Anin vitro test using cholesterol metabolism of macrophages to determine drug sensitivity and resistance ofMycobacterium leprae

Macrophages that have ingested liveMycobacterium leprae show a preferential accumulation of cholesterol ester. Such an accumulation is not seen, on the ingestion of dead bacteria. Among the macrophages that ingest liveMycobacterium leprae, the presence of dapsone or rifampicin prevents largely the alteration in the anticipated increase in the cholesterol ester indicating the sensitivity of the bacteria to the drug. In the small number of relapsed patients, the presence of dapsone did not reduce the cholesterol ester increase, suggesting that theMycobacterium leprae present are either resistant or escaped detection. The method provides a rapid drug screening system foranti-Mycobacterium leprae activity of known and unknown compounds     

In vitro drug screening system using membrane alteration in macrophages byMycobacterium leprae

The observation that liveMycobacterium leprae on entry into macrophages from lepromatous leprosy patients reduced the number of EA rosetting macrophages, was extended to macrophages from Swiss white mice also. Further, the fact that deadMycobacterium leprae do not bring about such a change in macrophages from mice, allowed us to develop this into a bacterial viability testing system. Thus drug treated macrophages in the presence ofMycobacterium leprae showed normal rosetting ability ifMycobacterium leprae are inactivated by the drug, but showed reduced level of rosetting when bacteria were not susceptible to the drug. It was shown that a drug like dapsone, does act onMycobacterium leprae based on its permeability, quantity available inside the macrophages and inhibition of its action by Para amino benzoic acid. The inactivation ofMycobacterium leprae by sulphone and rifampicin was also proved by the flourescence diacetate method, which showed poorly viable bacteria after exposure to drugs. Thus it has been possible to develop a rapid drug screening method for testing the activity of unknown compound againstMycobacterium leprae.     

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.     

Absence of Nceh1 augments 25-hydroxycholesterol-induced ER stress and apoptosis in macrophages

An excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis.     

Superoxide production from macrophages of leprosy patients after stimulation withMycobacterium leprae

The macrophages from peripheral blood of normal healthy individuals respond to live or killedMycobacterium leprae by producing superoxide. On the other hand, the macrophages from bacteriologically positive (B +LL) or long term treated bacteriologically negative (B -LL) and tuberculoid leprosy patients are unable to produce superoxide when stimulated with liveMycobacterium leprae. While killedMycobacterium leprae induce superoxide with the cells from tuberculoid andB(-)LL patients, cells fromB(+)LL patients fail to respond. The deficiency inB(-)LL patients to produce superoxide appears to be specific withMycobacterium leprae and the defect can be counteracted by the addition of colchicine. These observations indicate a preexisting membrane disposition which does not favour superoxide production. A similar situation is seen in the cells from tuberculoid leprosy patients. Thus it appears that both cured and active lepromatous leprosy patients have defective macrophages, unable to respond to liveMycobacterium leprae to produce superoxide anion, in contrast to the situation with the cells from normal healthy individuals.     

Photobiomodulation therapy promotes the ATP-binding cassette transporter A1-dependent cholesterol efflux in macrophage to ameliorate atherosclerosis

Atherosclerosis is a chronic inflammatory disease related to a massive accumulation of cholesterol in the artery wall. Photobiomodulation therapy (PBMT) has been reported to possess cardioprotective effects but has no consensus on the underlying mechanisms. Here, we aimed to investigate whether PBMT could ameliorate atherosclerosis and explore the potential molecular mechanisms. The Apolipoprotein E (ApoE)^−/− mice were fed with western diet (WD) for 18 weeks and treated with PBMT once a day in the last 10 weeks. Quantification based on Oil red O-stained aortas showed that the average plaque area decreased 8.306 ± 2.012% after PBMT (P < .05). Meanwhile, we observed that high-density lipoprotein cholesterol level in WD + PBMT mice increased from 0.309 ± 0.037 to 0.472 ± 0.038 nmol/L (P < .05) compared with WD mice. The further results suggested that PBMT could promote cholesterol efflux from lipid-loaded primary peritoneal macrophages and inhibit foam cells formation via up-regulating the ATP-binding cassette transporters A1 expression. A contributing mechanism involved in activating the phosphatidylinositol 3-kinases/protein kinase C zeta/specificity protein 1 signalling cascade. Our study outlines that PBMT has a protective role on atherosclerosis by promoting macrophages cholesterol efflux and provides a new strategy for treating atherosclerosis.     

巨噬细胞胆固醇转运相关蛋白研究进展

动脉粥样斑块中泡沫细胞的形成与巨噬细胞胆固醇的转运密切相关,巨噬细胞胆固醇转运是胆固醇逆转运中的一个重要过程,它可清除外周组织过多的胆固醇,对维持细胞内胆固醇稳定、延缓动脉粥样硬化的发生发展有着重要意义.这个过程涉及到许多转运相关蛋白的作用,如三磷酸腺苷结合盒转运体A1/G1、载脂蛋白A-Ⅰ、胆固醇脂转运蛋白、卵磷脂胆固醇酰基转移酶等.本文就巨噬细胞胆固醇转运过程中相关蛋白的作用做一综述,以期为动脉粥样硬化相关疾病的防治研究提供新的思路.     

Purification and characterization of extracellular cholesterol oxidase from <Emphasis Type="Italic">Enterobacter</Emphasis> sp.

The objective of this work is to obtain an abundant source of cholesterol oxidases for industrial and medicinal needs. Thirteen bacterial strains that express high level of inducible extracellular cholesterol oxidase (COX) were isolated from carnivore feces. One of these strains, named COX8-9, belonging to the genus Enterobacter, was found to produce the highest level of cholesterol oxidase. COX from strain COX8-9 was purified from the culture supernatant by ultrafiltration followed with two consecutive Q-Sepharose chromatographies at different pH values, and then by Superdex-75 gel filtration. The purified enzyme was a monomer with a molecular weight of 58 kDa, and exhibited maximum absorption at 280 nm. The K _m value for oxidation of cholesterol by this enzyme was 1.2 × 10⁻⁴ M, with optimum activity at pH 7.0. Enzymatic activity of COX was enhanced 3-fold in the presence of metal ion Cu²⁺, and the enzyme was stable during long-term aqueous storage under various temperatures, indicating its potential as a clinical diagnostic reagent. Preparation and characterization of cholesterol oxidases from the other selected strains are under way. Deping Ye and Jiahong Lei are contributed equally to this work.     

Contribution of GM-CSF on the enhancement of the T cell-stimulating activity of macrophages

Mycobacterium leprae is an intracellular parasitic organism that multiplies in macrophages (MØ). It inhibits the fusion of mycobacterial phagosome with lysosome and induces interleukin (IL)-10 production from macrophages. However, macrophages are heterogenous in various aspects. We examined macrophages that differentiated from monocytes using either recombinant (r) granulocyte-MØ colony-stimulating factor (GM-CSF) (these MØ are named as GM-MØ) or rMØ colony-stimulating factor (M-CSF) (cells named as M-MØ) in terms of the T cell-stimulating activity. Although both macrophages phagocytosed the mycobacteria equally, GM-MØ infected with M. leprae and subsequently treated with IFN-γ- and CD40 ligand (L) stimulated T cells to produce interferon-gamma (IFN-γ), but M-MØ lacked the ability to stimulate T cells. While M-MØ mounted a massive IL-10 production, GM-MØ did not produce the cytokine on infection with M. leprae. M. leprae-infected, IFN-γ- and CD40L-treated GM-MØ expressed a higher level of HLA-DR and CD86 Ags than those of M-MØ, and expressed one of the dominant antigenic molecules of M. leprae, Major Membrane Protein-II on their surface. These results indicate that GM-CSF, but not M-CSF, contributes to the up-regulation of the T cell-stimulating activity of M. leprae-infected macrophages.     

Correlation of bacterial viability with uptake of [14C] acetate into phenolic glycolipid-1 ofMycobacterium leprae within Schwannoma cells

The viability ofMycobacterium leprae, maintained within 33B Schwannoma cells, was estimated in terms of incorporation of [¹⁴C] acetate into its specific phenolic glycolipid-1. This measure of viability was correlated with two other assays,viz., fluorescein diacetate/ethidium bromide staining and mouse footpad growth. Observation of a 2-fold increase in the number of intracellularMycobacterium leprae over an experimental period of 12 days also corroborated this contention. Furthermore, on addition of anti-leprosy drugs to these intracellularMycobacterium leprae there was significant decrease in phenolic glycolipid-1 synthesis indicative of loss of viability of the organisms. This study also established the importance of the host cell for active bacillary metabolism, asMycobacterium leprae maintained in cell-free conditions showed no incorporation into phenolic glycolipid-1. Moreover, compromising the host’s protein synthesis capacity with cycloheximide, also led to reduction in bacillary metabolism. As this system measures the metabolic synthesis of a uniqueMycobacterium leprae component, it would be useful for development and screening of compounds acting against specific bacillary targets.     

Effects of high-density lipoprotein2 on cholesterol transport and acyl-coenzyme A:cholesterol acyltransferase activity in P388D1 macrophages

High-density lipoproteins are the putative vehicles for cholesterol removal from monocyte-derived macrophages, which are an important cell type in all stages of atherosclerosis. The role of HDL₂, an HDL subclass that accounts for most variation in plasma HDL-cholesterol concentration, in cholesterol metabolism in monocyte-derived macrophages is not known. In this study, the dose-dependent effects of HDL₂ on cellular cholesterol mass, efflux, and esterification, and on cellular cholesteryl ester (CE) hydrolysis using the mouse macrophage P388D1 cell line was investigated. HDL₂ at low concentrations (40 μg protein/ml) decreased CE content without affecting cellular free cholesterol content (FC), CE hydrolysis, or cholesterol biosynthesis. In addition, HDL₂ at low concentrations reduced cellular acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and increased FC efflux from macrophages. Thus, HDL₂ has two potential roles in reverse cholesterol transport. In one, HDL₂ is an acceptor of macrophage FC. In the other, more novel role, HDL₂ increases the availability of macrophage FC through the inhibition of ACAT. Elucidation of the mechanism by which HDL₂ inhibits ACAT could identify new therapeutic targets that enhance the transfer of cholesterol from macrophages to the liver.     

Sterol metabolism and oral epithelial cell growth

Summary Previous studies have demonstrated that as the density of cultured oral epithelial cells increases, there is a concomitant increase in phospholipids and cholesterol ester synthesis and a decrease in that of cholesterol and sterol precursors. Other studies have suggested that the effects of exogenous cholesterol sulfate may be similar to growth responses and influence metabolic steps related to cell density. To further examine this possibility, in the present study lipid synthesis was monitored in hamster cheek pouch epithelial cells in cultures established at different cells densities and in the presence of varying amounts of exogenous cholesterol sulfate. Cell [¹⁴C]acetate incorporation into lipids was measured in cultures established at four densities ranging from very subconfluent to very dense (postconfluent) in two media, Dulbecco’s modified Eagle’s medium (DMEM) with 5% fetal bovine serum and KSFM, a non-serum containing keratinocyte medium. Results indicated that the relative proportion of radiolabel incorporated into different lipid classes changed with cell density. In DMEM, the percentage of radiolabel incorporated into total phospholipids and fatty acids increased significantly with increasing cell density whereas percent incorporation into cholesterol, sterol precursors, and cholesterol esters significantly decreased. In KSFM cultures, proportionate phospholipids labeling was significantly increased in more dense cultures whereas cholesterol and cholesterol esters labeling was significantly decreased. In subconfluent and confluent cultures exposed to 10 or 25μM cholesterol sulfate, the relative proportions of phospholipid labeling also increased significantly compared to dimethyl sulfoxide (solvent) controls, whereas sterol precursors, fatty acids, and cholesterol esters labeling was signifcantly decreased. These results indicate that cholesterol sulfate can affect cellular lipid synthesis in a manner similar to that which occurs with increasing cell density, and strengthen the hypothesis that cholesterol sulfate may regulate lipid metabolic pathways related to growth and differentiation.     

Absence of endogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.     

Acyl coenzyme A: cholesterol acyltransferase types 1 and 2: structure and function in atherosclerosis

Two enzymes are responsible for cholesterol ester formation in tissues, acyl coenzyme A:cholesterol acyltransferase types 1 and 2 (ACAT1 and ACAT2). The available evidence suggests different cell locations, membrane orientations, and metabolic functions for each enzyme. ACAT1 and ACAT2 gene disruption experiments in mice have shown complementary results, with ACAT1 being responsible for cholesterol homeostasis in the brain, skin, adrenal, and macrophages. ACAT1 -/- mice have less atherosclerosis than their ACAT1 +/+ counterparts, presumably because of the decreased ACAT activity in the macrophages. By contrast, ACAT2 -/- mice have limited cholesterol absorption in the intestine, and decreased cholesterol ester content in the liver and plasma lipoproteins. Almost no cholesterol esterification was found when liver and intestinal microsomes from ACAT2 -/- mice were assayed. Studies in non-human primates have shown the presence of ACAT1 primarily in the Kupffer cells of the liver, in non-mucosal cell types in the intestine, and in kidney and adrenal cortical cells, whereas ACAT2 is present only in hepatocytes and in intestinal mucosal cells. The membrane topology for ACAT1 and ACAT2 is also apparently different, with ACAT1 having a serine essential for activity on the cytoplasmic side of the endoplasmic reticulum membrane, whereas the analogous serine is present on the lumenal side of the endoplasmic reticulum for ACAT2. Taken together, the data suggest that cholesterol ester formation by ACAT1 supports separate functions compared with cholesterol esterification by ACAT2. The latter enzyme appears to be responsible for cholesterol ester formation and secretion in lipoproteins, whereas ACAT1 appears to function to maintain appropriate cholesterol availability in cell membranes.     

Carboxyl ester lipase expression in macrophages increases cholesteryl ester accumulation and promotes atherosclerosis

Carboxyl ester lipase (CEL, also called cholesterol esterase or bile salt-dependent lipase) is a lipolytic enzyme capable of hydrolyzing cholesteryl esters, triacylglycerols, and phospholipids in a trihydroxy bile salt-dependent manner but hydrolyzes ceramides and lysophospholipids via bile salt-independent mechanisms. Although CEL is synthesized predominantly in the pancreas, a low level of CEL expression was reported in human macrophages. This study used transgenic mice with macrophage CEL expression at levels comparable with that observed in human macrophages to explore the functional role and physiological significance of macrophage CEL expression. Peritoneal macrophages from CEL transgenic mice displayed a 4-fold increase in [(3)H]oleate incorporation into cholesteryl [(3)H]oleate compared with CEL-negative macrophages when the cells were incubated under basal conditions in vitro. When challenged with acetylated low density lipoprotein, cholesteryl ester accumulation was 2.5-fold higher in macrophages expressing the CEL transgene. The differences in cholesteryl ester accumulation were attributed to the lower levels of ceramide and lysophosphatidylcholine in CEL-expressing cells than in CEL-negative cells. CEL transgenic mice bred to an atherosclerosis susceptible apoE(-/-) background displayed an approximate 4-fold higher atherosclerotic lesion area than apoE(-/-) mice without the CEL transgene when both were fed a high fat/cholesterol diet. Plasma level of the atherogenic lysophosphatidylcholine was lower in the CEL transgenic mice, but plasma cholesterol level and lipoprotein profile were similar between the two groups. These studies documented that CEL expression in macrophages is pro-atherogenic and that the mechanism is because of its hydrolysis of ceramide and lysophosphatidylcholine in promoting cholesterol esterification and decreasing cholesterol efflux.     

Macrophage‐mediated cholesterol handling in atherosclerosis

Formation of foam cells is a hallmark at the initial stages of atherosclerosis. Monocytes attracted by pro‐inflammatory stimuli attach to the inflamed vascular endothelium and penetrate to the arterial intima where they differentiate to macrophages. Intimal macrophages phagocytize oxidized low‐density lipoproteins (oxLDL). Several scavenger receptors (SR), including CD36, SR‐A1 and lectin‐like oxLDL receptor‐1 (LOX‐1), mediate oxLDL uptake. In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In the endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase‐1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out‐flowed from macrophages by cholesterol ATP‐binding cassette (ABC) transporters ABCA1 and ABCG1 and SR‐BI. In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells.     

Clofazimine Modulates the Expression of Lipid Metabolism Proteins in Mycobacterium leprae-Infected Macrophages

Mycobacterium leprae (M. leprae) lives and replicates within macrophages in a foamy, lipid-laden phagosome. The lipids provide essential nutrition for the mycobacteria, and M. leprae infection modulates expression of important host proteins related to lipid metabolism. Thus, M. leprae infection increases the expression of adipophilin/adipose differentiation-related protein (ADRP) and decreases hormone-sensitive lipase (HSL), facilitating the accumulation and maintenance of lipid-rich environments suitable for the intracellular survival of M. leprae. HSL levels are not detectable in skin smear specimens taken from leprosy patients, but re-appear shortly after multidrug therapy (MDT). This study examined the effect of MDT components on host lipid metabolism in vitro, and the outcome of rifampicin, dapsone and clofazimine treatment on ADRP and HSL expression in THP-1 cells. Clofazimine attenuated the mRNA and protein levels of ADRP in M. leprae-infected cells, while those of HSL were increased. Rifampicin and dapsone did not show any significant effects on ADRP and HSL expression levels. A transient increase of interferon (IFN)-β and IFN-γ mRNA was also observed in cells infected with M. leprae and treated with clofazimine. Lipid droplets accumulated by M. leprae-infection were significantly decreased 48 h after clofazimine treatment. Such effects were not evident in cells without M. leprae infection. In clinical samples, ADRP expression was decreased and HSL expression was increased after treatment. These results suggest that clofazimine modulates lipid metabolism in M. leprae-infected macrophages by modulating the expression of ADRP and HSL. It also induces IFN production in M. leprae-infected cells. The resultant decrease in lipid accumulation, increase in lipolysis, and activation of innate immunity may be some of the key actions of clofazimine.     

Tripterygium regelii decreases the biosynthesis of triacylglycerol and cholesterol in HepG2 cells

In the course of screening to find a plant material decreasing the activity of triacylglycerol and cholesterol, we identified Tripterygium regelii (TR). The methanol extract of TR leaves (TR-LM) was shown to reduce the intracellular lipid contents consisting of triacylglycerol (TG) and cholesterol in HepG2 cells. TR-LM also downregulated the mRNA and protein expression of the lipogenic genes such as SREBP-1 and its target enzymes. Consequently, TR-LM reduced the TG biosynthesis in HepG2 cells. In addition, TR-LM decreased SREBP2 and its target enzyme HMG-CoA reductase, which is involved in cholesterol synthesis. In this study, we evaluated that TR-LM attenuated cellular lipid contents through the suppression of de novo TG and cholesterol biosynthesis in HepG2 cells. All these taken together, TR-LM could be beneficial in regulating lipid metabolism and useful preventing the hyperlipidemia and its complications, in that liver is a crucial tissue for the secretion of serum lipids.     

Abrogation of neutral cholesterol ester hydrolytic activity causes adrenal enlargement

We have previously demonstrated that neutral cholesterol ester hydrolase 1 (Nceh1) regulates foam cell formation and atherogenesis through the catalytic activity of cholesterol ester hydrolysis, and that Nceh1 and hormone-sensitive lipase (Lipe) are responsible for the majority of neutral cholesterol ester hydrolase activity in macrophages. There are several cholesterol ester-metabolizing tissues and cells other than macrophages, among which adrenocortical cells are also known to utilize the intracellular cholesterol for steroidogenesis. It has been believed that the mobilization of intracellular cholesterol ester in adrenal glands was facilitated solely by Lipe. We herein demonstrate that Nceh1 is also involved in cholesterol ester hydrolysis in adrenal glands. While Lipe deficiency remarkably reduced the neutral cholesterol ester hydrolase activity in adrenal glands as previously reported, additional inactivation of Nceh1 gene completely abrogated the activity. Adrenal glands were enlarged in proportion to the degree of reduced neutral cholesterol ester hydrolase activity, and the enlargement of adrenal glands and the accumulation of cholesterol esters were most pronounced in the Nceh1/Lipe double-deficient mice. Thus Nceh1 is involved in the adrenal cholesterol metabolism, and the cholesterol ester hydrolytic activity in adrenal glands is associated with the organ enlargement.     

Inhibition of apoptosis, activation of NKT cell and upregulation of CD40 and CD40L mediated by M. leprae antigen(s) combined with Murabutide and Trat peptide in leprosy patients

Protective immunity against intracellular pathogen Mycobacterium leprae is dependent on the activation of T cells. Repeated stimulation of T cells by M. leprae antigens MLCwA (M. leprae total cell wall antigen) and ManLAM (mannose-capped lipoarabinomannan), may lead to apoptosis in leprosy patients. In the present study, inhibition of the Fas-induced apoptosis of peripheral blood mononuclear cells of leprosy patients was investigated using above M. leprae antigen(s), in combination with immunomodulators murabutide (MB) and a Trat peptide in particulate form (liposome). Incubation of the cells with antigen containing the two immunomodulators in particulate form (liposomes) led to decrease in percentage of propidium iodide positive cells and T cells expressing Fas–FasL as well as decreased caspase-8/-3 activities in lepromatous patients, thereby inhibiting apoptosis, while converse was true upon stimulation with soluble antigen. Concurrently, there was an upregulation of antiapoptotic protein Bcl-x_L in lepromatous patients, leading to the inhibition of apoptosis. It was also observed that same formulation upregulated the expression of CD40 on B cells and monocytes-macrophages and CD40L on T cells of lepromatous leprosy patients. The same liposomal formulation significantly increased the expression of CD1b and CD1d on monocytes-macrophages as well as percentage of NKT cells secreting IFN-γ in lepromatous leprosy patients. Thus, the liposomal formulation of antigen with the immunomodulators in vitro promoted the activation of CD40:CD40L pathways and NKT cell function involved in providing cell-mediated immunity to these patients. The same formulation also caused reversal of T cell anergy by inhibiting apoptosis through decreased expression of death receptors (Fas–FasL) and caspase activities (3 and 8) and increased expression of antiapoptotic protein Bcl-x_L in these patients.