IL-12 and IL-23 modulate plasticity of FoxP3+ regulatory T cells in human Leprosy.

Leprosy is a bacterial disease caused by M. leprae. Its clinical spectrum reflects the host's immune response to the M. leprae and provide an ideal model to investigate the host pathogen interaction and immunological dysregulation. Tregs are high in leprosy patients and responsible for immune suppression of the host by producing IL-10 and TGF-ß cytokines. In leprosy, plasticity of Tregs remain unstudied. This is the first study describing the conversion of Tregs into Th1-like and Th17-like cells using in vitro cytokine therapy in leprosy patients. Peripheral blood mononuclear cells from leprosy patients were isolated and stimulated with M. leprae antigen (MLCwA), rIL-12 and rIL-23 for 48h. Expression of FoxP3 in CD4+CD25+ Tregs, intracellular cytokines IFN-γ, TGF-ß, IL-10 and IL-17 in Tregs cells were evaluated by flow cytometry (FACS) after stimulation. rIL-12 treatment increases the levels of pStat4 in Tregs and IFN-γ production. In the presence of rIL-23, pStat3+ and IL-17A+ cells increase. rIL-12 and r-IL-23 treatment downregulated the FoxP3 expression, IL-10 and TGF-ß production by Tregs and enhances the expression of co-stimulatory molecules (CD80, CD86). In conclusion rIL-12 converts Tregs into IFN-γ producing cells through STAT-4 signaling while rIL-23 converts Tregs into IL-17 producing cells through STAT-3 signaling in leprosy patients. This study may helpful to provide a new avenue to overcome the immunosuprression in leprosy patients using in vitro cytokine.

Increased IL-35 producing Tregs and CD19+IL-35+ cells are associated with disease progression in leprosy patients.

BACKGROUND: The clinical forms of leprosy consist of a spectrum that reflects the host's immune response to the M. leprae; it provides an ideal model to study the host pathogen interaction and immunological dysregulation in humans. IL-10 and TGF-ß producing Tregs are high in leprosy patients and responsible for immune suppression and M. leprae specific T cells anergy. In leprosy, involvement of IL-35 producing Tregs and Bregs remain unstudied. OBJECTIVE: To study the role of IL-35 producing Tregs and Bregs in the human leprosy. METHODS: Peripheral blood mononuclear cells from leprosy patients were isolated and stimulated with M. leprae antigen (MLCwA) for 48h. Intracellular cytokine IL-35 was evaluated in CD4+CD25+ Tregs, CD19+ cells by FACS. Expression of PD-1 on CD4+CD25+ Tregs, CD19+ cells and its ligand (PD-L1) on B cells, CD11c cells were evaluated by flow cytometry (FACS). Serum IL-35 level was estimated by ELISA. RESULTS: The frequency of IL-35 producing Tregs and Bregs cells were found to be high in leprosy patients (p<0.0001) as compared to healthy controls. These cells produced suppressive cytokine IL-35 which showed positive correlation with bacteriological index (BI) and TGF-ß producing Tregs, indicating its suppressive nature. We found higher expression of PD-1 on Tregs, B cell and its ligand (PD-L1) on antigen presenting cells in leprosy patients. CONCLUSION: This study point out a shift in our understanding of the immunological features that mediate and regulate the immune suppression and the disease progression in leprosy patients with a new paradigm (IL-35 producing Tregs and Bregs) that is beyond TGF-ß and IL-10 producing Treg cells.

Association of TNF-α-(308(GG)), IL-10(-819(TT)), IL-10(-1082(GG)) and IL-1R1(+1970(CC)) genotypes with the susceptibility and progression of leprosy in North Indian population.

Leprosy is an infectious disease caused by M. leprae. We analyzed 48 cytokine polymorphisms in 13 (pro as well as anti-inflammatory) cytokine genes using PCR-SSP assay in 102 leprosy patients and 120 healthy controls with intent to find out a link between cytokine polymorphisms and disease susceptibility. TNF-α (-308) GG, IL-10 (-819) TT, IL-10 (-1082) GG and IL1R (+1970) CC genotypes are found to be predominant (p=0.01, p=0.02, p=0.0001 and p=0.001, respectively) in both tuberculoid as well as lepromatous leprosy patients. This observation suggests these genotypes as play the central role(s) in the progression of disease. CBA assay demonstrates the varied serum concentration of these cytokines with respect to their genotypes. The above genotypes appeared as high producer genotypes in our study. Even in presence of high produce genotypes, TNF-α level are found to be affected/masked by the presence of IL-10 in leprosy patients. Expressional masking of TNF-α is associated with the expression of IL-10 in these patients. This is one the negative impact of SNP-SNP interaction in leprosy patients. Therefore, we can conclude that cytokine gene polymorphisms determine the predisposition to the leprosy progression.

FoxP3 provides competitive fitness to CD4⁺CD25⁺ T cells in leprosy patients via transcriptional regulation.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. FoxP3 have been shown to have important implications in various diseases. The present study describes the mechanism of action of FoxP3 in CD4⁺CD25⁺ T cells derived from leprosy patients. Increased molecular interactions of FoxP3 with histone deacetylases 7/9 in the nucleus of CD4⁺CD25⁺ T cells derived from borderline lepromatous leprosy/lepromatous leprosy (BL/LL) patients were found to be responsible for FoxP3-driven immune suppression activities during the progression of leprosy. Further, downregulation of CTLA-4 and CD25 genes in siFoxP3-treated PBMCs derived from BL/LL patients elucidated the transcription-activating nature of FoxP3. This observation was supported by direct binding of FoxP3 to the promoter region of the CTLA-4 and CD25 genes, and FoxP3's molecular interaction with histone acetyl transferases. The study also revealed that the increased expression of miR155 in CD4⁺CD25⁺ cells from BL/LL governs the competitive fitness of these cells. Again, reduced Annexin V & propidium iodide staining and Nur77 expression, and concomitantly increased Ki-67 positivity suggested that CD4⁺CD25⁺ cells derived from BL/LL patients are more competitively fit than those from borderline tuberculoid leprosy/tuberculoid leprosy and healthy controls. Taken together, the study shows the orchestration of FoxP3 leading to competitive fitness of Treg cells in leprosy.

CD4+CD25+ T regs with acetylated FoxP3 are associated with immune suppression in human leprosy.

Leprosy is a chronic human disease that results from infection of Mycobacterium leprae. T reg cells have been shown to have important implications in various diseases. However, in leprosy, it is still unclear whether T regs can mediate immune suppression during progression of the disease. In the present study, we have proposed the putative mechanism leading to high proportion of T reg cells and investigated its significance in human leprosy. High levels of TGF-ß followed by adaptation of FoxP3(+) naive and memory (CD4(+)CD45RA(+)/RO(+)) T cells were observed as the principal underlying factors leading to higher generation of T reg cells during disease progression. Furthermore, TGF-ß was found to be associated with increased phosphorylation-mediated-nuclear-import of SMAD3 and NFAT towards BL/LL pole to facilitate FoxP3 expression in these cells, the same as justified after using nuclear inhibitors of SMAD3 (SIS3) and NFAT (cyclosporin A) in CD4(+)CD25(+) cells in the presence of TGF-ß and IL-2. Interestingly, low ubiquitination of FoxP3 in T reg cells of BL/LL patients was revealed to be a major driving force in conferring stability to FoxP3 which in turn is linked to suppressive potential of T regs. The present study has also pinpointed the presence of CD4(+)CD25(+)IL-10(+) sub class of T regs (Tr1) in leprosy.

IL-10 production from dendritic cells is associated with DC SIGN in human leprosy.

The defective antigen presenting ability of antigen presenting cells (APCs) modulates host cytokines and co-stimulatory signals that may lead to severity of leprosy. In the present study, we sought to evaluate the phenotypic features of APCs along with whether DC SIGN (DC-specific intercellular adhesion molecule-grabbing nonintegrin) influences IL-10 production while moving from tuberculoid (BT/TT) to lepromatous (BL/LL) pole in leprosy pathogenesis. The study revealed an increased expression of DC SIGN on CD11c⁺ cells from BL/LL patients and an impaired form of CD83 (∼50 kDa). However, the cells after treatment with GM-CSF+IL-4+ManLAM showed an increased expression of similar form of CD83 on DCs. Upon treatment with ManLAM, DCs were found to show increased nuclear presence of NF-κB, thus leading to higher IL-10 production. High IL-10 production from ManLAM treated PBMCs further suggested the role of DC SIGN in subverting the DCs function towards BL/LL pole of leprosy. Anti-DC SIGN treatment resulting in restricted nuclear ingression of NF-κB as well as its acetylation along with enhanced T cell proliferation validated our findings. In conclusion, Mycobacterium leprae component triggers DC SIGN on DCs to induce production of IL-10 by modulating intracellular signalling pathway at the level of transcription factor NF-κB towards BL/LL pole of disease.

Th3 immune responses in the progression of leprosy via molecular cross-talks of TGF-ß, CTLA-4 and Cbl-b.

Leprosy is a chronic human disease; primarily affecting skin, peripheral nerves, eyes, testis etc. Comprehensive-expressional-profiling of Th1-Th2-Th3 associated markers (84 genes) using qRT-PCR array, negated the previously prevailing notion, Th2 bias towards multibacillary stage of leprosy. High production TGF-ß further supported the dearth of any immune response(s) in leprosy progression. Over expression of Cbl-b, could emerge as plausible reason for contributing T cell hyporesponsiveness, possibly by degradation of T cells signaling molecules. Anti-TGF-ß treatments further confirm the TGF-ß-dependent-Cbl-b overexpression in multibacillary patients. Diminished Cbl-b expression in CTLA-4 knockout studies using siRNA, provided other evidence towards T cell hyporesponsiveness. Further, high T cell proliferation and IL-2 production in PBMC cultures treated with anti-TGF-ß and siRNA offers here a strategy to revert T cell hyporesponsiveness by downregulating Cbl-b expression in leprosy. Thus, this study negates Th2 bias and substantiates molecular cross-talk amongst TGF-ß-CTLA-4-Cbl-b eventually leads to M. leprae persistence.

Disruption of HLA-DR raft, deregulations of Lck-ZAP-70-Cbl-b cross-talk and miR181a towards T cell hyporesponsiveness in leprosy.

Leprosy, a chronic human disease, results from infection of Mycobacterium leprae. Defective CMI and T cell hyporesponsiveness are the major hallmark of M. leprae pathogenesis. The present study demonstrates immunological-deregulations that eventually lead to T cell anergy/hyporesponsiveness in M. lepare infection. We firstly, evaluated the membrane fluidity and antigen-presenting-lipid-raft (HLA-DR) on macrophages of leprosy patients using fluorescence anisotropy and confocal microscopy, respectively. Increased membrane fluidity and raft-out localizations of over-expressed HLA-DR towards BL/LL pole are pinpointed as major defects, may be leading to defective antigen presentation in leprosy. Furthermore, altered expression and localization of Lck, ZAP-70, etc. and their deregulated cross talks with negative regulators (CD45, Cbl-b and SHP2) turned out to be the major putative reason(s) leading to T cell hyporesponsiveness in leprosy. Deregulations of Lck-ZAP-70 cross-talk in T cells were found to be associated with cholesterol-dependent-dismantling of HLA-DR rafts in macrophages in leprosy progression. Increased molecular interactions between Cbl-b and Lck/ZAP-70 and their subsequent degradation via ubiquitinization pathway, as result of high expression of Cbl-b, were turned out to be one of the principal underlying reason leading to T cell anergy in leprosy patients. Interestingly, overexpression of SHP2 due to gradual losses of miR181a and subsequent dephosphorylation of imperative T cell signaling molecules were emerged out as another important reason associated with prevailing T cell hyporesponsiveness during leprosy progression. Thus, this study for the first time pinpointed overexpression of Cbl-b and expressional losses of miR-181 as important hallmarks of progression of leprosy.

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-xL 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-gamma 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-xL in these patients.

Liposomal delivery of Mycobacterium leprae antigen(s) with murabutide and Trat peptide inhibits Fas-mediated apoptosis of peripheral blood mononuclear cells derived from 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 particulate mode of antigen presentation led to both decreased percentage of propidium iodide (PI) positive cells and T cells expressing Fas-FasL, as well as decreased caspase-8/-3 activities in the lepromatous patients, thereby inhibiting apoptosis, while converse was true with stimulation with soluble antigen. Concurrently, there was an upregulation of antiapoptotic protein Bcl-X(L) in the lepromatous patients, thereby inhibiting apoptosis. Thus, the liposomal formulation of antigen promoted proliferation of anergized T cell by inhibiting apoptosis through decreased expression of death receptors and caspase activities and increased expression of anti-apoptotic protein Bcl-X(L) in these patients.

Alterations in T cell signal transduction by M. leprae antigens is associated with downregulation of second messengers PKC, calcium, calcineurin, MAPK and various transcription factors in leprosy patients.

Mycobacterium leprae, the causative agent of leprosy, challenges host defense mechanism by impairing the signal transduction of T cells which leads to downregulation of T cell proliferation, mainly as a consequence of interference with IL-2 production. In this study we sought to identify how soluble forms of M. leprae antigen(s) or particulate (liposome) delivery of the same antigens with two immunomodulators Murabutide and T cell peptide of Trat protein influence the transcription of IL-2 gene in anergic T cells of lepromatous patients. It was demonstrated that MLCwA/ManLAM stimulated cells of BL/LL patients showed defects in both jun-NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activities there by resulting in decreased AP-1 activity. Additionally these cells showed reduced calcium levels, PKC activity and calcineurin (CN) activity. This led to impaired nuclear translocation of NFkappaB and NFAT in these patients. In contrast, when same M. leprae antigen(s) were incorporated with the two immunomodulators in liposomal form, increased transcription of IL-2 gene was observed especially in BL/LL patients which appears to be due to, at least in part, to increased expression of AP-1 Fos and Jun family members, NFkappaB and NFAT1 proteins. The increased expression of these transcription factors correlated with increased ERK/JNK, PKC and CN activities in these patients. Since activation of ERK/JNK/PKC kinases and CN phosphatase are required for stimulation of IL-2 transcription, these data provide a molecular explanation for the block in IL-2 production by M. leprae antigens. Thus the above study revealed suppression of all the three distinct biochemical pathways, viz. Ca-CN-NFAT pathway, PKC-NF-kappaB pathway, and MAPK-AP-1 pathway by M. leprae antigen(s) in anergized T cells of lepromatous patients which were activated by liposomal delivery of M. leprae antigens containing the two immunomodulators leading to optimal induction of IL-2 gene expression, which was required for the activation, and proliferation of T cells in lepromatous patients.

Expression of costimulatory molecules (CD80, CD86, CD28, CD152), accessory molecules (TCR alphabeta, TCR gammadelta) and T cell lineage molecules (CD4+, CD8+) in PBMC of leprosy patients using Mycobacterium leprae antigen (MLCWA) with murabutide and T cell peptide of Trat protein.

In leprosy, cell-mediated immunity (CMI) is more significant than humoral response to eliminate intracellular pathogen. T cell defect is a common feature in lepromatous leprosy (LL) patients as compared to tuberculoid type (TT) patients. For efficient initiation of CD4+, T cell response requires T cell receptor (TCR) activation and costimulation provided by molecules on antigen-presenting cells (APC) and their counter receptors on T cells. In our previous study, the defective T cell function in LL patients was restored to a proliferating state with the release of TH1 type cytokines using mycobacterial antigen(s) with two immunomodulators (Murabutide (MDP-BE) and T cell epitope of Trat protein of Escherichia coli) by presenting the antigen in particulate form in vitro to PBMC derived from leprosy patients. This observation prompted us to study the expression of the costimulatory molecules (CD80, CD86, CD28, CD152), other accessory molecules (TCR alphabeta/gammadelta) and T cell lineage molecules (CD4+ and CD8+) during constitutive and activated state of peripheral blood mononuclear cells (PBMC) derived from normal and leprosy individuals using different formulations of Mycobacterium leprae total cell wall antigen (MLCWA), Trat and MDP-BE using flow cytometric analysis. An increased surface expression of CD80, CD86 and CD28 but decreased CD152 expression was observed when PBMC of normal, BT/TT (tuberculoid) and BL/LL (lepromatous) patients were stimulated in vitro with MLCWA+MDP-BE+Trat peptide using liposomal mode of antigen delivery, while opposite results were obtained with the antigen alone. Antibody inhibition study using antihuman CD80 or CD86 completely abolished the T cell lymphoproliferation, thereby reconfirming the importance of these costimulatory molecules during T cell activation/differentiation. Though the liposome-entrapped antigen formulation has no effect on expression of alphabeta/gammadelta T cell receptor, the constitutive levels of TCR gammadelta were high in lepromatous patients. Thus, TCR bearing gammadelta appears to have a negligible regulatory role in peripheral blood of leprosy patients. The percentage of cells positive for CD4+ are increased in inducible state in all the three groups, while CD8+-positive cells were decreased in LL patients, thereby reconfirming the fact that priming of CD4+ cells are necessary for producing final effector functions. Lastly, intracellular cytokine staining experiment indicated that CD4+ cells are the major producers of IFN-gamma but not NK cells. The study highlights the reversal of T cell anergy especially in lepromatous patients through the modulation of costimulatory molecule expression under the influence of Th1 cytokines, i.e., IL-2 and IFNgamma.

Reversal of T cell anergy in leprosy patients: in vitro presentation with Mycobacterium leprae antigens using murabutide and Trat peptide in liposomal delivery.

Mycobacterium leprae, the causative agent of leprosy resides and multiplies within the host monocytes and macrophages, thereby evading host immune system. Cell-mediated immune response (CMI) plays a vital role as evidenced from the high CMI in BT/TT (borderline and tuberculoid) patients and conversely low in BL/LL (borderline and lepromatous) patients. In the present study, an attempt was made to immunomodulate the anergized T cells of lepromatous leprosy patients by presenting the mycobacterial antigen in combination with T cell adjuvant, murabutide (active analog of muramyl' dipeptide, MDP-BE) and a Trat peptide (T cell epitope of Integral membrane protein (Trat) from Escherichia coli) in particulate form (liposomes) or soluble form (media). PBMNC of normal, BT/TT and BL/LL were stimulated in vitro with five mycobacterial antigens (Ag) in the following formulations, Ag, Ag+murabutide, Ag+murabutide+Trat peptide either in liposomes or in medium. All the five antigen(s) when delivered in liposomes containing murabutide and Trat peptide showed a very high lymphoproliferative response (p<0.001) in all the three groups. IFN-gamma and IL-2 were significantly (p<0.001) high in these culture supernatants compared to IL-10 and IL-4 confirming a shift from CD4+Th2 to Th1 response in leprosy patients with particulate mode of antigen presentation. Interestingly, PBMNC derived from lepromatous patients also showed consistent T cell proliferation with all the formulations. Further, the mechanism of liposomal processing of antigens was studied using different inhibitors that interfere at different stages of antigen presentation. Results indicate that this study may pave way for an immunotherapeutic approach for reverting the anergic T cells of lepromatous patients to proliferating T cells with the release of Th1 cytokines thereby restoring the CMI response in these patients.

New age adjuvants and delivery systems for subunit vaccines.

The dramatic advancements in the field of vaccinology has led to the formulation of chemically well defined vaccines composed of synthetic peptides and recombinant proteins derived from the immunologically dominant regions of the pathogens. Though these subunit vaccines are safer compared to the traditional vaccines they are known to be poorly immunogenic. This necessitates the use of adjuvants to enhance the immunogenicity of these vaccine formulations. The most common adjuvant for human use is alum. Research in the past has focused on the development of systemic immunity using conventional immunization protocols. In the present are, the emphasis is on the development and formulation of alternative adjuvants and delivery systems in generating systemic as well as mucosal immunity. This review mainly focuses on a variety of adjuvants (particulate as well as non-particulate) used with protective antigens of HIV, malaria, plague, leprosy using modified delivery vehicles. The experience of our laboratory and other researchers in this field clearly proves that these new age adjuvants and delivery systems undoubtedly generate enhanced immune response-both humoral and cell mediated. The choice of antigens, the nature of adjuvant used and the mode of delivery employed have a profound effect on the type of immune response generated. Besides the quantity, the quality of the antibodies generated also play a vital role in protection against these diseases. Some of the adjuvants and delivery systems used promoted high titre and affinity antibodies, which were shown to be cytophilic in nature, an important criteria in providing protection to the host. Thus the studies on these adjuvants/delivery systems with respect to various infectious diseases indicate their active role in efficient modulation of immune response along with safety and permissibility.

Quantitative assessment of tuftsin receptor expression and second messenger during in vitro differentiation of peripheral blood derived monocytes of leprosy patients.

Tuftsin, a tetrapeptide (Thr-Lys-Pro-Arg) is known to potentiate the immunogenic activity of antigen-fed macrophages. The present study describes the mechanism of action of tuftsin in leprosy patients throughout the spectrum of the disease in vitro as a function of culture age in terms of (A) involvement of second messengers cAMP, cGMP and [Ca2+]i and (B) number of tuftsin binding sites/and their relative affinities on the monocytes/macrophages. There is apparently no direct involvement of either cAMP or cGMP while comparing the stimulated and unstimulated cultures during in vitro differentiation of monocytes (days 1, 3 and 7) or with the spectrum of the disease. Inhibition of superoxide anion release either by verapamil or with Quin 2 clearly demonstrated the involvement of [Ca2+]i as a second messenger during activation of monocytes/macrophages with tuftsin. Scatchard analysis of radiolabelled tuftsin binding data showed only one type of tuftsin receptor (low affinity) on BL/ LL monocytes/macrophages and normal and BT/TT cultures showed a gradual change in receptor number and affinities (low to high) with the maturation of monocytes to macrophages in contrast to BL/LL groups which displayed significantly less number of receptors. This study elicits a model which depicts that the biological responses/metabolic functions of early monocytes of normal and BT/TT gradually increase with the age of the culture till day 3 and tapers off thereafter in the older (day 7) cultures, whereas the monocytes/macrophages of BL/LL group are metabolically active only on day 1. The present study thereby implies that the clearance of leprosy bacilli from lepromatous leprosy lesions as a consequence of local or systemic immunotherapy (in the present study, the macrophage modulation by tuftsin) depends on the influx of new competent macrophages, rather than the local activation of resident lepromatous macrophages.

Release of reactive nitrogen intermediates from the peripheral blood-derived monocytes/macrophages of leprosy patients stimulated in vitro by tuftsin.

The production of reactive nitrogen intermediates (RNI) by macrophages is critical to host defence, particularly for exerting the bactericidal and tumoricidal properties. Nitric oxide (NO) were measured in the peripheral blood-derived monocytes/macrophages of normal and leprosy patients (BT/TT and BL/LL) in the presence and absence of 'tuftsin' as a function of in vitro culture age (on 1, 3, 7 days). Macrophages from both groups of leprosy patients were able to produce NO during the unstimulated state but only BL/LL macrophages could be activated by tuftsin to produce significantly high levels of NO. This increase was highest on day 1, then gradually decreased with in vitro culture age. Surprisingly, tuftsin was unable to enhance the NO production in normal macrophages above the basal level. Further, normal and BT/TT macrophages had only Cu-Zn derived superoxide dismutase (SOD) activity whereas BL/LL cultures has Cu-Zn and Mn derived SOD activity. These studies indicate that in BL/LL cultures: a, apart from tuftsin, some additional signal is required to activate nitric oxide synthase (NOS) gene for NO production; and b, Mn-SOD produced by Mycobacterium leprae is playing a defensive role against toxic-free radicals. The final outcome of this mechanism is the survival of M. leprae inside the macrophages.

A comparative study of the redox-cycling of a quinone (rifamycin S) and a quinonimine (rifabutin) antibiotic by rat liver microsomes.

Rifamycin S and rifabutin are clinical drugs used to treat tuberculosis and leprosy. The formation of reactive oxygen species during the redox-cycling of rifamycin S (quinone) and rifabutin (quinonimine) was evaluated. The semiquinone (or semiquinonimine) and hydroquinone (or hydroquinonimine) formed during the reduction of the parent molecules by microsomal electron transfer in the presence of nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) or nicotinamide-adenine dinucleotide, reduced (NADH) reoxidizes in air to generate superoxide radical and hydrogen peroxide. In the presence of added iron, hydroxyl radicals, formed by the Fenton reaction, were detected using 5,5'-dimethyl-1-pyroline-N-oxide as the spin-trap. Rifamycin S, a quinone, redox cycles more efficiently than rifabutin, a quinonimine, as approximately five times the concentration of hydroxyl radical adduct of 5,5'-dimethyl-1-pyroline-N-oxide (DMPO) was detected, when compared with rifabutin. The NADPH-dependent microsomal production of hydroxyl radical in the presence of rifamycin S was somewhat higher than the NADH-rifamycin S system with most iron chelators. However, with rifabutin, NADH-dependent microsomal production of hydroxyl radical was higher than that found with the NADPH-rifabutin system. An exception was the iron chelator, diethylene-triamine-pentacetic acid (DTPA), in which NADPH-dependent rates exceeded the rates with NADH with both antibiotics. Rat liver sub-mitochondrial particles also generated hydroxyl radical in the presence of NADH and either rifamycin S or rifabutin. The electron transport chain inhibitors such as rotenone and antimycin A enhanced the signal intensity of DMPO-OH, suggesting NADH dehydrogenase (complex I) as the major component involved in the reduction of rifamycin S. Rifamycin S was shown to be readily reduced to rifamycin SV, the corresponding hydroquinone by Fe(II); under similar conditions Fe(II) did not reduce rifabutin. Using optical spectroscopy, we determined that rifamycin S forms a complex with Fe(II). The stoichiometry of the complex was Fe(rifamycin S)3 in phosphate buffer at pH 7.4. Rifabutin did not form a detectable complex with Fe(II). The redox cycling of rifamycin S and rifabutin did not cause microsomal lipid peroxidation. In fact, the Fe:ATP induced lipid peroxidation was completely inhibited by these two molecules. These results indicate that rifamycin S and rifabutin can interact with rat liver microsomes to undergo redox-cycling, with the subsequent production of hydroxyl radicals when iron complexes are present. Compared to NADPH, NADH is almost as effective (rifamycin S) or even more effective (rifabutin) in promoting these interactions. These interactions may play a role in the hepatotoxicity associated with the use of these antibiotics.

Modulation of peripheral blood derived monocytes/macrophages from leprosy patients using 'tuftsin' for production of reactive oxygen intermediates.

Phagocytic cells respond to a variety of membrane stimulants by producing reactive oxygen intermediates (ROI), i.e. O2-, H2O2 and OH.metabolites. Plasma membrane activation is associated with superoxide generating NADPH oxidase, thereby causing the production of these toxic species. Stimulation of phagocytic cells also results in activation of purine catabolism, which directs the metabolic flux through xanthine oxidase to produce the superoxide anion. We previously observed that BL/LL macrophages (M phi) exhibited a premature inability to undergo tuftsin stimulated phagocytosis and microbicidal activity. The present study was undertaken to measure ROI levels in the absence and presence of 'tuftsin' pulsing as a function of in vitro culture age and also correlated these levels with adenosine deaminase (ADA) activity. The latter is known to be a contributor of O2- generation and is also involved in the maturation of the monocyte/macrophage system. The behaviour of normal and tuberculoid monocytes/macrophages were more or less the same, either in the presence or absence of tuftsin, i.e. they showed a progressive increase in ROI production until day 3, then tapered off in older cultures by day 7. In contrast, after day 1, the lepromatous macrophages were unable to undergo tuftsin mediated stimulation for the production of ROI and ADA activity. These findings indicate a defective M phi function in lepromatous patients towards tuftsin pulsing, thereby supporting our earlier observations. Thus BL/LL M phi behaved as if they were aged after 1 day of in vitro culture, which may account for an inability to handle Mycobacterium leprae for efficient killing.

Enzyme immunoassay of phagocytosis stimulating tetrapeptide "tuftsin" in normal and leprosy sera.

The serum concentrations of the phagocytosis stimulating the tetrapeptide, tuftsin, were determined by competitive enzyme immunoassay in borderline tuberculoid/tuberculoid (BT/TT, 16 cases), borderline lepromatous/lepromatous (BL/LL, 16 cases), and in healthy controls (20 cases). Using checkerboard titration, 10 ng/well of diphtheria toxoid-p-aminophenylacetyl tuftsin (DTPT) conjugate when incubated with tuftsin antisera at 1:15,000 dilution with a preincubation time of 60 min with the competitor (tuftsin) followed by a further 60-min incubation onto the DTPT-coated wells gave consistent results with a sensitivity of 5 ng/well tuftsin. The mean serum tuftsin concentration was significantly lower in BL/LL patients (134.42 +/- 48.7 ng/ml, p less than 0.01) than in healthy controls (262.86 +/- 59.8 ng/ml), while BT/TT sera (210.94 +/- 75.5 ng/ml) showed slightly decreased levels than did normals, which was not statistically significant. The mean serum IgG levels in BL/LL and BT/TT patients (37.26 +/- 10.99 mg/ml; 28.08 +/- 6.57 mg/ml, respectively) showed significantly (p less than 0.001) higher concentrations than did healthy controls (12.3 +/- 3.6 mg/ml). These observations on the serum concentrations of tuftsin and IgG in leprosy individuals suggest that there is splenic dysfunction in BL/LL patients in terms of the processing of leukokinin to release the free, active molecule tuftsin.

Modulation of human lepromatous monocyte-macrophage functions in vitro by tuftsin.

Human peripheral blood monocytes/macrophages derived from normal donors, patients of tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) were assayed for stimulated phagocytic responses to the potent macrophage stimulator "Tuftsin" (NH2-Thr-Lys-Pro-Arg-OH) after varying periods (6 h to 14 days) of culture in vitro. The assays consisted of visual scoring of ingested Mycobacterium leprae and radiometric measurement of ingested 14C-acetate labelled Staphylococcus aureus and Mycobacterium tuberculosis (H37Ra). While normal and BT/TT macrophages showed a progressively increasing ability for tuftsin-stimulated phagocytosis with increasing age of culture in vitro, BL/LL macrophages showed the opposite response so that 14-day cultures were refractory to a stimulatory dose of up to 7.0 microM (10 to 20 times the optimal dose for normal and BT/TT macrophages). The 14-day BL/LL macrophage cultures were, however, responsive to 35 microM tuftsin (100 times the optimal dose for normal macrophages). Analysis of the dose-response curves also indicates that BT/TT cultures despite exhibiting an apparent similarity to normal macrophages demonstrate a rightward shift for a maximal stimulated phagocytosis. Finally SEM photo-micrographs of 14-day macrophage cultures of the three groups revealed that while normal and BT/TT cultures demonstrated an increase in membrane ruffling and filopodia on stimulation with 0.8 microM tuftsin, BL/LL cultures exhibited none of the features associated with stimulation. From the above findings, we conclude that lepromatous macrophages may display an aberrant differentiation profile leading to a terminal state of unresponsiveness and that the defect may possibly lie at the level of tuftsin receptor expression or transmembrane signal transduction.