Inhibition of apoptosis by ionomycin and zinc in peripheral blood mononuclear cells (PBMC) of leprosy patients.

PBMC from tuberculoid (BT/TT) and lepromatous leprosy (BL/LL) leprosy patients showed spontaneous apoptosis when cultured in the absence of mitogen for 24 h, which was inhibited by anti-tumour necrosis factor-alpha (TNF-alpha) antibodies. Apoptosis was also inhibited by ionomycin and zinc, which also increased IL-2 and decreased TNF-alpha production. The increase in IL-2 production suggests a mechanism whereby dietary supplements with zinc might alter the cell-mediated immunity response in leprosy patients.

Immunological defect in leprosy patients: altered T-lymphocyte signals.

The early events of activation were studied in paucibacillary (TT/BT) and multibacillary (BL/LL) leprosy patients by stimulation of their lymphocytes with mitogenic agents (calcium ionophore A23187/PMA) and Micobacterium leprae antigen (PGL-1). Maximum proliferation in response to PMA/A23187 and PGL-1 was observed in the BT/TT patients and the control group, respectively. Inositol triphosphate (IP3) and calcium were constitutively elevated in BT/TT and LL/BL patients. PMA/A23187 caused an increase in both IP3 and [Ca2+]i in BT/TT patients and controls. PGL-1 marginally increased IP3 levels in BT/TT patients. In the LL/BL patients, although PMA/A23187 increased IP3 levels, but no change was seen in [Ca2+]i, PGL-1 had no effect. Protein kinase C levels were seen to be associated with particulate fractions in BT/TT patients and were found to increase further in response to PMA/A23187. PGL-1 did not increase translocation of protein kinase C in controls or LL/BL patients. A preactivated and sensitised state of T-lymphocytes was observed in BT/TT patients, responsive to antigen and mitogens, whereas the cells of LL/BL patients were unresponsive to PGL-1. The altered signal transduction events characterised in the MB patients thus correlate well with the anergic state of their cells.

Spontaneous apoptosis in peripheral blood mononuclear cells of leprosy patients: role of cytokines.

Peripheral blood mononuclear cells from leprosy patients underwent spontaneous apoptosis upon culture for 24 h. The apoptosis was inhibited by anti-TNFalpha antibodies and to a certain extent by anti-IL-1alpha and IL-6, thus showing that T(H)2-type cytokines (mainly TNFalpha) are responsible for inducing apoptosis. This cytokine-mediated apoptosis could be inhibited by ionomycin and zinc, thereby suggesting that these metal ions can be used to decrease the levels of these inflammatory cytokines in various diseases.

Alterations in early biochemical events following T cell activation in leprosy patients.

The early events of activation and cytokine profiles (IL-2, 4, and 6) were studied in lymphocytes of paucibacillary (TT/BT) and multibacillary (BL/LL) leprosy patients after stimulation with PMA/A23187 and Mycobacterium leprae antigen (PGL-1). Lymphocytes from BT/TT patients showed proliferation in response to both PMA/A23187 and PGL-1 compared to BL/LL. The levels of early activation signaling molecules such as IP3, calcium, and protein kinase C (PKC) in the particulate fraction were found to be elevated in BT/TT and BL/LL patients and showed a further significant increase after stimulation with PMA/A23187 in BT/TT patients. PGL-1 marginally increased the IP3 levels in BT/TT patients, whereas in BL/LL patients, it had no effect. The levels of IL-2 were enhanced in lymphocytes of BT/TT leprosy patients and were further augmented by PPD and PGL-1, while the levels of IL-4 and IL-6 were increased in LL/BL lymphocytes and further augmented by PGL-1. Thus PGL-1 seems to be a major culprit in inducing the TH2-type cytokine response observed in lepromatous leprosy patients.

Urinary excretion of renal brush border membrane enzymes in leprosy patients--effect of multidrug therapy.

Renal function at the brush border membrane level has been studied using characteristic enzymes, such as alkaline phosphatase, leucine-aminopeptidase and gamma-glutamyl transpeptidase. Urinary enzyme studies were performed using leprosy patients, classified on the basis of bacteriological index (BI>3; n=20, BI<3; n=12, BI-ve; n=10) and compared with control subjects (n=10). The role of enzymuria in monitoring WHO-recommended multidrug therapy (MDT) has been evaluated in these patients. A significant increase in the enzyme activities (p<0.01), as well as significant (p<0.01) proteinurea in 24-hour urine samples of both the smear positive groups (BI>3, BI<3) prior to therapy compared to control subjects, indicates proximal tubular functional impairment at brush border membrane level. In the smear negative (BI-ve) group, no significant difference was observed in enzyme activities as compared with the control group. In a follow-up study (BI>3;n=13, BI<3; n=4) the activities of all the enzymes decreased significantly in all the groups when compared to a corresponding untreated group. The follow-up study was not carried out on the smear negative group. The surprising finding was the differential behaviour of r-glutamyl transpeptidase, whose activity increased significantly (p<0.01) even after therapy in BI>3 group when compared with untreated patients. However in a detailed work-up including hepatic and renal function tests, the serum biochemistry was found to be normal both before and after therapy. Urinary excretion of brush border enzymes seems to be related to bacterial load, and their potential in studying the effect of MDT remains unclear.

Role of reactive oxygen species in renal damage in experimental leprosy.

Renal involvement is known to occur in leprosy. In the present study the possible role of reactive oxygen species (ROS) in causation of renal damage in mice infected with Mycobacterium leprae has been investigated. At least six animals from each group (control and infected) were killed at 0 day, 3, 6 and 9 months postinfection. The results showed a significant increase in the chemiluminescence (CL) response of peritoneal macrophages which was maximum between 3 and 6 months. No significant increase was observed in CL response of blood neutrophils. A significant increase in lipid peroxidation was observed at 3 and 6 months as evident by an increase in malondialdehyde levels. The increased ROS production might be the cause of lipid peroxidation. The renal damage is alos evident by decrease in the activity of renal brush border membrane enzymes, namely, alkaline phosphatase, leucine aminopeptidase and r-glutamyl transpeptidase. Thus ROS might play a role during early stages of M. leprae infection but in the later stages other immunological mechanisms may overpower the effect of ROS.

Transport of amino-acids across renal brush border membrane vesicles in Mycobacterium leprae infected Swiss albino mice--effect of Convit vaccine.

Brush border membrane vesicles prepared from kidneys of Mycobacterium leprae infected (non-vaccinated) and vaccinated-infected Swiss albino mice were used to assess the effect of Convit's combined vaccine (BCG + M. leprae) on amino acid transport activity across the tubular basement membrane. The protective effect of Convit's vaccine was more pronounced with respect to the uptake of L-alanine than L-aspartate. Uptake of L-lysine showed no significant difference in the different groups. Footpad counts followed characteristic growth curves in the non-vaccinated infected group but showed a lag in the development of peak levels in the vaccinated group. Further Convit's vaccine appeared to have a protective effect on renal impairment in the mouse model of leprosy in the initial stages of infection only, as indicated by the transient reversal of amino acid uptake and a diminution in the footpad counts induced by M. leprae infection. No significant (P > 0.05) protective effect of the vaccine was found in the advanced disease state.

Bacillaemia and Mycobacterium leprae cell wall antigen in paucibacillary leprosy.

A study was undertaken to estimate bacillaemia and M. leprae antigen detection in 54 paucibacillary leprosy patients (TT, BT). Acid-fast bacilli were detected in the blood of 14.8% patients of borderline tuberculoid (BT) leprosy. M. leprae antigen was demonstrated in 48.2% patients of BT leprosy. Slit-skin smears were negative in all these patients. At the end of treatment (6 months of WHO-MDT) all the follow-up blood samples were negative for both bacillaemia and M. leprae antigen in the serum.

Suppression of ADCC by immune complexes formed in vitro in Mycobacterium leprae-infected mice.

Antibody-dependent cellular cytotoxicity (ADCC) was assessed in mice infected experimentally with Mycobacterium leprae and injected simultaneously with in vitro-formed immune complexes (IC). Significant decrease in the ADCC function was observed in animals given IC at zero day (0dIC) and 3 months (3mIC) post inoculation with M. leprae, when ADCC activity was assessed at 3, 6 and 9 months period. From the data obtained we believe that ADCC is suppressed by IC formed in vitro.

Adenosine triphosphate content of Mycobacterium leprae by percoll buoyant density centrifugation.

Thirty nine untreated patients of bacilliferous leprosy with a mean bacteriological index of 4.8 and morphological index of 1.3% formed the study group. Adenosine triphosphate assay was carried out by (i) enzyme treatment method in 18 patients and (ii) percoll buoyant density gradient method in 21 patients. ATP content obtained by percoll buoyant density gradient method was significantly higher than that obtained by enzyme treatment method. Percoll buoyant density centrifugation for purification and isolation of bacilli from human leproma is simplier, quicker and can serve as an alternate method of enzyme treatment.

Detection of a Mycobacterium leprae cell wall antigen in the urine of untreated and treated patients.

A total of 90 leprosy patients, 12 household contacts and 10 normal subjects were studied for the detection of Mycobacterium leprae cell wall antigen in urine using monoclonal antibody (ML30A2 IgG). In untreated multibacillary leprosy (BL-LL) the M. leprae cell wall antigen could be demonstrated in the urine of 14 (64%) patients by immunofluorescence (IF) and 22 (100%) by ELISA. In untreated paucibacillary leprosy (TT-BT), it could be demonstrated in 3 (11.5%) and in 13 (50%) patients by IF and ELISA methods respectively. All but 1 household contact (later confirmed to have BL leprosy) and all 10 normal subjects' urine was negative for M. leprae cell wall antigen by both methods. The same antigen was, however, demonstrated in urine of 50% paucibacillary patients who had received 6 months of treatment and in 68% multibacillary patients who had received 24 months of WHO recommended multidrug therapy. M. leprae cell wall antigen assays in urine will not be useful in the follow-up of leprosy patients on multidrug therapy.