The potential role of nicotinamide on Leishmania tropica: An assessment of inhibitory effect, cytokines gene expression and arginase profiling.

Leishmaniasis represents a major health concern worldwide which has no effective treatment modality. Nicotinamide (NAm) has been used for a wide range of applications from anticancer to antimicrobial usage. This study aimed to assess the effect of NAm combination on Leishmania tropica Inhibition, as well as on cytokines gene expression and arginase (ARG) activity in L. tropica-infected macrophages in an in vitro model. The leishmanicidal effects of NAm and Glucantime (meglumine antimoniate, MA) alone and in combination (NAm/MA) were evaluated using a colorimetric assay and macrophage model. Additionally, immunomodulatory effects and enzymatic activity were assessed by analyzing Th1 and Th2 cytokines gene expression and ARG level, respectively, in infected macrophages treated with NAm and MA, alone and in combination. Findings indicated that the NAm/MA combination demonstrated greater inhibitory effects on L. tropica promastigotes and amastigotes compared with each drug individually. Docking results proved the affinity of NAm to IFN-γ, which can affirm the increased levels of IFN-γ, IL-12p40 and TNF-α as well as reductions in IL-10 secretion with a dose-response effect, especially in the combination group. The NAm/MA combination also showed a significant reduction in the level of ARG activity at all concentrations used compared to each drug individually. These findings indicate higher effectiveness of NAm plus MA in reducing parasite growth, promoting immune response and inhibiting ARG level. This combination should be considered as a potential therapeutic regimen for treatment of volunteer patients with anthroponotic cutaneous leishmaniasis (ACL) in future control programs.

Targeting of antitumor immune responses with live-attenuated Leishmania strains in breast cancer model.

BACKGROUND: Cancer is a major cause of death worldwide and most of the therapeutic approaches are relatively ineffective in eliminating cancer especially due to drug resistance. As an alternative, therapy with live microorganisms can induce a robust proinflammatory and anti-cancer immune response in the microenvironment of the tumor. In the present study, we aimed to establish a model for taking the advantages of immune responses against intracellular protozoan parasites for cancer treatment. METHODS: Leishmania infantum and L. tropica were used in our study as agents of visceral and cutaneous forms of the infection, respectively. After establishing 4T1 breast cancer in mice groups, live-attenuated L. infantum (At-Li) and live-attenuated L. tropica (At-Lt) treatments were performed and results were evaluated according to tumor volume, immune markers and histological examination. RESULTS: Live-attenuated Leishmania strains regressed 4T1-breast cancer in mice and are nonpathogenic, and these strains induce an immune response against 4T1 breast cancer. It is shown that At-Lt is found to be more effective than At-Li in breast cancer treatment using different methods included in the study as analyses of immune parameters, and histopathological examination in tumor tissue besides spleen cells. The tumor grew more slowly by the immune-stimulant effect of live-attenuated Leishmania parasites. CONCLUSION: This promising therapy should be investigated for optimization in further studies with different cancer types and L. tropica may be designed to express antigens to enhance tumor antigen-specific responses, which may further improve efficacy and immune memory development.

Camel nanobodies: Promising molecular tools against leishmaniasis.

AIM: To characterize several anti-Leishmania tropica nanobodies and to investigate their effect on Leishmania infection. METHODS: Several immunological tests were implied to characterize five different (as confirmed by sequencing) anti-L tropica nanobodies (NbLt05, NbLt06, NbLt14, NbLt24 and NbLt36) against parasite lysates or intact cells from different stages, promastigotes and amastigotes. Direct inhibitory effect of these nanobodies on parasite infection cycle on macrophages was tested in cell culture. RESULTS: All the five nanobodies (with distinguished characteristics) were more specific to L tropica than to L major, but could equally recognize the lysate and the outer surface of the intact cells from the two main stages of the parasite. Nanobodies recognized several leishmania antigens (majorly between 75 and 63 kDa), and their proteinaceous nature was confirmed. Because of its role in leishmania life cycle, gp63 was considered a potential antigen candidate for nanobodies, and bioinformatics predicted such interaction. All nanobodies have a negative effect on the infectivity of L tropica, as they decreased the number of infected macrophages and the amastigotes inside those macrophages. CONCLUSION: Such anti-leishmania nanobodies, with outstanding characteristics and important target, can be of great use in the development of promising treatment strategies against leishmaniasis.

Immunodetection and molecular determination of visceral and cutaneous Leishmania infection using patients' urine.

The diagnosis of leishmaniasis relies mainly on the use of invasive processes, to collect the biological material for detecting Leishmania parasites. Body fluids, which can be collected by non-invasive process, would greatly facilitate the leishmaniasis diagnosis. In the present study, we investigated the potency of urine immunoblotting to diagnose cutaneous and visceral leishmaniasis and we compared with routine molecular methods. A total of 80 samples, including 40 sera and their 40 corresponding urine samples were collected from 37 suspected patients with cutaneous and visceral leishmaniasis, and 3 healthy individuals (as control), in Ilam and Ardabil provinces of Iran. All sera and urine samples were analyzed, using immunoblotting. The confirmation of leishmaniasis infection was performed, using conventional and quantitative PCRs as well as by sequencing the amplicons. Among 37 suspected patients, 23 patients presented cutaneous lesions (CL) and 14 exhibited clinical symptoms reminiscent of visceral leishmaniasis (L. infantum). Among cutaneous patients, 15 were positive for zoonotic cutaneous leishmaniasis (L. major), and eight for anthroponotic cutaneous leishmaniasis (L. tropica). Molecular quantification of Leishmania parasites was performed on sera, urines and cutaneous biopsies of CL and VL patients, demonstrating that parasite load is lower in urines, compared to sera or biopsy. DNA can be detected in 20 out of 23 (86.9%) CL urine samples and in 13 out of 14 (92.8%) VL urine samples. Immunodetection analysis demonstrates that 22 out of 23 (95.6%) sera from CL patients and all patients suspected with VL are positive. For urine samples, 18 out of 23 (78.2%) urine of CL patients and 13 out of 14 (92.8%) urine of VL patients were positive, using Western blot. Therefore, immunodetection and molecular analysis using urine samples can be used as a diagnostic tool for surveying cutaneous and visceral leishmaniasis.

Role of Mast Cells in clearance of Leishmania through extracellular trap formation.

Mast Cells (MCs) are one of the first immune cells encountered by invading pathogens. Their presence in large numbers in the superficial dermis, where Leishmania is encountered, suggests that they may play a critical role in immune responses to Leishmania. In this study the interactions of Leishmania donovani, the causative agent of visceral Leishmaniasis, and Leishmania tropica, the causative agent of cutaneous Leishmaniasis with MCs were studied. Co-culture of Leishmania with Peritoneal Mast Cells (PMCs) from BALB/c mice and Rat Basophilic Leukaemia (RBL-2H3) MCs led to significant killing of L. tropica and to a lesser extent of L. donovani. Also, while there was significant uptake of L. tropica by MCs, L. donovani was not phagocytosed. There was significant generation of Reactive Oxygen Species (ROS) by MCs on co-culture with these species of Leishmania which may contribute to their clearance. Interactions of MCs with Leishmania led to generation of MC extracellular traps comprising of DNA, histones and tryptase probably to ensnare these pathogens. These results clearly establish that MCs may contribute to host defences to Leishmania in a differential manner, by actively taking up these pathogens, and also by mounting effector responses for their clearance by extracellular means.

The immune microenvironment in cutaneous leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis is an infection that has spread to non-endemic regions, stimulating recent interest for the enhanced understanding of this disease. Downregulation of the CD1a receptor on Langerhans cells has been described in various cutaneous infections. OBJECTIVE: In this study, the immune response across different Ridley patterns and parasitic indices is outlined in a case series of cutaneous leishmaniasis. METHODS: Skin punch biopsies from the interface of normal and lesional cutaneous leishmaniasis were collected from 33 patients with molecularly confirmed Leishmania tropica or L. major infection. Ridley patterns (2-5) were assessed for various clinicopathological features including age, gender, disease duration, parasitic index and constituents of the inflammatory infiltrate. CD1a, CD68, CD3, CD4, CD8, CD20 and CD138 stains were performed on normal skin tissue, cutaneous leishmaniasis biopsies and cytospin/cell block cytology preparations of cultured leishmania promastigotes. CD1a was quantified per mm2 in the epidermis and dermis. The remaining stains were graded according to a 4-tiered grading system [0 (0-4%); 1 (5-24%); 2 (25-49%); 3 (50-74%) and 4 (75-100%). RESULTS: Total CD1a expression significantly decreased (14-fold) from parasitic indices (0-2) to (5-6); (ρ < 0.001). CD1a expression in the epidermis was at least 5-fold lower than normal skin (58 vs. 400 cells/mm2), inversely correlating with the parasitic index. There was an increase in dermal CD1a Langerhans cells (33 vs. 0 cells/mm² in the dermis). CD1a and CD68 staining of amastigotes was strong and diffuse, whereas promastigotes were negative. The major inflammatory infiltrate, in all Ridley patterns, consisted of macrophages and double-negative CD3(+) CD4(-) CD8(-) T lymphocytes. The double-negative CD3 T cells formed a ring around the parasitic laden macrophages. Apart from CD1a, there was no significant difference in inflammatory markers between the various Ridley patterns and parasitic indices. Disease duration did not correlate with Ridley pattern. CONCLUSION: The significant decrease in CD1a expression is postulated by two mechanisms; either via direct CD1a receptor uptake by leishmania amastigotes and/or negative feedback inhibition of CD1a Langerhans cells by double-negative CD3 T-regulatory cells. Modulation of the immune microenvironment in cutaneous leishmaniasis represents a potential therapeutic and prophylactic target.

Human chorionic gonadotrophin (hCG) enhances immunity against L. tropica by stimulating human macrophage functions.

During pregnancy, there are important changes in hormone levels such as the huge production of human chorionic gonadotropin (hCG), which is supposed to influence the immune system. The aim of this study was to investigate the effect of hCG on immune response against Leishmania, through the evaluation of the functions of human macrophages infected with L. tropica. This study demonstrated that hCG significantly increased the NO production by rHu-IFNγ-primed macrophages then infected with L. tropica, which was correlated with decrease in the number of infected macrophages as well as the number of amastigotes per macrophage in a dose-dependent manner; however, the greatest effect was shown with the 250 U/mL concentration. The addition of the same concentration of hCG to rHu-IFNγ-primed macrophages caused also a major increase in both IL-6 and IL-12p40 production. In conclusion, hCG enhances different macrophage functions involved in immunity against L. tropica.

Comparative histological and immunohistochemical changes of dry type cutaneous leishmaniasis after administration of meglumine antimoniate, imiquimod or combination therapy.

BACKGROUND: This study compared histological and immunohistochemical changes of cutaneous leishmaniasis treated with meglumine antimoniate, imiquimod, and the combination of both therapies. METHODS: Single blind clinicopathological studies of fifteen patients with old world cutaneous leishmaniasis in Kerman, Iran were included. A total of four patients received a combination of imiquimod (5% cream) and intra-lesional meglumine antimoniate weekly for four weeks. Monotherapy with imiquimod was given to seven patients and four patients were treated with meglumine antimoniate intralesionally. Histological confirmation was performed before and during therapy. Semi-quantitative histological parameters such as numbers of mixed inflammatory cells (cells/mm(2)) and percentages of Langerhans cells (CD1a+), T-cells (CD3+), B-cells (CD20+), and macrophages (CD68+) were calculated immunohistochemically in the dermis and adjacent epidermis. RESULTS: Topical imiquimod significantly reduced mean histiocytic cellular aggregation size (P<0.05). Meglumine antimoniate reduced parasite load and infected activated histiocytes in the dermis (P<0.05). Meglumine antimoniate therapy decreased epidermal CD3+ lymphocytes but increased them in the dermis, within the granulomas (P<0.05). During topical application of imiquimod a depletion of CD1a+ dendritic cells in the epidermis (P<0.05) and slight predominance of dendritic cells in the dermis were observed. Combined therapy and imiquimod monotherapy decreased CD68+ macrophages in the dermis (P<0.05). CONCLUSION: Meglumine antimoniate decreases parasite load with considerable effect on up-regulation of T-cells, which demonstrates that meglumine antimoniate works as parasitocidal and immunomodulator, which could be a first line of treatment. Imiquimod accentuates the host immune response and reduces granuloma size which could be effective immunomodulator for combination therapy. Monotherapy of imiquimod is less effective than the two other regimens in decreasing parasite load, inflammation and congestion at the inoculated site.

Evaluation of localized and systemic immune responses in cutaneous leishmaniasis caused by Leishmania tropica: interleukin-8, monocyte chemotactic protein-1 and nitric oxide are major regulatory factors.

We have established Leishmania tropica as the causative agent of cutaneous leishmaniasis (CL) in the region of India where the disease is endemic. The association between localized and circulating levels of immune-determinants in CL patients was evaluated. Reverse transcription-polymerase chain reaction analysis revealed up-regulation of interferon-gamma (IFN-gamma), interleukin (IL)-1beta, IL-8, tumour necrosis factor-alpha (TNF-alpha), IL-10 and IL-4 in dermal lesions at the pretreatment stage (n = 31) compared with healthy controls (P < 0.001) and a significant down-regulation after treatment (n = 14, P < 0.05). The results indicated that an unfavourable clinical outcome in CL was not related to an inadequate T helper 1 (Th1) cell response, but rather to impairment in multiple immune functions. Comparative assessment of treatment regimes with rifampicin (RFM) or sodium antimony gluconate (SAG) revealed tissue cytokine levels to be significantly reduced after treatment with RFM (P < 0.005), while no significant decrease was evident in the levels of IFN-gamma, TNF-alpha and IL-10 (P > 0.05) as a result of treatment with SAG. Increased transcripts of monocyte chemoattractant protein-1 (MCP-1) (P < 0.001) and inducible nitric oxide synthase (iNOS) (P < 0.05) were evident before treatment in tissue lesions and remained high after treatment. Immunohistochemistry demonstrated strong expression of myeloperoxidase (MPO) and IL-8, and moderate expression of iNOS in dermal lesions. The expression levels of IL-8, MCP-1 and nitric oxide (NO) were high in patient sera before treatment, as determined using cytokine bead array and enzyme-linked immunosorbent assay (ELISA). At the post-treatment stage, the serum IL-8 levels had decreased; however, the levels of MCP-1 and NO remained high. These data suggest that IL-8 is an effector immune-determinant in the progression of CL, whereas NO facilitates the parasite killing by macrophages via MCP-1-mediated stimulation.

Correlation of parasitic load with interleukin-4 response in patients with cutaneous leishmaniasis due to Leishmania tropica.

We have established the association between parasite burden and localized immune response in patients with cutaneous leishmaniasis (CL) caused by Leishmania tropica. Real-time PCR was used to measure parasitic load in tissue lesions of CL patients at the pretreatment (n=26) and at the post-treatment stage (n=10). Leishmania tropica was detected in all CL lesions with a mean value of 118 357 parasites g(-1) of dermal tissue. Following treatment, only one out of 10 patients showed residual parasites (100 parasites g(-1) tissue). Parasite load was high (mean, 306 000 parasites g(-1) tissue) in acute infections (early lesions) and low (mean, 1081 parasites g(-1) tissue) in chronic infections (late lesions). Intralesional transcripts of interferon-gamma, tumour necrosis factor-alpha, interleukin-1beta (IL-1beta), IL-8, IL-10 and IL-4 were investigated in early lesions (2 months, n=15) by reverse transcriptase-PCR, where IL-4 was found to be significantly upregulated in early lesions (P<0.02). Further, the levels of parasite burden and IL-4 were distinctly correlated in various clinical forms of CL. Other cytokines were at comparable levels in early/late lesions and in different clinical forms. Upregulation of IL-4 was correlated with a higher parasite burden in early lesions of CL, which may be involved in the pathogenesis of CL by inhibiting a protective immune response.

Leishmania tropica-induced cutaneous and presumptive concomitant viscerotropic leishmaniasis with prolonged incubation.

BACKGROUND: Leishmaniasis includes a spectrum of diseases caused by protozoan parasites belonging to the genus Leishmania. The disease is traditionally classified into visceral, cutaneous, or mucocutaneous leishmaniasis, depending on clinical characteristics as well as the species involved. Leishmania tropica is one of the causative agents of cutaneous leishmaniasis, with a typical incubation period of weeks to months. Observation We describe a 17-year-old Afghani girl who had lived in the United States for 4 years and who presented with a 6-month history of pretibial ulcerations, 9.1-kg weight loss, abdominal pain, splenomegaly, and extreme fatigue. Histopathologic examination and culture with isoenzyme electrophoresis speciation of her skin lesions confirmed the presence of L tropica. In addition, results of serum laboratory and serological studies were highly suggestive of concomitant visceral involvement. The patient was treated with a 28-day course of intravenous pentavalent antimonial compound sodium stibogluconate with complete resolution of her systemic signs and symptoms and improvement of her pretibial ulcerations. CONCLUSIONS: This is an exceptional case in that our patient presented with disease after an incubation period of years rather than the more typical weeks to months. In addition, this patient had confirmed cutaneous involvement, as well as strong evidence of viscerotropic disease caused by L tropica, a species that characteristically displays dermotropism, not viscerotropism.

Immune response to Leishmania antigen in anthroponotic cutaneous leishmaniasis.

BACKGROUND: Leishmania (L.) tropica is the causative agent of anthroponotic cutaneous leishmaniasis (ACL) in Iran. The disease often heals within a year; however, the non-healing forms of disease are also known. The immunologic responses to L. major infection have been studied in depth, however little is known about the immune status of L. tropica-infected patients. MATERIALS AND METHODS: This study was conducted to evaluate T-cell responses to Leishmania antigen in non-healing patients, patients with acute lesion, and healthy donors. Peripheral blood mononuclear cells (PBMC) were cultured with antigen and lymphoproliferative responses were determined. Cytokine profile including gamma interferon (IFN-gamma), interleukin (IL)-5, and IL-13 in supernatants of stimulated cells was also determined. RESULTS: The results showed PBMC from both groups of patients proliferated vigorously in response to Leishmania antigens. The levels of IFN-gamma and IL-13 were comparable between patients with acute lesions and non-healing patients. Non-healing patients had significantly higher median levels of IL-5 than patients with acute lesions. The cells from healthy individuals did not respond to Leishmania antigens. CONCLUSIONS: High levels of IFN-gamma, IL-5, and IL-13 in non-healing patients suggest a mixed Th1/Th2 response, whereas patients with acute lesion respond to infection by Th1-type response.

The effect of amphotericin b derivatives on Leishmania and immune functions.

The effects of a water-soluble amphotericin B (AmB)-arabinogalactan (AG) conjugate on several immune functions were investigated. The experiments measured the effects of AmB-AG on (1) release of tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), and interferon-gamma (IFN-gamma) from phagocytic cells and (2) cell-mediated immune responses. AmB-AG increased TNF-alpha release from mouse peritoneal macrophages and human monocytes but had no effect on IFN-gamma and NO release. A commercial preparation of nonconjugated AmB (Fungizone) also increased TNF-alpha production, but to a lesser extent than AmB-AG. AG alone had no effect on TNF-alpha production, proving that AmB caused the increased TNF-alpha production. AmB-AG and Fungizone were also tested for their effect on B- and T-cell proliferation. Neither compound altered T-lymphocyte responses to concanavalin A, but both inhibited the stimulation of B lymphocytes by lipopolysaccharides. However, Fungizone showed a stronger inhibitory effect on B cells. Allocytotoxicity was also inhibited by AmB-AG and more strongly by Fungizone. The increased production of TNF-alpha by cells treated with AmB-AG and the lower inhibitory effect of AmB-AG on lymphocyte stimulation and allocytotoxicity, as compared with Fungizone, explain the better therapeutic efficacy of the AmB-polysaccharide conjugate. AmB is active because of its preferential binding to ergosterol rather than cholesterol, the former sterol preferentially present in parasite surface membranes. This is also valid for the axenic amastigotes, which were sensitive to the AmB-AG. Overall, our results suggest that the antileishmanial activity of AmB-AG is mediated both directly and via modulation of immune functions.

The mechanism behind the antileishmanial effect of zinc sulphate. I. An in-vitro study.

In an attempt to determine the possible mechanism(s) behind the antileishmanial activity of zinc sulphate, promastigotes, axenic amastigotes and intracellular amastigotes of both Leishmania major and L. tropica were incubated with different concentrations of the compound. For each of the two Leishmania species, all three forms were found to be inhibited by the zinc sulphate, in a dose-dependent manner, the promastigotes being the most resistant form, followed by the axenic amastigotes. These results indicate that zinc sulphate has a direct antileishmanial effect. Compared with macrophages from starch-treated mice, the macrophages recovered from mice that had been injected intraperitoneally with zinc sulphate (daily for the 4 days prior to the macrophage collection) or BCG (once, 4 days before the cell collection) showed increased phagocytosis and increased killing of L. major and L. tropica. As the effects of the zinc sulphate were not statistically different from those of the known immunomodulating agent BCG, zinc sulphate appears to have an immunomodulating effect, in addition to its direct antileishmanial effect.

Leishmania priming of human dendritic cells for CD40 ligand-induced interleukin-12p70 secretion is strain and species dependent.

A major question in the study of leishmaniasis is what dictates clinical disease expression produced by different Leishmania species, i.e., cutaneous versus systemic and healing versus nonhealing. Animal models using a Leishmania species associated with self-limiting cutaneous disease (L. major) have revealed that protective immunity requires CD40/CD40 ligand (CD40L)-dependent, interleukin-12 (IL-12)-driven Th1 responses. We recently showed that L. major can prime human dendritic cells (DCs) for CD40L-triggered IL-12p70 secretion and that these cells can drive a Th1 response in autologous T cells from sensitized individuals. Here we show that in contrast to L. major, Leishmania species responsible for visceral disease (L. donovani), as well as species associated with persistent, cutaneous lesions and occasional systemic disease (L. tropica), did not induce CD40L-dependent IL-12p70 production, despite comparable levels of uptake by DCs. Up-regulated surface expression of CD40 did not correlate with IL-12p70 production, and appreciable CD40L-induced IL-12p40 secretion was observed in uninfected as well as infected DCs, regardless of species. Reverse transcription-PCR analysis confirmed that the production of heterodimeric IL-12 was limited by expression of IL-12p35 mRNA, which was dependent on both a microbial priming signal and CD40 engagement for its high-level induction. The intrinsic differences in the ability of Leishmania species to prime DCs for CD40L-dependent IL-12p70 secretion may account, at least in part, for the evolution of healing and nonhealing forms of leishmanial disease.

Identification and characterization of T cell-stimulating antigens from Leishmania by CD4 T cell expression cloning.

Persistent immunity against Leishmania: infections in humans is mediated predominantly by CD4(+) T cells of the Th1 phenotype. Herein we report the expression cloning of eight Leishmania: Ags using parasite-specific T cell lines derived from an immune donor. The Ags identified by this technique include the flagellar proteins alpha- and beta-tubulin, histone H2b, ribosomal protein S4, malate dehydrogenase, and elongation factor 2, as well as two novel parasite proteins. None of these proteins have been previously reported as T cell-stimulating Ags from Leishmania: beta-tubulin-specific T cell clones generated against Leishmania: major amastigotes responded to Leishmania:-infected macrophages and dendritic cells. IFN-gamma enzyme-linked immunospot analysis demonstrated the presence of T cells specific for several of these Ags in PBMC from self-healing cutaneous leishmaniasis patients infected with either Leishmania: tropica or L. major. The responses elicited by Leishmania: histone H2b were particularly striking in terms of frequency of histone-specific T cells in PBMC (1 T cell of 6000 PBMC) as well as the percentage of responding donors (86%, 6 of 7). Ags identified by T cells from immune donors might constitute potential vaccine candidates for leishmaniasis.

Cytologic features of "dry-type" cutaneous leishmaniasis.

Exfoliative cytology smears from the lesions of 179 patients with cutaneous leishmaniasis due to Leishmania tropica were studied with specific reference to cellular reactions and their effect on the parasite. Aggregates of the parasite (so-called Leishmania Donovan bodies) were present within macrophages and in some fibroblasts. The nature of the inflammatory reaction to the disease was studied by performing differential counts of the inflammatory cells present in the smears. These were correlated with the number of Leishman Donovan bodies. There was an inverse relationship between the number of Leishman Donovan bodies and the percentage of small lymphocytes, neutrophils, and type I macrophages. It is postulated that aggregates of activated macrophages (designated types II and III) and the Leishmanian milieu (sticky matrix) protect the amastigote Leishmania parasites from being eradicated by the inflammatory and immune reaction. The cytoplasmic blebbing of the parasitophorous vacuoles and cell to cell connection of the activated histiocytes could be shown by the CD-68 immunostaining of the tissue biopsy.

Interleukin-7 enhances antimicrobial activity against Leishmania major in murine macrophages.

Recently, it has been shown that interleukin-7 (IL-7) is able to induce secretion of cytokines and tumoricidal activity by human monocytes. This study shows that treatment of murine macrophages infected with Leishmania major with IL-7 without any other stimulus reduced the percentage of infected cells, as well as the parasite burden per cell, in a dose-dependent manner to a limited degree (45% reduction of the number of amastigotes per 100 macrophages). Simultaneous treatment of macrophages with gamma interferon and IL-7 led to nearly complete (> 99%) elimination of amastigotes. Addition of anti-tumor necrosis factor alpha or N omega-monomethyl-L-arginine acetate reversed the leishmanicidal effects of IL-7, and production of nitric oxide was induced in the presence of IL-7.

Targeted activation of CD8 cells and infection of beta 2-microglobulin-deficient mice fail to confirm a primary protective role for CD8 cells in experimental leishmaniasis.

CD8+ T cells play an important role in the immunologic control of intracellular pathogens, particularly viruses. Leishmania are obligate intracellular parasites of macrophages in the mammalian host, and previous studies using deletion of CD8+ cells by administration of mAb to infected animals have suggested a protective role for these cells. Two complementary approaches were used to define more carefully the role of CD8+ cells in leishmaniasis. In BALB/c mice susceptible to Leishmania major (L. major) infection, targeted activation of CD8+ T cells was attempted by immunization with nonapeptides derived from the conserved major outer surface protein of the organism, gp63, that contained the consensus binding motif for MHC class I H-2Kd molecules. Two of the nonapeptides induced CTL activity in subsequently infected BALB/c mice that could be elicited against P815 cells pulsed either with peptide or lysates of L. major. Purified CD8+ T cells from immunized mice had elevated levels of IFN-gamma mRNA transcripts as compared to unimmunized mice. Despite evidence for activation of CD8+ cells, none of the mice immunized with nine different peptides alone or in combination were protected from progressive disease. In a second series of experiments, beta 2-microglobulin deficient mice that lack CD8+ cells were infected with L. major and the course of infection monitored. These mice cured disease as rapidly as beta 2-m +/- and +/+ littermates, and cure was associated with comparable levels of IFN-gamma mRNA in the draining lymph node population. Neither of these approaches was able to confirm a substantive role for CD8+ T cells in the primary protective response to L. major.

Pre-exposure of murine macrophages to lipopolysaccharide inhibits the induction of nitric oxide synthase and reduces leishmanicidal activity.

Murine macrophages produce nitric oxide (NO) from L-arginine on stimulation with lipopolysaccharide (LPS), alone or with interferon-gamma (IFN-gamma). The effect of incubation of macrophages with low concentrations of LPS on NO synthesis on subsequent stimulation was investigated, using a murine macrophage cell line, J774, and peritoneal macrophages from CBA mice. Cells which had been incubated with LPS produced significantly lower amounts of NO, and expressed lower levels of NO synthase activity, following stimulation with IFN-gamma and LPS, or with a high concentration of LPS. This effect was not reversed by tumor necrosis factor-alpha. The ability of CBA macrophages to kill the intracellular parasite Leishmania major was markedly reduced by pre-incubation with LPS. Reduced NO production by macrophages previously exposed to LPS is a manifestation of endotoxin tolerance, and may represent an important means of regulation of NO synthesis and thus a survival mechanism for intracellular parasites.