miR-548d-3p Is Up-Regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth in Macrophages.

Visceral leishmaniasis caused by Leishmania (Leishmania) infantum in Latin America progress with hepatosplenomegaly, pancytopenia, hypergammaglobulinemia, and weight loss and maybe lethal mainly in untreated cases. miRNAs are important regulators of immune and inflammatory gene expression, but their mechanisms of action and their relationship to pathogenesis in leishmaniasis are not well understood. In the present study, we sought to quantify changes in miRNAs associated with immune and inflammatory pathways using the L. (L.) infantum promastigote infected- human monocytic THP-1 cell model and plasma from patients with visceral leishmaniasis. We identified differentially expressed miRNAs in infected THP-1 cells compared with non-infected cells using qPCR arrays. These miRNAs were submitted to in silico analysis, revealing targets within functional pathways associated with TGF-ß, chemokines, glucose metabolism, inflammation, apoptosis, and cell signaling. In parallel, we identified differentially expressed miRNAs in active visceral leishmaniasis patient plasma compared with endemic healthy controls. In silico analysis of these data indicated different predicted targets within the TGF-ß, TLR4, IGF-I, chemokine, and HIF1α pathways. Only a small number of miRNAs were commonly identified in these two datasets, notably with miR-548d-3p being up-regulated in both conditions. To evaluate the potential biological role of miR-548d-3p, we transiently transfected a miR-548d-3p inhibitor into L. (L.) infantum infected-THP-1 cells, finding that inhibition of miR-548d-3p enhanced parasite growth, likely mediated through reduced levels of MCP-1/CCL2 and nitric oxide production. Further work will be required to determine how miR-548d-3p plays a role in vivo and whether it serves as a potential biomarker of progressive leishmaniasis.

Staphylococcus aureus Protection-Related Type 3 Cell-Mediated Immune Response Elicited by Recombinant Proteins and GM-CSF DNA Vaccine.

Staphylococcus aureus mastitis remains a major challenge for dairy farming. Here, 24 mice were immunized and divided into four groups: G1: control; G2: Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) DNA vaccine; G3: F0F1 ATP synthase subunit α (SAS), succinyl-diaminopimelate (SDD), and cysteinyl-tRNA synthetase (CTS) recombinant proteins; and G4: SAS+SDD+CTS plus GM-CSF DNA vaccine. The lymphocyte subpopulations, and the intracellular interleukin-17A (IL-17A) and interferon-γ production in the draining lymph node cells were immunophenotyped by flow cytometry. The immunophenotyping and lymphocyte proliferation was determined in spleen cells cultured with and without S. aureus stimulus. Immunization with S. aureus recombinant proteins generated memory cells in draining lymph nodes. Immunization with the three recombinant proteins plus GM-CSF DNA led to an increase in the percentage of IL-17A+ cells among overall CD44+ (memory), T CD4+, CD4+ T CD44+ CD27-, γδ TCR, γδ TCR+ CD44+ CD27+, and TCRVγ4+ cells. Vaccination with S. aureus recombinant proteins associated with GM-CSF DNA vaccine downregulated TH2 immunity. Immunization with the three recombinant proteins plus the GM-CSF DNA led to a proliferation of overall memory T, CD4+, and CD4+ TEM cells upon S. aureus stimulus. This approach fostered type 3 immunity, suggesting the development of a protective immune response against S. aureus.

Pleiotropic Effect of Hormone Insulin-Like Growth Factor-I in Immune Response and Pathogenesis in Leishmaniases.

Leishmaniases are diseases caused by several Leishmania species, and many factors contribute to the development of the infection. Because the adaptive immune response does not fully explain the outcome of Leishmania infection and considering that the initial events are crucial in the establishment of the infection, we investigated one of the growth factors, the insulin-like growth factor-I (IGF-I), found in circulation and produced by different cells including macrophages and present in the skin where the parasite is inoculated. Here, we review the role of IGF-I in leishmaniasis experimental models and human patients. IGF-I induces the growth of different Leishmania species in vitro and alters the disease outcome increasing the parasite load and lesion size, especially in L. major- and L. amazonensis-infected mouse leishmaniasis. IGF-I affects the parasite interacting with the IGF-I receptor present on Leishmania. During Leishmania-macrophage interaction, IGF-I acts on the arginine metabolic pathway, resulting in polyamine production both in macrophages and Leishmania. IGF-I and cytokines interact with reciprocal influences on their expression. IL-4 is a hallmark of susceptibility to L. major in murine leishmaniasis, but we observed that IGF-I operates astoundingly as an effector element of the IL-4. Approaching human leishmaniasis, patients with mucosal, disseminated, and visceral diseases presented surprisingly low IGF-I serum levels, suggesting diverse effects than parasite growth. We observed that low IGF-I levels might contribute to the inflammatory response persistence and delayed lesion healing in human cutaneous leishmaniasis and the anemia development in visceral leishmaniasis. We must highlight the complexity of infection revealed depending on the Leishmania species and the parasite's developmental stages. Because IGF-I exerts pleiotropic effects on the biology of interaction and disease pathogenesis, IGF-I turns up as an attractive tool to explore biological and pathogenic processes underlying infection development. IGF-I pleiotropic effects open further the possibility of approaching IGF-I as a therapeutical target.

Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection

BACKGROUND Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Unusual manifestation of genital cutaneous leishmaniasis in an immunocompetent patient from São Paulo, Brazil: A case report

Abstract A 31-year-old male patient developed an ulcer on the glans penis that evolved for three months without healing. We diagnosed it as leishmaniasis using polymerase chain reaction. No immunosuppression or associated diseases were observed. The patient was treated with meglumine antimoniate that cured the lesion in a month post-treatment. Here, we report this case of cutaneous leishmaniasis lesion at the unusual location of glans penis in an immunocompetent individual. The lesion likely developed due to the bite of a vector, highlighting the need for considering cutaneous leishmaniasis among differential diagnosis of sexually transmitted diseases in areas endemic for leishmaniasis.

Macrophages From Subjects With Isolated GH/IGF-I Deficiency Due to a GHRH Receptor Gene Mutation Are Less Prone to Infection by Leishmania amazonensis.

Isolated growth hormone (GH) deficiency (IGHD) affects approximately 1 in 4,000 to 1 in 10,000 individuals worldwide. We have previously described a large cohort of subjects with IGHD due to a homozygous mutation in the GH releasing hormone (GHRH) receptor gene. These subjects exhibit throughout the life very low levels of GH and its principal mediator, the Insulin Growth Factor-I (IGF-I). The facilitating role of IGF-I in the infection of mouse macrophages by different Leishmania strains is well-known. Nevertheless, the role of IGF-I in Leishmania infection of human macrophages has not been studied. This study aimed to evaluate the behavior of Leishmania infection in vitro in macrophages from untreated IGHD subjects. To this end, blood samples were collected from 14 IGHD individuals and 14 age and sex-matched healthy controls. Monocytes were isolated and derived into macrophages and infected with a strain of Leishmania amazonensis. In addition, IGF-I was added to culture medium to evaluate its effect on the infection. Cytokines were measured in the culture supernatants. We found that macrophages from IGHD subjects were less prone to Leishmania infection compared to GH sufficient controls. Both inflammatory and anti-inflammatory cytokines increase only in the supernatants of the control macrophages. Addition of IGF-I to the culture medium increased infection rates. In conclusion, we demonstrated that IGF-I is crucial for Leishmania infection of human macrophages.

ATP6V0d2 controls Leishmania parasitophorous vacuole biogenesis via cholesterol homeostasis.

V-ATPases are part of the membrane components of pathogen-containing vacuoles, although their function in intracellular infection remains elusive. In addition to organelle acidification, V-ATPases are alternatively implicated in membrane fusion and anti-inflammatory functions controlled by ATP6V0d2, the d subunit variant of the V-ATPase complex. Therefore, we evaluated the role of ATP6V0d2 in the biogenesis of pathogen-containing vacuoles using ATP6V0d2 knock-down macrophages infected with the protozoan parasite Leishmania amazonensis. These parasites survive within IFNγ/LPS-activated inflammatory macrophages, multiplying in large/fusogenic parasitophorous vacuoles (PVs) and inducing ATP6V0d2 upregulation. ATP6V0d2 knock-down decreased macrophage cholesterol levels and inhibited PV enlargement without interfering with parasite multiplication. However, parasites required ATP6V0d2 to resist the influx of oxidized low-density lipoprotein (ox-LDL)-derived cholesterol, which restored PV enlargement in ATP6V0d2 knock-down macrophages by replenishing macrophage cholesterol pools. Thus, we reveal parasite-mediated subversion of host V-ATPase function toward cholesterol retention, which is required for establishing an inflammation-resistant intracellular parasite niche.

Insulin-Like Growth Factor-I as an Effector Element of the Cytokine IL-4 in the Development of a Leishmania major Infection.

Certain cytokines modulate the expression of insulin-like growth factor- (IGF-) I. Since IL-4 and IGF-I promote growth of the protozoan Leishmania major, we here addressed their interaction in downregulating the expression of Igf-I mRNA using small interfering RNA (siRNA) in Leishmania major-infected macrophages. Parasitism was decreased in the siRNA-treated cells compared with the nontreated cells, reversed by the addition of recombinant IGF-I (rIGF-I). In IL-4-stimulated macrophages, parasitism and the Igf-I mRNA amount were increased, and the effects were nullified upon siRNA transfection. IGF-I downregulation inhibited both parasite and macrophage arginase activation even in IL-4-stimulated cells. Searching for intracellular signaling components shared by IL-4 and IGF-I, upon siRNA transfection, phosphorylated p44, p38, and Akt proteins were decreased, affecting the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. In L. major-infected C57BL6-resistant mice, the preincubation of the parasite with rIGF-I changed the infection profile to be similar to that of susceptible mice. We conclude that IGF-I constitutes an effector element of IL-4 involving the PI3K/Akt pathway during L. major infection.

CD4+ T Cells Alter the Stromal Microenvironment and Repress Medullary Erythropoiesis in Murine Visceral Leishmaniasis.

Human visceral leishmaniasis, a parasitic disease of major public health importance in developing countries, is characterized by variable degrees of severity of anemia, but the mechanisms underlying this change in peripheral blood have not been thoroughly explored. Here, we used an experimental model of visceral leishmaniasis in C57BL/6 mice to explore the basis of anemia following infection with Leishmania donovani. 28 days post-infection, mice showed bone marrow dyserythropoiesis by myelogram, with a reduction of TER119+ CD71-/+ erythroblasts. Reduction of medullary erythropoiesis coincided with loss of CD169high bone marrow stromal macrophages and a reduction of CXCL12-expressing stromal cells. Although the spleen is a site of extramedullary erythropoiesis and erythrophagocytosis, splenectomy did not impact the extent of anemia or affect the repression of medullary hematopoiesis that was observed in infected mice. In contrast, these changes in bone marrow erythropoiesis were not evident in B6.Rag2-/- mice, but could be fully reconstituted by adoptive transfer of IFNγ-producing but not IFNγ-deficient CD4+ T cells, mimicking the expansion of IFNγ-producing CD4+ T cells that occurs during infection in wild type mice. Collectively, these data indicate that anemia during experimental murine visceral leishmaniasis can be driven by defects associated with the bone marrow erythropoietic niche, and that this represents a further example of CD4+ T cell-mediated immunopathology affecting hematopoietic competence.

Insulin-like growth factor-I induces arginase activity in Leishmania amazonensis amastigote-infected macrophages through a cytokine-independent mechanism.

Leishmania (Leishmania) amazonensis exhibits peculiarities in its interactions with hosts. Because amastigotes are the primary form associated with the progression of infection, we studied the effect of insulin-like growth factor (IGF)-I on interactions between L. (L.) amazonensis amastigotes and macrophages. Upon stimulation of infected macrophages with IGF-I, we observed decreased nitric oxide production but increased arginase expression and activity, which lead to increased parasitism. However, stimulation of amastigote-infected macrophages with IGF-I did not result in altered cytokine levels compared to unstimulated controls. Because IGF-I is present in tissue fluids and also within macrophages, we examined the possible effect of this factor on phosphatidylserine (PS) exposure on amastigotes, seen previously in tissue-derived amastigotes leading to increased parasitism. Stimulation with IGF-I induced PS exposure on amastigotes but not on promastigotes. Using a PS-liposome instead of amastigotes, we observed that the PS-liposome but not the control phosphatidylcholine-liposome led to increased arginase activity in macrophages, and this process was not blocked by anti-TGF-ß antibodies. Our results suggest that in L. (L.) amazonensis amastigote-infected macrophages, IGF-I induces arginase activity directly in amastigotes and in macrophages through the induction of PS exposure on amastigotes in the latter, which could lead to the alternative activation of macrophages through cytokine-independent mechanisms.

In vivo assessment of antiretroviral therapy-associated side effects

Antiretroviral therapy has been associated with side effects, either from the drug itself or in conjunction with the effects of human immunodeficiency virus infection. Here, we evaluated the side effects of the protease inhibitor (PI) indinavir in hamsters consuming a normal or high-fat diet. Indinavir treatment increased the hamster death rate and resulted in an increase in triglyceride, cholesterol and glucose serum levels and a reduction in anti-oxLDL auto-antibodies. The treatment led to histopathological alterations of the kidney and the heart. These results suggest that hamsters are an interesting model for the study of the side effects of antiretroviral drugs, such as PIs.

Cell-to-cell transfer of Leishmania amazonensis amastigotes is mediated by immunomodulatory LAMP-rich parasitophorous extrusions.

The last step of Leishmania intracellular life cycle is the egress of amastigotes from the host cell and their uptake by adjacent cells. Using multidimensional live imaging of long-term-infected macrophage cultures we observed that Leishmania amazonensis amastigotes were transferred from cell to cell when the donor host macrophage delivers warning signs of imminent apoptosis. They were extruded from the macrophage within zeiotic structures (membrane blebs, an apoptotic feature) rich in phagolysosomal membrane components. The extrusions containing amastigotes were selectively internalized by vicinal macrophages and the rescued amastigotes remain viable in recipient macrophages. Host cell apoptosis induced by micro-irradiation of infected macrophage nuclei promoted amastigotes extrusion, which were rescued by non-irradiated vicinal macrophages. Using amastigotes isolated from LAMP1/LAMP2 knockout fibroblasts, we observed that the presence of these lysosomal components on amastigotes increases interleukin 10 production. Enclosed within host cell membranes, amastigotes can be transferred from cell to cell without full exposure to the extracellular milieu, what represents an important strategy developed by the parasite to evade host immune system.

Microbial translocation induces an intense proinflammatory response in patients with visceral leishmaniasis and HIV type 1 coinfection.

BACKGROUND: Leishmania infection is a cofactor in the heightened cellular activation observed in patients with American visceral leishmaniasis and human immunodeficiency virus type 1 (HIV) infection, with or without progression to AIDS (AVL/HIV). Thus, the persistence of a high parasite load despite antileishmanial therapy could be responsible for the continued immune stimulation. METHODS: CD8(+) T cells expressing CD38, parasite load, lipopolysaccharide (LPS), soluble CD14, macrophage migration inhibitory factor (MIF), intestinal fatty acid-binding protein (IFABP), and proinflammatory cytokines (interleukin 1ß, interleukin 6, interleukin 8, interleukin 17, interferon γ, and tumor necrosis factor) were measured in 17 patients with AVL/HIV, 16 with HIV, and 14 healthy subjects (HS). RESULTS: Lower Leishmania parasitemia was observed after antileishmanial and antiretroviral therapies. However, higher levels of CD38(+) on CD8(+) T cells were observed in both clinical phases of leishmaniasis, compared with HIV cases. AVL/HIV and HIV patients showed higher levels of LPS and IFABP than HS. Proinflammatory cytokine levels were significantly augmented in patients with active coinfection, as well as those with remission of Leishmania infection. LPS levels and Leishmania infection were positively correlated with CD38 expression on CD8(+) T cells and with IL-6 and IL-8 levels. CONCLUSIONS: LPS levels along with the immune consequences of Leishmania infection were associated with elevated cellular activation in coinfected patients. As a consequence, secondary chemoprophylaxis for leishmaniasis or even the use of antiinflammatory drugs or antibiotics may be considered for improving the prognosis of AVL/HIV.

Cutaneous and mucocutaneous leishmaniasis.

Tegumentary leishmaniases are caused by approximately 15 species of protozoa of the genus Leishmania. They prevail in tropical and subtropical areas of the Old and New World but human mobility also makes them a medical problem in nonendemic areas. Clinical manifestations may comprise cutaneous and mucocutaneous forms that may be localized, disseminated, or diffuse in distribution and may differ in Old and New World leishmaniases. Diagnosis and treatment vary according to the clinical manifestations, geographic area, and Leishmania species involved. This article highlights the diversity and complexity of tegumentary leishmaniases, which are worsened by human immunodeficiency virus/Leishmania coinfection.

Protein disulfide isomerase and host-pathogen interaction.

Reactive oxygen species (ROS) production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals) functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i) pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation) and (ii) phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI) family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER) and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections.

Infecção por leptospiras em ovinos e caracterização da resposta inflamatória renal / Leptospirosis infection in sheep and characterization of the renal inflammatory response

As Leptospiroses são zoonoses graves de distribuição mundial que afetam o homem e outros animais. A infecção em animais, geralmente, é inaparente, ou os sintomas quando presentes são similares a outras infecções. Neste estudo foram colhidos soros de 119 ovinos e seus respectivos rins durante abate em feiras livres no município de Teresina-Piauí. Pela técnica de soroaglutinação microscópica (SAM) obtiveram-se 34 amostras sorológicas positivas para um ou mais sorovares de Leptospira spp., com taxa de ocorrência de 28,6 por cento de anticorpos anti-leptospiras, sendo 23 casos de infecção para um único sorovar e 11 com coaglutinações para dois ou mais sorovares. Dentre os sorovares patogênicos, o de maior ocorrência foi o Autumnalis (29,4 por cento). A análise histopatológica de 36 fragmentos de rins revelou alterações túbulo intersticiais em 33 (91,7 por cento) animais soro-reagentes. Lesões tubulares foram observadas em 20 (55,5 por cento) animais soro-reagentes. A presença de leptospiras, pela técnica de Warthin Starry, foi observada em 8 (22,20 por cento) amostras positivas. Pela técnica de imunoperixidase, de 20 casos analisados, foi verificada a presença de leptospira em 12 (60 por cento) de 20 amostras positivas. Nos animais soro-reagentes, o infiltrado inflamatório foi significantemente mais evidente na região córtico-medular e cortical do que na região medular (p=0,000), mas não houve diferença entre animais soro-reagentes e soro não-reagentes. Cilindros hialinos nos túbulos proximais estavam presentes em quantidade significantemente maior nos animais soro-reagentes comparados aos não-reagentes (p=0,0001). Em glomérulos, foi observada lesão discreta. Os resultados deste estudo mostram que ovinos soro-reagentes para leptospiras apresentam lesões renais túbulo intersticiais, com presença de leptospiras nos túbulos, o que confere a esses animais a condição de disseminadores da infecção.

Lepitospirosis is a serious worldwide distribution disease which affects man and other animals. The infection is generally asymptomatic in animals. In cases whose symptoms are present, symptoms are similar to other infections. In the present study serum samples from 119 sheep and their kidneys were collected during their slaughter in outdoor markets in the city of Teresina, Piauí, Brazil. The Microscopic Agglutination Test (MAT) obtained 34 positive serological samples for one or more Leptospira spp. serovar with occurrence rate of 28.6 percent of leptospiral antibodies. There were 23 cases of infection for a single serovar, and 11 cases with coagglutination for two or more serovars. Autumnalis had the highest occurrence (29.4 percent) among the pathogenic serovars. The histopathological analysis of 36 kidney fragments revealed tubulo-interstitial alterations in 33 (91.7 percent) positive animals. Tubular lesions were observed in 20 (55.5 percent) positive animals. The Warthin Starry staning revealed the presence of Leptospira in 8 (22.20 percent) of the 36 positive samples. The immunoperoxidase staining revealed the presence of Leptospira in 12 (60 percent) of 20 positive samples. The inflammatory infiltrate in the positive animals was significantly more evident in the cortical-medullar and cortical regions than in the medullar region (p=0.000), however, there was no difference between positive and negative animals. The presence of hyaline casts in the proximal tubules was significantly higher in positive animals compared to the negative ones (p=0.0001). Discrete lesion was observed in glomeruli. In conclusion, the results from this study showed that sheep which are positive for Leptospira present tubulo-intersticial renal lesions with the presence of Leptospira in the tubules, conferring to these animals the condition of asymptomatic carriers.

High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load.

BACKGROUND: Concomitant infections may influence HIV progression by causing chronic activation leading to decline in T-cell function. In the Americas, visceral (AVL) and tegumentary leishmaniasis (ATL) have emerged as important opportunistic infections in HIV-AIDS patients and both of those diseases have been implicated as potentially important co-factors in disease progression. We investigated whether leishmaniasis increases lymphocyte activation in HIV-1 co-infected patients. This might contribute to impaired cellular immune function. METHODS: To address this issue we analyzed CD4+ T absolute counts and the proportion of CD8+ T cells expressing CD38 in Leishmania/HIV co-infected patients that recovered after anti-leishmanial therapy. RESULTS: We found that, despite clinical remission of leishmaniasis, AVL co-infected patients presented a more severe immunossupression as suggested by CD4+ T cell counts under 200 cells/mm3, differing from ATL/HIV-AIDS cases that tends to show higher lymphocytes levels (over 350 cells/mm3). Furthermore, five out of nine, AVL/HIV-AIDS presented low CD4+ T cell counts in spite of low or undetectable viral load. Expression of CD38 on CD8+ T lymphocytes was significantly higher in AVL or ATL/HIV-AIDS cases compared to HIV/AIDS patients without leishmaniasis or healthy subjects. CONCLUSIONS: Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4+ T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.

Diminished CD4+/CD25+ T cell and increased IFN-gamma levels occur in dogs vaccinated with Leishmune in an endemic area for visceral leishmaniasis.

The Leishmune vaccine has been used in endemic areas to prevent canine visceral leishmaniasis in Brazil, but cytokine production induced by vaccination has rarely been investigated in dogs. This study aimed to evaluate the immune response of dogs vaccinated with Leishmune FML vaccine (Fort Dodge) against total antigen of Leishmania (Leishmania) chagasi (TAg) and FML. Twenty healthy dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniasis area, received three consecutive subcutaneous injection of Leishmune vaccine at 21-day intervals. PBMC were isolated before and 10 days after completing vaccination and lymphoproliferative response and antibody production against FML or total promastigote antigen were tested. Cytokines IFN-gamma, IL-4 and TNF-alpha were measured in culture supernatant and CD4+/CD25+ and CD8+/CD25+ T cell presence was determined. Analysis of the data indicated that the vaccine conferred humoral responses (100%) against both antigens and cellular immunity to FML (85%) and total antigen (80%), the supernatant of cultured cells stimulated with TAg and FML showed an increase in IFN-gamma (P<0.05), and the vaccine reduced CD4+/CD25+ T cell presence compared to that observed before vaccination. These responses may constitute part of the immune mechanism induced by Leishmune.