Murine Leishmaniasis Research Articles

Interleukin-4 Responsive Dendritic Cells Are Dispensable to Host Resistance Against Leishmania mexicana Infection.

IL-4 and IL-13 cytokines have been associated with a non-healing phenotype in murine leishmaniasis in L. mexicana -infected BALB/c mice as demonstrated in IL-4−/−, IL-13−/− and IL-4Rα-/- global knockout mouse studies. However, it is unclear from the studies which cell-type-specific IL-4/IL-13 signaling mediates protection to L. mexicana. Previous studies have ruled out a role for IL-4-mediated protection on CD4+ T cells during L. mexicana infections. A candidate for this role may be non-lymphocyte cells, particularly DCs, as was previously shown in L. major infections, where IL-4 production drives dendritic cell-IL-12 production thereby mediating a type 1 immune response. However, it is unclear if this IL-4-instruction of type 1 immunity also occurs in CL caused by L. mexicana, since the outcome of cutaneous leishmaniasis often depends on the infecting Leishmania species. Thus, BALB/c mice with cell-specific deletion of the IL-4Rα on CD11c+ DCs (CD11ccreIL-4Rα-/lox) were infected with L. mexicana promastigotes in the footpad and the clinical phenotype, humoral and cellular immune responses were investigated, compared to the littermate control. Our results show that CL disease progression in BALB/c mice is independent of IL-4Rα signaling on DCs as CD11ccreIL-4Rα-/lox mice had similar footpad lesion progression, parasite loads, humoral responses (IgE, IgG1, IgG 2a/b), and IFN-γ cytokine secretion in comparison to littermate controls. Despite this comparable phenotype, surprisingly, IL-4 production in CD11ccreIL-4Rα-/lox mice was significantly increased with an increasing trend of IL-13 when compared to littermate controls. Moreover, the absence of IL-4Rα signaling did not significantly alter the frequency of CD4 and CD8 lymphocytes nor their activation, or memory phenotype compared to littermate controls. However, these populations were significantly increased in CD11ccreIL-4Rα-/lox mice due to greater total cell infiltration into the lymph node. A similar trend was observed for B cells whereas the recruitment of myeloid populations (macrophages, DCs, neutrophils, and Mo-DCs) into LN was comparable to littermate IL-4Rα-/lox mice. Interestingly, IL-4Rα-deficient bone marrow-derived dendritic cells (BMDCs), stimulated with LPS or L. mexicana promastigotes in presence of IL-4, showed similar levels of IL-12p70 and IL-10 to littermate controls highlighting that IL-4-mediated DC instruction was not impaired in response to L. mexicana. Similarly, IL-4 stimulation did not affect the maturation or activation of IL-4Rα-deficient BMDCs during L. mexicana infection nor their effector functions in production of nitrite and arginine-derived metabolite (urea). Together, this study suggests that IL-4 Rα signaling on DCs is not key in the regulation of immune-mediated protection in mice against L. mexicana infection.

Morphological alterations of Thymus during the early murine leishmaniasis.

Leishmaniasis is a neglected and endemic disease that affects poorest population mainly in developing countries. Thymus provides an essential complex environment for T cell maturation and differentiation during leishmania infection. The aim of this study was to investigate the pathological alterations of the Thymus during early Leishmania amazonensis murine infection. BALB/c mice were infected with 105 amastigotes for 24 h, 3 days, 7 days, 15 days or 30 days. At different times of infection, the relative weight of the Thymus was obtained, and the Thymus cellularity was determined by counting total cells of one thymic lobe. The thymic lobe was, alternatively, processed for standard Haematoxylin and Eosin protocol. Our results suggest thymic alteration during the early days of BALB/c mice infection with L. amazonensis. The thymic hypertrophy was accompanied by histological alterations in Thymus architecture with thickening cortex at 3 days p.i. and loss of an evident delimitation between the cortex and medulla at 7 days p.i. when compared to the control mice. That is the first time that Thymus hypertrophy was observed during the early leishmaniasis. However, how it may contribute to infection susceptibility requires further investigation.

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.

Inoculation of the Leishmania infantum HSP70-II Null Mutant Induces Long-Term Protection against L. amazonensis Infection in BALB/c Mice.

Leishmania amazonensis parasites are etiological agents of cutaneous leishmaniasis in the New World. BALB/c mice are highly susceptible to L. amazonensis challenge due to their inability to mount parasite-dependent IFN-γ-mediated responses. Here, we analyzed the capacity of a single administration of the LiΔHSP70-II genetically-modified attenuated L. infantum line in preventing cutaneous leishmaniasis in mice challenged with L. amazonensis virulent parasites. In previous studies, this live attenuated vaccine has demonstrated to induce long-protection against murine leishmaniasis due to Old World Leishmania species. Vaccinated mice showed a reduction in the disease evolution due to L. amazonensis challenge, namely reduction in cutaneous lesions and parasite burdens. In contrast to control animals, after the challenge, protected mice showed anti-Leishmania IgG2a circulating antibodies accompanied to the induction of Leishmania-driven specific IFN-γ systemic response. An analysis performed in the lymph node draining the site of infection revealed an increase of the parasite-specific IFN-ϒ production by CD4+ and CD8+ T cells and a decrease in the secretion of IL-10 against leishmanial antigens. Since the immunity caused by the inoculation of this live vaccine generates protection against different forms of murine leishmaniasis, we postulate LiΔHSP70-II as a candidate for the development of human vaccines.

Evaluation of Leishmanization Using Iranian Lizard Leishmania Mixed With CpG-ODN as a Candidate Vaccine Against Experimental Murine Leishmaniasis.

Background and Objectives: The live non-pathogenic Leishmania tarantolae has recently provided a promising approach as an effective vaccine candidate against experimental leishmaniasis (ILL). Here, we evaluated the immunoprotective potential of the live Iranian Lizard Leishmania mixed with CpG adjuvant against L. major infection in BALB/c mice.Methods: Four groups of female BALB/c mice were included in the study. The first and second groups received PBS and CpG, respectively. The immunized groups received 2 × 105 ILL promastigotes and the CpG-mixed ILL (ILL+CpG). Injections were performed subcutaneously in the right footpad. Three weeks later, all mice were challenged with 2 × 105 metacyclic promastigotes of Leishmania majorEGFP; inoculation was done in the left footpad. The measurement of footpad swelling and in vivo fluorescent imaging were used to evaluate disease progress during infection course. Eight weeks after challenge, all mice were sacrificed and the cytokines levels (IFN-γ, IL-4, and IL-10) and sera antibodies concentrations (IgG2a and IgG1) using ELISA assay, nitric oxide production using Griess assay, and arginase activity in cultured splenocytes, were measured. In addition, direct fluorescent microscopy analysis and qPCR assay were used to quantify the splenic parasite burden.Result: The results showed that mice immunized with ILL+CpG were protected against the development of the dermal lesion. Moreover, they showed a significant reduction in the parasite load, in comparison to the control groups. The observed protection was associated with higher production of IFN-γ, as well as a reduction in IL-4 level. Additionally, the results demonstrated that arginase activity was decreased in ILL+CpG group compared to other groups.Conclusion: Immunization using ILL+CpG induces a protective immunity; indicating that ILL with an appropriate adjuvant would be a suitable choice for vaccination against leishmaniasis.

Induction of Regulatory T Cells in Leishmania major‒Infected BALB/c Mice Does Not Require Langerin+ Dendritic Cells

Cutaneous leishmaniasis is a disease caused by the protozoan parasite Leishmania major and transferred to humans by infected phlebotomine sand flies. Cutaneous leishmaniasis manifests in lesion formation of the infected skin and poses a severe health burden with 1 million cases reported in the past 5 years. In healthy individuals and murine experimental leishmaniasis of resistant C57BL/6 mice, the cutaneous disease can be controlled through T helper (Th)1‒ and/or cytotoxic T-lymphocyte‒mediated immune responses that are characterized by IFN-γ–driven macrophage activation and parasite killing, which induces live-long memory and protection.

Sugar-based colloidal nanocarriers for topical meglumine antimoniate application to cutaneous leishmaniasis treatment: Ex vivo cutaneous retention and in vivo evaluation

The leishmaniases are a group of diseases caused by protozoan parasites from Leishmania species. Effectiveness therapies for cutaneous leishmaniasis (CL), the most common form, are still needed to be developed since the available drugs such as meglumine antimoniate (MA) present severe adverse reactions. Here, we develop and characterize maltodextrin polymeric colloidal nanocarriers containing MA (PCN-MA) for topical CL treatment. PCN-MA is composed of 5 to 8% maltodextrin, 0.3% NaCl, 1% MA in 21% of water as aqueous-internal phase, containing or no 3% Kolliphor® P-188, and 10% SF1540 dispersed in a silicone-based external phase. It formed a colloidal system dispersed in silicone with high encapsulation efficiency (87% to 92%) and composite spherical-shaped particles with the smooth and regular surface within the nanosized scale, which was confirmed by scanning electron microscopy (SEM) and dynamic light scattering (DLS) analysis. Ex vivo cutaneous retention studies using pig ears skin on Franz diffusion cells revealed that the MA cutaneous retention is improved when delivered by PCN. Topical PCN-MA evaluation in murine leishmaniasis model showed similar efficacy than the intraperitoneal injection of the reference medicine (Glucantime®) regarding parasite titer reduction and superior healing activity in terms of collagen area deposition. Our results suggest that this sugar-based PCN is a promising agent for topical delivery of meglumine antimoniate.

Broad Spectrum and Safety of Oral Treatment with a Promising Nitrosylated Chalcone in Murine Leishmaniasis.

The oral efficacy and safety of a leishmanicidal nitrochalcone (CH8) were studied in BALB/c mouse infections with Leishmania amazonensis and Leishmania infantum Although 10-fold-higher doses of CH8 were needed to produce the same antiparasitic effect as that seen with the reference drug miltefosine, the latter was nephrotoxic, whereas CH8 restored disease toxicity markers to normal. This study shows the therapeutic potential of an orally active and hepato-/nephroprotective chalcone against cutaneous and visceral leishmaniases.

T cell exhaustion during murine cutaneous and visceral leishmaniasis.

Abstract Leishmaniasis is a chronic progressive disease usually controlled by TH1-type IFN-γ producing cells. Human or murine leishmaniasis can progress despite the presence of T cells capable of producing IFN-γ. It is reported that CD8 cells in humans with visceral leishmaniasis (VL) express markers of T cell exhaustion. We hypothesized that myeloid cells play a role in exhausting T cells at the local sites of Leishmania species infection inhibiting proper adaptive responses. We addressed this hypothesis in BALB/c models of cutaneous leishmaniasis (CL) or VL caused by L. major or L. donovani infection, respectively. We examined inhibitory receptors PD1, LAG3, CTLA4 and TIM3 on lymphoid cells, and counter-receptors PDL1, MHCII, and CD80 in myeloid cell by flow cytometry throughout 4 weeks of infection. Both models showed significant increases in PD-1 in circulating dendritic cells after 1 week of infection. However, after 3–4 weeks when infection was chronic, PDL-1 was expressed on neutrophils in the blood and at the local sites of infection (infected ear tissue and draining lymph nodes of L. major infected mice; spleen cells of L. donovani-infected mice). Furthermore, PD1 was expressed on both CD4 and CD8 T cells in blood and lymph nodes of mice infected with L. major at the same time. In contrast, only CD4 T cells, but not CD8 T cells) displayed higher PD-1 in blood and spleens of mice infected with L. donovani. In conclusion, both disease models elicited the exhaustion counter-receptor PDL1 on neutrophils in the blood and the local site of parasite growth. The T cell exhaustion marker PD1 was expressed on T cells at the same sites, although both CD4 and CD8 T cells displayed PD1 in the CL model, whereas PD1 expression was more profound on CD4 T cells in the VL model.

Concentration of 2′C-methyladenosine triphosphate by Leishmania guyanensis enables specific inhibition of Leishmania RNA virus 1 via its RNA polymerase

Leishmania is a widespread trypanosomatid protozoan parasite causing significant morbidity and mortality in humans. The endobiont dsRNA virus Leishmania RNA virus 1 (LRV1) chronically infects some strains, where it increases parasite numbers and virulence in murine leishmaniasis models, and correlates with increased treatment failure in human disease. Previously, we reported that 2'-C-methyladenosine (2CMA) potently inhibited LRV1 in Leishmania guyanensis (Lgy) and Leishmania braziliensis, leading to viral eradication at concentrations above 10 μm Here we probed the cellular mechanisms of 2CMA inhibition, involving metabolism, accumulation, and inhibition of the viral RNA-dependent RNA polymerase (RDRP). Activation to 2CMA triphosphate (2CMA-TP) was required, as 2CMA showed no inhibition of RDRP activity from virions purified on cesium chloride gradients. In contrast, 2CMA-TP showed IC50 values ranging from 150 to 910 μm, depending on the CsCl density of the virion (empty, ssRNA-, and dsRNA-containing). Lgy parasites incubated in vitro with 10 μm 2CMA accumulated 2CMA-TP to 410 μm, greater than the most sensitive RDRP IC50 measured. Quantitative modeling showed good agreement between the degree of LRV1 RDRP inhibition and LRV1 levels. These results establish that 2CMA activity is due to its conversion to 2CMA-TP, which accumulates to levels that inhibit RDRP and cause LRV1 loss. This attests to the impact of the Leishmania purine uptake and metabolism pathways, which allow even a weak RDRP inhibitor to effectively eradicate LRV1 at micromolar concentrations. Future RDRP inhibitors with increased potency may have potential therapeutic applications for ameliorating the increased Leishmania pathogenicity conferred by LRV1.

Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites.

Activated natural killer (NK) cells release interferon (IFN)-γ, which is crucial for the control of intracellular pathogens such as Leishmania. In contrast to experimental murine leishmaniasis, the human NK cell response to Leishmania is still poorly characterized. Here, we investigated the interaction of human blood NK cells with promastigotes of different Leishmania species (Leishmania major, Leishmania mexicana, Leishmania infantum, and Leishmania donovani). When peripheral blood mononuclear cells or purified NK cells and monocytes (all derived from healthy blood donors from Germany without a history of leishmaniasis) were exposed to promastigotes, NK cells showed increased surface expression of the activation marker CD69. The extent of this effect varied depending on the Leishmania species; differences between dermotropic and viscerotropic L. infantum strains were not observed. Upregulation of CD69 required direct contact between monocytes and Leishmania and was partly inhibitable by anti-interleukin (IL)-18. Unexpectedly, IL-18 was undetectable in most of the supernatants (SNs) of monocyte/parasite cocultures. Confocal fluorescence microscopy of non-permeabilized cells revealed that Leishmania-infected monocytes trans-presented IL-18 to NK cells. Native, but not heat-treated SNs of monocyte/Leishmania cocultures also induced CD69 on NK cells, indicating the involvement of a soluble heat-labile factor other than IL-18. A role for the NK cell-activating cytokines IL-1β, IL-2, IL-12, IL-15, IL-21, and IFN-α/β was excluded. The increase of CD69 was not paralleled by NK cell IFN-γ production or enhanced cytotoxicity. However, prior exposure of NK cells to Leishmania parasites synergistically increased their IFN-γ release in response to IL-12, which was dependent on endogenous IL-18. CD1c+ dendritic cells were identified as possible source of Leishmania-induced IL-12. Finally, we observed that direct contact between Leishmania and NK cells reduced the expression of CD56 mRNA and protein on NK cells. We conclude that Leishmania activate NK cells via trans-presentation of IL-18 by monocytes and by a monocyte-derived soluble factor. IL-12 is needed to elicit the IFN-γ-response of NK cells, which is likely to be an important component of the innate control of the parasite.

Cutaneous Manifestations of Human and Murine Leishmaniasis.

The leishmaniases are diseases caused by pathogenic protozoan parasites of the genus Leishmania. Infections are initiated when a sand fly vector inoculates Leishmania parasites into the skin of a mammalian host. Leishmania causes a spectrum of inflammatory cutaneous disease manifestations. The type of cutaneous pathology is determined in part by the infecting Leishmania species, but also by a combination of inflammatory and anti-inflammatory host immune response factors resulting in different clinical outcomes. This review discusses the distinct cutaneous syndromes described in humans, and current knowledge of the inflammatory responses associated with divergent cutaneous pathologic responses to different Leishmania species. The contribution of key hematopoietic cells in experimental cutaneous leishmaniasis in mouse models are also reviewed and compared with those observed during human infection. We hypothesize that local skin events influence the ensuing adaptive immune response to Leishmania spp. infections, and that the balance between inflammatory and regulatory factors induced by infection are critical for determining cutaneous pathology and outcome of infection.

Nanoliposomal Buparvaquone Immunomodulates Leishmania infantum-Infected Macrophages and Is Highly Effective in a Murine Model.

Visceral leishmaniasis is a fatal parasitic neglected disease affecting 1.5 million people worldwide. Based on a drug repositioning approach, the aim of this work was to investigate the in vitro immunomodulatory potential of buparvaquone (BPQ) and to establish a safe regimen to evaluate the in vivo efficacy of BPQ entrapped by negatively charged nanoliposomes (BPQ-LP) in Leishmania infantum-infected hamsters. Small-angle X-ray scattering, dynamic light scattering, and the ζ-potential were applied in order to study the influence of BPQ on the liposome structure. Our data revealed that BPQ was located in the polar-apolar interface, snorkeling the polar region, and protected against aggregation inside the lipophilic region. The presence of BPQ also decreased the Z-average hydrodynamic diameter and increased the surface charge. Compared to intravenous and intramuscular administration, a subcutaneous route was a more effective route for BPQ-LP; at 0.4 mg/kg, BPQ-LP reduced infection in the spleen and liver by 98 and 96%, respectively. Treatment for 5 days resulted in limited efficacy, but 10 days of treatment resulted in an efficacy similar to that of a 15-day regimen. The nanoliposomal drug was highly effective, with a mean 50% effective dose of 0.25 mg/kg, reducing the parasite load in bone marrow by 80%, as detected using quantitative PCR analysis. In addition, flow cytometry studies showed that BPQ upregulated cytokines as tumor necrosis factor, monocyte chemoattractant protein 1, interleukin-10 (IL-10), and IL-6 in Leishmania-infected macrophages, eliminating the parasites via a nitric oxide-independent mechanism. This new formulation proved to be a safe and effective treatment for murine leishmaniasis that could be a useful candidate against visceral leishmaniasis.

Myeloid cells do not contribute to gender-dependent differences in disease outcome in murine cutaneous leishmaniasis

Gender-associated differences in the outcome of infections are well known. Apart from behavior-released differences in their incidence, immunological factors also contribute to disease outcome. The underlying mechanisms are often unknown. Here, we show that in murine experimental leishmaniasis, female mice develop larger skin lesions that harbor significantly more parasites, exhibit increased parasite dissemination to visceral organs associated with a shift towards T helper (Th) 2 immunity with increased levels of IL-4. Antigen presenting cells (APC) responsible for T cell priming, such as macrophages or dendritic cells, were not involved in the process. Additionally, in adoptive transfer experiments, we show that differences in the lymphoid lineage are also not critical for mediating these gender-dependent effects. In summary, neither myeloid nor lymphoid cells contribute to disease outcome against this important human pathogen, but stromal cells influenced by e.g. hormonal effects in addition to other parts of the immune system might play a role.

An 8-hydroxyquinoline-containing polymeric micelle system is effective for the treatment of murine tegumentary leishmaniasis.

The current treatment of leishmaniasis has been hampered due to the high toxicity of the available drugs and long duration protocols, which often lead to its abandonment. In the present study, a poloxamer 407-based delivery system was developed, and a molecule, 8-hydroxyquinoline (8-HQN), was incorporated with it, leading to an 8-HQN/micelle (8-HQN/M) composition. Assays were performed to evaluate the in vitro antileishmanial activity of 8-HQN/M against Leishmania amazonensis stationary promastigotes. The cytotoxicity in murine macrophages and in human red cells, as well as the efficacy of the treatment in macrophages infected by parasites, was also assessed. This product was also evaluated for the treatment of murine tegumentary leishmaniasis, using L. amazonensis-infected BALB/c mice. To evaluate the in vivo efficacy of the treatment, the average lesion diameter (area) in the infected tissue, as well as the parasite load at the site of infection (skin), spleen, liver and draining lymph nodes were examined. Non-incorporated micelle (B-8-HQN/M) and the free molecule (8-HQN) were used as controls, besides animals that received only saline. The parasite burden was evaluated by limiting dilution and quantitative real-time PCR (qPCR) techniques, and immunological parameters associated with the treatments were also investigated. In the results, the 8-HQN/M group, when compared to the others, presented more significant reductions in the average lesion diameter and in the parasite burden in the skin and all evaluated organs. These animals also showed significantly higher levels of parasite-specific IFN-γ, IL-12, and GM-CSF, associated with low levels of IL-4 and IL-10, when compared to the saline, 8-HQN/M, and B-8-HQN groups. A predominant IL-12-driven IFN-γ production, against parasite proteins, mainly produced by CD4+ T cells, was observed in the treated animals, post-infection. In conclusion, 8-HQN/M was highly effective in treating L. amazonensis-infected BALB/c mice and can be considered alone, or combined with other drugs, as an alternative treatment for tegumentary leishmaniasis. Graphical Abstract Therapeutic scheme and immunological and parasitological parameters developed in the present study.

Mast cells have no impact on cutaneous leishmaniasis severity and related Th2 differentiation in resistant and susceptible mice.

The genus leishmania comprises different protozoan parasites which are causative agents of muco-cutaneous and systemic, potentially lethal diseases. After infection with the species Leishmania major, resistant mice expand Th1 cells which stimulate macrophages for Leishmania destruction. In contrast, susceptible mice generate Th2 cells which deactivate macrophages, leading to systemic spread of the pathogens. Th-cell differentiation is determined within the first days, and Th2 cell differentiation requires IL-4, whereby the initial IL-4 source is often unknown. Mast cells are potential sources of IL-4, and hence their role in murine leishmaniasis has previously been studied in mast cell-deficient Kit mutant mice, although these mice display immunological phenotypes beyond mast cell deficiency. We therefore readdressed this question by infecting Kit-independent mast cell-deficient mice that are Th1 (C57BL/6 Cpa(Cre) ) or Th2 (BALB/c Cpa(Cre) ) prone with L. major. Using different parasite doses and intra- or subcutaneous infection routes, the results demonstrate no role of mast cells on lesion size development, parasite load, immune cell phenotypes expanding in draining lymph nodes, and cytokine production during murine cutaneous leishmaniasis. Thus, other cell types such as ILCs or T cells have to be considered as primary source of Th2-driving IL-4.

Presence ofLeishmaniaRNA Virus 1 inLeishmania guyanensisIncreases the Risk of First-Line Treatment Failure and Symptomatic Relapse

Treatment failure and symptomatic relapse are major concerns in American tegumentary leishmaniasis (TL). Such complications are seen frequently in Leishmania guyanensis infections, in which patients respond variously to first-line antileishmanials and are more prone to develop chronic cutaneous leishmaniasis. The factors underlying this pathology, however, are unknown. Recently, we reported that a double-stranded RNA virus, Leishmania RNA virus 1 (LRV1), nested within L. guyanensis parasites is able to exacerbate experimental murine leishmaniasis by inducing a hyperinflammatory response. This report investigates the prevalence of LRV1 in human L. guyanensis infection and its effect on treatment efficacy, as well as its correlation to symptomatic relapses after the completion of first-line treatment. In our cohort of 75 patients with a diagnosis of primary localized American TL, the prevalence of LRV1-positive L. guyanensis infection was elevated to 58%. All patients infected with LRV1-negative L. guyanensis were cured after 1 dose (22 of 31 [71%]) or 2 doses (31 of 31 [100%]) of pentamidine. In contrast, 12 of 44 LRV1-positive patients (27%) presented with persistent infection and symptomatic relapse that required extended therapy and the use of second-line drugs. Finally, LRV1 presence was associated with a significant increase in levels of intra-lesional inflammatory markers. In conclusion, LRV1 status in L. guyanensis infection is significantly predictive (P = .0009) of first-line treatment failure and symptomatic relapse and has the potential to guide therapeutic choices in American TL.

Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice.

BackgroundHighly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs).Methodology/Principal FindingsThree different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid.Conclusion/SignificanceThe results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis.

Modulation of inflammation response to murine cutaneous Leishmaniosis by homeopathic medicines: Thymulin 5cH

In previous studies, we observed that thymulin 5cH could modulate BCG (Bacillus Calmette-Guerin) induced chronic inflammation by increasing peritoneal B1 stem cells differentiation into phagocytes and improving phagocytosis efficiency. We used the same protocol to study the effects of thymulin 5cH in the experimental murine Leishmaniasis, in order to elucidate some aspects of the parasite-host relation under this homeopathic treatment. Male Balb/c mice were orally treated with thymulin 5cH or vehicle during 60 days, after the subcutaneous inoculation of 2 × 10(6) units of Leishmania (L.) amazonensis into the footpad. Washied inflammatory cell suspension from peritoneal cavity, spleen, local lymph node and infected subcutaneous tissue were harvested after 2 and 60 days from infection to quantify the inflammation cells by flow cytometry and histometry methods. After a transitory increase of peritoneal T reg cells, treated mice presented, chronically, increase in the peritoneal and spleen B1 cells percentage (p = 0.0001) in relation to other cell types; more organized and exuberant inflammation response in the infection site, and decrease in the number of parasites per field inside the primary lesion (p = 0.05). No difference was seen in local lymph node histology. Thymulin 5cH is able to improve B1 cell activation and Leishmania (L) amazonensis phagocytosis efficiency in mice, similarly to that observed previously in BCG experimental infection.

267 : Challenging the Th1 paradigm: A detrimental role for IFN-gamma and IFN-regulated CD64 in human leishmaniasis

Our previous findings suggested a deleterious role for IFN-beta in human leishmaniasis, in contrast to murine leishmaniasis. In addition, an antagonistic effect of IFN-beta upon IFN-gamma bactericidal activity has been recently shown in human leprosy, another cutaneous pathology caused by an intracellular pathogen. Here we show a similar IFN-beta/gamma antagonism for both leishmanicidal activity and monocyte/macrophage CD64 expression, suggesting that high/low CD64 expression, reflecting IFN-gamma vs. IFN-beta activity, respectively, would be associated to a positive vs negative clinical outcome in human leishmaniasis. We found that ex vivo monocyte CD64 level was significantly elevated, but, unexpectedly, positively correlated to therapeutic failure in two independent cutaneous leishmaniasis cohorts ( p p = 0.003). To further explore a possible IFN-gamma/beta gene signature and its relationship to parasite burden in vivo , we employed nCounter technology to quantify >700 human and Leishmania RNAs in situ in biopsies from the whole clinical spectrum of human cutaneous leishmaniasis. IFN-gamma mRNA level (G6PD-normalized) was significantly and positively correlated to macrophage IFN-regulated CD64, CD80 and HLA-DR mRNAs. In contrast, IFN-beta mRNA was undetectable/low in most biopsies. Neither IFN-gamma nor IFN-beta mRNA levels were significantly correlated to parasite burden, suggesting the deleterious role of increased IFN-gamma/CD64 might be related to an exaggerated pro-inflammatory response in situ . Next, we found that CD64-targeted immunotoxins selectively induce apoptosis and decrease parasite survival in Leishmania -infected human macrophages in vitro . Finally, CD64-immunotoxin treatment in vivo decreases lesion size, parasite load and inflammation in infected HuCD64-transgenic mice, but not control WT mice. In conclusion, our results reveal the therapeutic potential of CD64-immunotoxin treatment in cutaneous leishmaniasis, but also challenge the clinical relevance of Th1 boosting, as suggested in established murine models, for therapeutic and vaccination strategies in human leishmaniasis.