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Abstract: The purinergic P2X7 receptor is a membrane protein of leucocytes involved in the clearance of intracellular bacteria such as Chlamydia and Mycobacterium. In this work, we investigated the role and modulation of macrophage P2X7R in intracellular infection with the protozoan parasite Leishmania amazonensis. Upon infection, isolated murine macrophages displayed enhanced expression of P2X7R and were significantly more responsive to extracellular ATP (ATPe)-induced pore opening, as demonstrated by the increased uptake of Lucifer Yellow. This was extended to the in vivo situation, where cells from established cutaneous lesions were more sensitive to ATPe than cells from uninfected mice. ATP treatment of infected macrophages inhibited parasite growth, and this was prevented by pre-treatment with oxidized ATP, a selective antagonist of P2X7R. Parasite killing was unlikely due to induction of nitric oxide production or cytolysis of infected macrophage, as those functions were unaltered with parasite-effective ATPe concentrations. A direct drug effect is also unlike, as ATPe enhanced axenic parasite growth. We found that leishmanial infection rendered wild-type but not P2X7R-deficient macrophages more prone to ATP-induced apoptosis. These results show that macrophage infection with L. amazonensis leads to enhanced expression of functional P2X7R, that upon ligation with ATPe helps in the elimination of the parasites by an as yet unclear mechanism possibly involving host cell apoptosis. [Copyright &y& Elsevier]

Leishmania spp. depend on effective macrophage infection to establish and develop in mammalian hosts. Both metacyclic promastigotes and amastigotes are able to infect host cells, and thus they rely on several ligands that, when recognized by macrophage receptors, mediate parasite uptake. During macrophage primary infection with metacyclic forms from the insect vector and during amastigote dissemination via macrophage rupture, both infective stages have to cope with the host extracellular microenvironment, including extracellular matrix molecules. Glycosaminoglycans are abundant in the extracellular matrix and many of these molecules are able to interact with the parasite and the host cell, mediating positive and negative effects for the infection, depending on their structure and/or location. In addition, glycosaminoglycans are present at the surface of macrophages as proteoglycans, playing important roles for parasite recognition and uptake. In this review, we discuss glycosaminoglycans in the context of Leishmania infection as well as the possible applications of the current knowledge regarding these molecules for the development of new therapeutic strategies to control parasite dissemination. [ABSTRACT FROM AUTHOR]

Background: Previously, we demonstrated that unlike subcutaneous or intramuscular vaccination, intranasal vaccination of BALB/c mice with whole Leishmania amazonensis antigens leads to protection against cutaneous leishmaniasis. Here, the role of parasite serine proteases in the protective immunity was investigated. Findings: Serine Proteases were partially purified from both soluble (LaSP-Sol) and extracellular (LaSP-Ex) Leishmania amazonensis promastigote extracts by aprotinin-agarose chromatography. BALB/c mice were intranasally immunized with LaSP-Sol and LaSP-Ex prior to infection with L. amazonensis. LaSP-Ex but not LaSP-Sol vaccination led to significantly smaller lesions and parasite burdens as compared with non-vaccinated controls. Protection was accompanied by systemic Th1 polarization with increased IFN-ã and decreased IL-4 and IL-10 splenic production. Likewise, increased production of IFN-ã, IL-12 and IL-4 concomitant with decreased TGF-â and TNF-á was locally observed in the infected footpad. Conclusion: This study indicates that extracellular serine proteases of L. amazonensis are strong candidates for a more defined intranasal vaccine against cutaneous leishmaniasis. [ABSTRACT FROM AUTHOR]

Summary Nucleotides are released into the extracellular milieu from infected cells and cells at inflammatory sites. The extracellular nucleotides bind to specific purinergic (P2) receptors and thereby induce a variety of cellular responses including anti-parasitic effects. Here we investigated whether extracellular nucleotides affect leishmanial infection in macrophages, and found that UTP reduces strongly the parasite load in peritoneal macrophages. Ultrastructural analysis of infected cells revealed that UTP induced morphological damage in the intracellular parasites. Uridine nucleotides also induced dose-dependent apoptosis of macrophages and production of ROI and RNI only in infected macrophages. The intracellular calcium measurements of infected cells showed that the response to UTP, but not UDP, increased the sensitivity and amplitude of cytosolic Ca2+ changes. Infection of macrophages with Leishmania upregulated the expression of P2Y2 and P2Y4 receptor mRNA. The data suggest indirectly that Leishmania amazonensis infection induces modulation and heteromerization of P2Y receptors on macrophages. Thus UTP modulates the host response against L. amazonensis infection. UTP and UTP homologues should therefore be considered as novel components of therapeutic strategies against cutaneous leishmaniasis. [ABSTRACT FROM AUTHOR]

Abstract: We previously demonstrated that intradermal and intramuscular vaccination with Leishmania amazonensis promastigote antigens (LaAg) increases the susceptibility of BALB/c mice to cutaneous leishmaniasis. In this study, we investigated the role played by serine and cysteine proteases as disease-promoting components of LaAg. Mice were immunized by the intramuscular route with LaAg that was pre-treated with a pool of serine or cysteine protease inhibitors (SPi and CPi, respectively) prior to infection with L. amazonensis. Neutralization of either enzyme type reversed the disease-promoting effect of LaAg, as seen by the slower lesion development. However, the parasite burden was only effectively controlled in mice receiving SPi-treated LaAg. Protection was associated with diminished production of TGF-β and particularly IL-10 in response to parasite antigens by the lesion-draining lymph node cells of vaccinated mice relative to control. In vitro, soluble proteases isolated from LaAg (LaSP-Sol) directly activated IL-4, IL-10 and TGF-β production by immune cells. Like native LaAg, vaccination with LaSP-Sol primed mice to respond to parasite challenge with a strong Jones-Mote cutaneous hypersensitivity reaction, and increased susceptibility to infection. Furthermore, neutralization of serine but not cysteine proteases blocked the capacity of LaAg to sensitize mice for Jones-Mote reaction. Together, these results indicate that soluble serine proteases are key components of LaAg responsible for its disease-promoting immunity. [Copyright &y& Elsevier]

Abstract Leishmaniasis is considered a serious public health problem and the current available therapy has several disadvantages, which makes the search for new therapeutic targets and alternative treatments extremely necessary. In this context, this review focuses on the importance of parasite proteases as target drugs against Leishmania parasites, as a chemotherapy approach. Initially, we discuss about the current scenario for the treatment of leishmaniasis, highlighting the main drugs used and the problems related to their use. Subsequently, we describe the inhibitors of major proteases of Leishmania already discovered, such as Compound s9 (aziridine-2,3-dicarboxylate), Compound 1c (benzophenone derivative), Au2Phen (gold complex), AubipyC (gold complex), MDL 28170 (dipeptidyl aldehyde), K11777, Hirudin, diazo-acetyl norleucine methyl ester, Nelfinavir, Saquinavir, Nelfinavir, Saquinavir, Indinavir, Saquinavir, GNF5343 (azabenzoxazole), GNF6702 (azabenzoxazole), Benzamidine and TPCK. Next, we discuss the importance of the protease gene to parasite survival and the aspects of the validation of proteases as target drugs, with emphasis on gene disruption. Then, we describe novel important strategies that can be used to support the research of new antiparasitic drugs, such as molecular modeling and nanotechnology, whose main targets are parasitic proteases. And finally, we discuss possible perspectives to improve drug development. Based on all findings, proteases could be considered potential targets against leishmaniasis. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Highlights • Leishmania amazonensis infection induced IL-10 production by follicular B cells. • Infected Xid mice had delayed lesion growth and lower levels of IL-10 production than WT. • Infected Xid presented lower number of total follicular B cells than WT. • Infected Xid mice presented lower levels of serum IgM, IgG. IgG1, IgG2a and IgG2b antibodies. • Infected Xid exhibited similar production of IL-10 by T Cells but less by B cells. Abstract This work aims to study the immunomodulation of B lymphocytes during L. amazonensis infection. We demonstrated in this study that follicular B cells from draining lymph nodes of infected wild type BALB/c mice are the major source of IL-10 during infection. We infected BALB/Xid mice that developed smaller lesions in comparison with the control, but the parasite load obtained from the infected tissues was similar in both groups. We observed a reduction in the number of follicular B cells from BALB/Xid mice in relation to WT mice and, consequently, lower levels of IgM, IgG, IgG1, IgG2a and IgG2b in the serum of BALB/Xid when compared with wild type mice. BALB/Xid mice also presented lower levels of IL-10 in the infected footpad, draining lymph nodes and in the spleen when compared with WT infected tissues. We did not detect differences in the number of IL-10 producing CD4+ and CD8+ T cells between WT and BALB/Xid mice; however, a strong reduction of IL-10 producing follicular B cells was noted in BALB/Xid mice. When analyzed together, our data indicate that B cells are related with lesion pathogenesis through the production of antibodies and IL-10. [ABSTRACT FROM AUTHOR]

Background: We have previously demonstrated that intranasal vaccination of highly susceptible BALB/c mice with whole Leishmania amazonensis antigens (LaAg) leads to protection against murine cutaneous leishmaniasis. Here, we evaluate the response of partially resistant C57BL/6 mice to vaccination as a more representative experimental model of human cutaneous leishmaniasis. Methods: C57BL/6 mice from different animal facilities were infected with L. amazonensis (Josefa strain) to establish the profile of infection. Intranasal vaccination was performed before the infection challenge with two doses of 10 μg of LaAg alone or associated with the adjuvant ADDAVAX® by instillation in the nostrils. The lesion progression was measured with a dial caliper and the parasite load by limited dilution assay in the acute and chronic phases of infection. Cytokines were quantified by ELISA in the homogenates of infected footpads. Results: C57BL/6 mice from different animal facilities presented the same L. amazonensis infection profile, displaying a progressive acute phase followed by a controlled chronic phase. Parasites cultured in M199 and Schneider’s media were equally infective. Intranasal vaccination with LaAg led to milder acute and chronic phases of the disease. The mechanism of protection was associated with increased production of IFN-gamma in the infected tissue as measured in the acute phase. Association with the ADDAVAX® adjuvant did not improve the efficacy of intranasal LaAg vaccination. Rather, ADDAVAX® reduced vaccination efficacy. Conclusion: This study demonstrates that the efficacy of adjuvant-free intranasal vaccination with LaAg is extendable to the more resistant C57Bl/6 mouse model of infection with L. amazonensis, and is thus not exclusive to the susceptible BALB/c model. These results imply that mucosal immunomodulation by LaAg leads to peripheral protection irrespective of the genetic background of the host. [ABSTRACT FROM AUTHOR]