatting an infectious disease: The efficacy of a tryptophan-enriched diet in the treatment of leprosy.

Deoxyfructosylserotonin (DFS), a stable derivative of serotonin, was shown to have an anti-leprosy effect (Mester and Antia). Therefore, a tryptophan-enriched food (NAL) was devised to increase the concentration of free tryptophan as serotonin precursor in blood.32 multibacillary lepromatous leprosy patients were divided in 3 groups receiving: 1) MDT (multi-drug therapy, control - 8 patients), 2) NAL (50 g/day - 13 patients) and 3) NAL + 1/2 MDT (11 patients). The clinical improvement (clinical score) was 24.2% for 1), 19.9% for 2) and 30.4% for 3). The loss of viability of theM. leprae bacilli in the mouse foot-pad test was 66% for 1), 75% for 2) and 70% for 3). The improvement by serodiagnosis (ELISA) was 19.4% for 1), 25.1% for 2) and 23.3% for 3).These preliminary results (6 months) suggest that the NAL-food is efficient against leprosy and apparently not very different from the control MDT in that respect.

Delipidified cell components of Mycobacterium leprae and its applications.

Delipidified cell components (DCC) of Mycobacterium leprae obtained as an insoluble material consist of several proteins. This preparation, DCC, has ability to differentially bind to sera from lepromatous leprosy patients and antibodies to this complex get reduced as patients improve under chemotherapy. The antigenic complex has no ability to bind to proteins of sera from normal healthy individuals or tuberculoid leprosy patients. The DCC is antigenic and is recognised by immune deficient cells of lepromatous leprosy patients, leading to lymphocyte proliferation, production of Interleukin II and interferon gamma, and resulting in activation of the phagocytes to initiate killing of endocytosed M.leprae through reactive oxygen intermediates, primarily superoxide. The DCC has also immunomodulatory properties to protect mice against M.leprae infection. Experiments with mice and isolated peripheral blood cells from patients have indicated the probable molecular mechanism of immunomodulation by DCC.

Antigenic protein from Mycobacterium leprae released in macrophages in vitro as indicator of viability of bacteria.

Peritoneal macrophages from randombred, Swiss white mice, when cultured and infected with Mycobacterium leprae for 24 hours, are able to show the presence of antigen(s) with binding affinity to antibodies present in the sera of bacteriologically positive, lepromatous leprosy patients. Such antibodies are not seen in sera from normal and healthy persons, tuberculoid leprosy patients, or long-term-treated, bacteriologically negative, lepromatous leprosy patients. The production of the antigen(s) is blocked by the anti-M leprae drug rifampin. Other mycobacteria when incubated with macrophages from mice show very little antigens in the lysate but the antigens have an equal affinity for antibodies in sera from both normal individuals and lepromatous patients. Only the lysates from macrophages exposed to live M. leprae could discriminate and could exhibit differential binding to sera from leprosy patients compared to sera from normal individuals. This antigen(s) does not have any binding ability to the monoclonal antibodies available to the antigens of M. leprae identified at present and shown to be specific to M. leprae. This indicates a separate identity of this product which has potential for further exploitation in exploring host-pathogen interactions related specifically to the leprosy infection and the tolerance of M. leprae inside cells.

An antigenic complex that restores ability in leprosy patients to kill Mycobacterium leprae--the probable molecular events identified by in vitro experiments.

The delipidified cell components of Mycobacterium leprae (DCC) obtained as an insoluble material was presented as an antigen by the macrophages of lepromatous leprosy patients. This resulted in in vitro lymphocyte proliferation and production of lymphokines, like IL-2 and IFN-gamma. This DCC induced culture supernatant was capable of activating patient macrophages through changes induced in the membrane, as monitored by same specific markers, before and after exposure to the supernatant. The activated macrophages could recognise M. leprae as an antigen to initiate cell mediated immunity and also recognise the bacilli as a pathogen to produce superoxide leading to the killing of phagocytosed M. leprae. Based on these observations, it is indicated that DCC could be a potent immunomodulatory restoring in the phagocytes of leprosy patients to kill M. leprae like normal resistant individuals.

Viability of Mycobacterium leprae inside macrophages from different strains of mice and possible genetic control.

Peritoneal macrophages from Swiss white mice in vitro tolerated Mycobacterium leprae and allowed metabolism of the bacteria leading to release of bacteria-specific antigenic protein. This was associated with the maintenance of viability of the bacilli inside the cells. Macrophages from C57BL mice reduced viability of M. leprae after phagocytosis, and this was associated with the production of superoxide. Blockage of superoxide production resulted in maintaining viability of the cells of these mouse strains. Associated with loss of viability of the bacilli is the absence of the production of antigenic protein in the lysate. Interestingly, the maintenance of viability or loss of viability and the factors controlling such viability in the macrophages of Swiss white and C57BL mice, respectively, appeared to be genetically controlled.

In vitro and in vivo experiments with the new inhibitor of mycobacterium leprae brodimoprim alone and in combination with dapsone.

The antibacterial effect of brodimoprim alone and in combination with dapsone has been studied in vitro in cell-free systems and in whole mycobacterial cells as well as in vivo in mice and humans. The obtained inhibitory effects in vitro and in vivo against model mycobacterial strains and M. leprae, the pharmacokinetic properties in human and its synergistic effect with the most used drug in the chemotherapy of leprosy, dapsone, make brodimoprim a promising candidate in the therapy of leprosy.

A complex component modulating immune-deficient cells in leprosy patients leading to loss of viability of Mycobacterium leprae--a possible vaccine.

Macrophages from peripheral blood of leprosy patients, both multi-bacillary and paucibacillary are unable to kill phagocytosed Mycobacterium leprae due to their inability to produce superoxide (O2-) and hydroxyl radicals (OH.). The macrophages from healthy individuals are able to kill M. leprae along with release of O2- and OH. radicals. The deficiency in the macrophages of both types of leprosy patients is removed by activation of these cells when exposed to a culture supernatant obtained after stimulation of peripheral blood mononuclear cells from the same patients with delipidified cell components of M. leprae which are most likely cell wall proteins. The activation of macrophages also leads to recognition of whole live M. leprae as an antigen by cells from lepromatous patients. This activation of the phagocytes by delipidified cell components is blocked by cyclosporin A, indicating the possible role of several steps involved in immune activation of cells. The observations thus indicate the significant ability of delipidified cell components to eliminate the deficiencies in the macrophages from leprosy patients and restore them to behave like the cells from healthy individuals. Considering all these, it is suggested that delipidified cell components could be potential modulators, and are probably capable of functioning as a vaccine for leprosy.

Reactive oxygen intermediates inactivate Mycobacterium leprae in the phagocytes from human peripheral blood.

Reactive oxygen intermediates such as hydrogen peroxide, superoxide, and hydroxyl radicals are important microbicidal components, and they could also play a role in an infection with Mycobacterium leprae. A comparative study of the level of hydrogen peroxide and superoxide produced by peripheral blood phagocytes from normal healthy individuals and lepromatous leprosy patients showed a deficiency in superoxide production in the patients. In the phagocytes from normal healthy individuals, there was good release of superoxide ions, and this mediated the killing of M. leprae. The lack of superoxide production allowed the viability of M. leprae inside the macrophages from leprosy patients. This deficiency could be rectified by the use of an immunomodulator, the delipidified cell wall of M. leprae. This modulation resulted in the ability of the patients' phagocytes to respond to M. leprae, to produce reactive oxygen intermediates such as superoxide, and also to kill the bacteria. These observations indicate that delipidified cell wall could have significant potential to positively modulate the immune-deficient cells of leprosy patients.

Correlation of in vitro Fc receptor assay with in vivo mouse foot pad method.

Resistant strains of M. leprae have been reported to the various antileprosy drugs. There is currently no accepted test to identify the susceptibility pattern of M. leprae to the drugs in a short period. The only accepted test is the mouse foot method which takes a long period to yield results. The Fc receptor assay using the ability of viable M. leprae to alter the membrane of the macrophage is well established. It takes only ten days and is inexpensive. In 6 cases of leprosy patients the susceptibility pattern was worked out both with the in vitro Fc receptor assay and the vivo in mouse foot method The results correlated very well leading to the fact that the assay system is reliable. Hence it can be used not only to study the status of a patient, but also to shortlist the number of compounds to be tested on the mouse foot pad as anti-leprosy drug candidates.

Effect of deoxyfructoserotonin (DFS) on lepromatous leprosy.

To examine the anti-leprosy effect of deoxyfructoserotonin the drug was given in a dose of 10 mg/kg for 6 months to 6 patients with active lepromatous leprosy, in accordance with the WHO-THELEP protocol. Clinical and histological assessment and mouse foot-pad studies suggest that the drug has some anti-leprosy effect.

A component of Mycobacterium leprae as immunomodulating agent for immune deficient cells of leprosy patients.

The delipidified component of the insoluble portion which presumably is the cell wall of Mycobacterium leprae (DCW) was able to induce lymphocyte proliferation in the leucocyte culture from the peripheral blood of lepromatous leprosy patients. Normally these cells show no lymphocyte proliferation in response to M. leprae or their sonicated extract. The delipidified component (DCW) appears to be proteinaceous and able to induce antibodies in rabbit. The DCW has affinity to the sera from lepromatous leprosy patients but not sera from normal healthy individuals or tuberculoid leprosy patients. The ability to induce lymphocyte proliferation is blocked by agglutination of DCW with patient sera, heat treatment of DCW or protease treatment of the component. Along with lymphocyte proliferation, DCW also induces ability in the macrophages to render phagocytosed M. leprae non viable. Thus it is proposed that DCW of M. leprae could be a potent immunomodulator for immunedeficient cells of leprosy patients. The efficacy of DCW as a probable immunoprotector for M. leprae infection in mice has already been demonstrated earlier.

A rapid method for viability and drug sensitivity of Mycobacterium leprae cultured in macrophages and using fluorescein diacetate.

The ability of viable M. leprae to hydrolyze Fluorescein diacetate and retain fluorescein inside the bacteria was used to identify viable M. leprae inside the cultured in vitro macrophages. The subjective microscopic count of the FDA test was demonstrated as useful routine test by confirming the results obtained therein with a quantitative and non subjective measurement of fluorescence in spectrofluorimeter. Using this method loss of viability of M. leprae in presence of dapsone and rifampicin was demonstrated. Such an assay, was well correlated with another in vitro assay, the Fc receptor test and also the in vivo mouse foot test. The drug resistance of clinical isolates of M. leprae demonstrated by mouse foot pad was also correlated with FDA test system. Thus we have reported a reliable, consistent and rapid in vitro test system for determining viability and drug sensitivity of M. leprae.