Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.
Animals ; Disease Models, Animal ; Female ; Leishmania major/*immunology ; Leishmania tropica/*immunology/*pathogenicity ; Leishmaniasis/*immunology/parasitology/*pathology ; Mice ; Mice, Inbred BALB C ; Treatment Outcome

Leishmania tropica and L. major are etiologic agents of human cutaneous leishmaniasis. Delayed type hypersensitivity (DTH) is an immunologic response that has been frequently used as a correlate for protection against or sensitization to leishmania antigen. In BALB/c mice, L. tropica infection results in non-ulcerating disease, whereas L. major infection results in destructive lesions. In order to clarify the immunologic mechanisms of these 2 different outcomes, we compared the ability of these 2 leishmania species in induction of DTH response in this murine model. BALB/c mice were infected with L. major or L. tropica, and disease evolution and DTH responses were determined. The results show that the primary L. major infection can exacerbate the secondary L. major infection and is associated with DTH response. Higher doses of the primary L. major infection result in more disease exacerbation of the secondary L. major infection as well as higher DTH response. L. tropica infection induces lower DTH responses than L. major. We have previously reported that the primary L. tropica infection induces partial protection against the secondary L. major infection in BALB/c mice. Induction of lower DTH response by L. tropica suggests that the protection induced against L. major by prior L. tropica infection may be due to suppression of DTH response.
Animals ; Disease Models, Animal ; Ear/pathology ; Female ; Foot/pathology ; *Hypersensitivity, Delayed ; Leishmania major/*immunology ; Leishmania tropica/*immunology ; Leishmaniasis, Cutaneous/*immunology/*parasitology/pathology ; Mice ; Mice, Inbred BALB C

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.
Animals ; Antigens, Protozoan/*chemistry/*isolation & purification ; Blotting, Western ; Chromatography, High Pressure Liquid ; Electrophoresis, Polyacrylamide Gel/methods ; Humans ; Leishmania major/growth & development/*immunology ; Protein Isoforms/chemistry/isolation & purification

Experimental murine models with high, intermediate and low levels of genetically based susceptibility to Leishmania major infection reproduce almost entire spectrum of clinical manifestations of the human disease. There are increasing non-comparative studies on immune responses against isolated antigens of L. major in different murine strains. The aim of the present study was to find out whether there is an antigen that can induce protective immune response in resistant and susceptible murine strains. To do that, crude antigenic extract of procyclic and metacyclic promastigotes of L. major was prepared and subjected to SDS-PAGE electrophoresis. Western-blotting was used to search for antigen(s) capable of raising high antibody level of IgG2a versus IgG1 in the sera of both infected resistant and susceptible strains. Two novel antigens from metacyclic promastigotes of L. major (140 and 152 kDa) were potentially able to induce specific dominant IgG2a responses in BALB/c and C57BL/6 mice. The 2 antigens also reacted with IgG antibody of cutaneous leishmaniasis patients. We confirm that 140 and 152 kDa proteins of L. major promastigotes are inducing IgG production in mice and humans.
Protozoan Proteins/immunology/*isolation & purification ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Life Cycle Stages/immunology ; Leishmaniasis, Cutaneous/immunology ; Leishmania major/*immunology ; Immunoglobulin G/*biosynthesis/blood ; Humans ; Female ; Blotting, Western/methods ; Antigens, Protozoan/immunology/*isolation & purification ; Animals

This study investigated whether trinitroglycerine (TNG) as nitric oxide (NO) releasing agent had anti-leishmanial effects and mediated pathology in BALB/c mice infected with Leishmania major. Cutaneous leishmaniasis (CL), a zoonotic infection caused by leishmania protozoa is still one of the health problems in the world and in Iran. NO is involved in host immune responses against intracellular L. major, and leishmania killing by macrophages is mediated by this substance. Moreover, application of CL treatment with NO-donors has been recently indicated. In our study, TNG was used for its ability to increase NO and to modify CL infection in mice, in order to evaluate NO effects on lesion size and formation, parasite proliferation inside macrophages, amastigote visceralization in target organs, and NO induction in plasma and organ suspensions. Data obtained in this study indicated that TNG increased plasma and liver-NO, reduced lesion sizes, removed amastigotes from lesions, livers, spleens, and lymph nodes, declined proliferation of amastigotes, hepatomegaly, and increased survival rate. However, TNG reduced spleen-NO and had no significant effects on spelenomegaly. The results show that TNG therapy reduced leishmaniasis and pathology in association with raised NO levels. TNG had some antiparasitic activity by reduction of positive smears from lesions, livers, spleens, and lymph nodes, which could emphasize the role of TNG to inhibit visceralization of L. major in target organs.
Animal Structures/parasitology ; Animals ; Antiprotozoal Agents/chemistry/*therapeutic use ; Female ; Leishmania major/*drug effects/immunology ; Leishmaniasis, Cutaneous ; Macrophages/parasitology ; Mice ; Mice, Inbred BALB C ; Nitric Oxide/blood/metabolism/*pharmacology ; Nitroglycerin/*analogs & derivatives/*therapeutic use ; Severity of Illness Index ; Skin/pathology ; Survival Analysis

For treating Leishmania major infection in BALB/c mice, we used thalidomide in conjunction with glucantime. Groups of mice were challenged with 5 x 10(3) metacyclic promastigotes of L. major subcutaneously. A week after the challenge, drug treatment was started and continued for 12 days. Thalidomide was orally administrated 30 mg/kg/day and glucantime was administrated intraperitoneally (200 mg/kg/day). It was shown that the combined therapy is more effective than single therapies with each one of the drugs since the foot pad swelling in the group of mice received thalidomide and glucantime was significantly decreased (0.9 +/- 0.2 mm) compared to mice treated with either glucantime, thalidomide, or carrier alone (1.2 +/- 0.25, 1.4 +/- 0.3, and 1.7 +/- 0.27 mm, respectively). Cytokine study showed that the effect of thalidomide was not dependent on IL-12; however, it up-regulated IFN-gamma and down-regulated IL-10 production. Conclusively, thalidomide seems promising as a conjunctive therapy with antimony in murine model of visceral leishmaniasis.
Time Factors ; Thalidomide/pharmacology/*therapeutic use ; Organometallic Compounds/pharmacology/*therapeutic use ; Mice, Inbred BALB C ; Mice ; Meglumine/pharmacology/*therapeutic use ; Leishmaniasis, Visceral/*drug therapy/immunology ; Leishmania major/*drug effects ; Interleukin-12/analysis/biosynthesis ; Interleukin-10/analysis/biosynthesis ; Interferon Type II/analysis/biosynthesis/drug effects ; Immunosuppressive Agents/pharmacology/*therapeutic use ; Female ; Drug Therapy, Combination ; Disease Progression ; Disease Models, Animal ; Cells, Cultured ; Antiprotozoal Agents/pharmacology/*therapeutic use ; Animals