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Diego M Santos1, Marcia W Carneiro1, Tatiana R de Moura1, Kiyoshi Fukutani1, Jorge Clarencio1, Manuel Soto2, Socorro Espuelas3,4, Claudia Brodskyn1,5, Aldina Barral1,5, Manoel Barral-Netto1,5, Camila I de Oliveira1,51Centro de Pesquisas Gonçalo Moniz, FIOCRUZ, Salvador, BA, Brazil; 2Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas, Departamento de Biologia Molecular, Universidad Autonoma de Madrid, Madrid; 3Departamento de Farmacia y Tecnología Farmacéutica, 4Instituto de Salud Tropical, Facultad de Farmacia, Universidad de Navarra, Pamplona, Spain; 5Instituto de Investigação em Imunologia, Salvador, BA, BrazilBackground: Vaccine development has been a priority in the fight against leishmaniases, which are vector-borne diseases caused by Leishmania protozoa. Among the different immunization strategies employed to date is inoculation of plasmid DNA coding for parasite antigens, which has a demonstrated ability to induce humoral and cellular immune responses. In this sense, inoculation of plasmid DNA encoding Leishmania kinetoplasmid membrane protein-11 (KMP-11) was able to confer protection against visceral leishmaniasis. However, recently the use of antigen delivery systems such as poly(lactic-co-glycolic acid) (PLGA) nanoparticles has also proven effective for eliciting protective immune responses.Methods: In the present work, we tested two immunization strategies with the goal of obtaining protection, in terms of lesion development and parasite load, against cutaneous leishmaniasis caused by L. braziliensis. One strategy involved immunization with plasmid DNA encoding L. infantum chagasi KMP-11. Alternatively, mice were primed with PLGA nanoparticles loaded with the recombinant plasmid DNA and boosted using PLGA nanoparticles loaded with recombinant KMP-11.Results: Both immunization strategies elicited detectable cellular immune responses with the presence of both proinflammatory and anti-inflammatory cytokines; mice receiving the recombinant PLGA nanoparticle formulations also demonstrated anti-KMP-11 IgG1 and IgG2a. Mice were then challenged with L. braziliensis, in the presence of sand fly saliva. Lesion development was not inhibited following either immunization strategy. However, immunization with PLGA nanoparticles resulted in a more prominent reduction in parasite load at the infection site when compared with immunization using plasmid DNA alone. This effect was associated with a local increase in interferon-gamma and in tumor necrosis factor-alpha. Both immunization strategies also resulted in a lower parasite load in the draining lymph nodes, albeit not significantly.Conclusion: Our results encourage the pursuit of immunization strategies employing nanobased delivery systems for vaccine development against cutaneous leishmaniasis caused by L. braziliensis infection.Keywords: vaccine, nanoparticle, Leishmania, kinetoplastid membrane protein-11

To examine the role of different immunoglobulin subclasses in the immune clearance of Trypanosoma cruzi, mice containing bloodstream trypomastigotes were injected intravenously with immune serum, IgG-depleted serum, or with the IgG11 or IgG2 fraelions and the rate of removal of the parasites from circulation was determined. Using IgG concentrations similar to those found in the immune serum, the rate of clearance mediated by IgG2 was six-fold higher than that obtained with IgG1. This difference did not appear to be due to differences in antibody specificity, as Western blotting showed that each isotype recognized a similar set of antigens extracted from the parasite.However, the T. cruzi specific antibody content of the IgG2 was approximately five-fold higher than IgG1 - When the dose of IgG was adjusted to equalize the antibody content, the clearance ability of the IgG1 and IgG2 was very similar. It is concluded that the two subclasses have a similar clearanceability. [ABSTRACT FROM AUTHOR]

Abstract Background Sand fly saliva contains potent and complex pharmacologic molecules that are able to modulate the host's hemostatic, inflammatory, and immune systems. In this study, we evaluated the effects of salivary gland sonicate (SGS) of Lutzomyia intermedia, the natural vector of Leishmania braziliensis, on monocytes obtained from the peripheral blood mononuclear cells (PBMC) of healthy volunteers. We investigated the effects of sand fly saliva on cytokine production and surface molecule expression of LPS-stimulated human monocytes uninfected or infected with L. braziliensis. Results Pre-treatment of non-infected human monocytes with L. intermedia SGS followed by LPS-stimulation led to a significant decrease in IL-10 production accompanied by a significant increase in CD86, CD80, and HLA-DR expression. Pre-treatment with SGS followed by LPS stimulation and L. braziliensis infection led to a significant increase in TNF-α, IL-6, and IL-8 production without significant alterations in co-stimulatory molecule expression. However, pre-treatment with L. intermedia SGS did not result in significant changes in the infection rate of human monocytes. Conclusion Our data indicate that L. intermedia saliva is able to modulate monocyte response, and, although this modulation is dissociated from enhanced infection with L. braziliensis, it may be associated with successful parasitism.

Abstract Background Leishmaniasis remains a serious public health problem in several parts of the developing world. Effective prophylactic measurements are hampered by imprecise comprehension of different aspects of the disease, including its immunoregulation. A better comprehension of immunoregulation in human VL may be useful both for designing and evaluating immunoprophylaxis. Methods To explore immunoregulatory mechanisms, 20 visceral leishmaniasis (VL) patients were evaluated during active disease and at different periods up to one year after treatment determining their plasma cytokine levels, clinical parameters (palpable spleen and liver) and antibody levels. Results Elevated plasma levels of IFN-γ and of IL-12 p40 were observed during active disease, significantly decreasing after treatment whereas in vitro Leishmania antigen-stimulated IFN-γ production by PBMC exhibited an inverse pattern being low during disease and increasing steadily thereafter. Absence of IFN-γ activity is a hallmark of VL. The main candidate for blunting IFN-γ activity is IL-10, a cytokine highly elevated in plasma with sharp decrease after treatment. Activity of IL-10 is inferred by high levels of anti-Leishmania specific IgG1 and IgG3. TGF-β had elevated total, but not of active, levels lessening the likelihood of being the IFN-γ counterpart. Spleen or liver size presented a steady decrease but return to normal values at only 120 days after treatment. Anti-Leishmania IgG (total and subclasses) levels and DTH or Leishmania-stimulated lymphocyte proliferation conversion to positive also present a slow decrease after treatment. IL-6 plasma levels were elevated in only a few patients. Conclusion Taken together our results suggest that IFN-γ and IL-10 are the molecules most likely involved in determining fate of disease. After treatment, there is a long delay before the immune profile returns to normal what precludes using plasma cytokine levels as criteria of cure as simpler clinical evaluations, as a palpable spleen or liver, can be used.

In the search for new biomarkers and therapeutic targets for infectious diseases, several molecules have been investigated. Small RNAs, known as microRNAs (miRs), are important regulators of gene expression, and have emerged as promising candidates for these purposes. MiRs are a class of small, endogenous non-coding RNAs that play critical roles in several human diseases, including host-pathogen interaction mechanisms. Recently, miRs signatures have been reported in different infectious diseases, opening new perspectives for molecular diagnosis and therapy. MiR profiles can discriminate between healthy individuals and patients, as well as distinguish different disease stages. Furthermore, the possibility of assessing miRs in biological fluids, such as serum and whole blood, renders these molecules feasible for the development of new non-invasive diagnostic and prognostic tools. In this manuscript, we will comprehensively describe miRs as biomarkers and therapeutic targets in infectious diseases and explore how they can contribute to the advance of existing and new tools. Additionally, we will discuss different miR analysis platforms to understand the obstacles and advances of this molecular approach and propose their potential clinical applications and contributions to public health., Competing Interests: All authors declare that they have no commercial or financial relationships that could be construed as a potential conflict of interest disclosure., (© 2024 The Authors.)

Skin ulcers of cutaneous leishmaniasis (CL) are characterized by a localized inflammatory response mediated by innate and adaptive immune cells, including dendritic cells (DC) and natural killer (NK) cells. Bidirectional interactions between DCs and NK cells contribute to tailor leishmaniasis outcome. Despite advances in the Leishmania biology field in recent decades, the mechanisms involved in DC/NK-mediated control of Leishmania sp. pathogenesis as well as the cellular and molecular players involved in such interaction remain unclear. The present study sought to investigate canonical pathways associated with CL arising from Leishmania braziliensis infection. Initially, two publicly available microarray datasets of skin biopsies from active CL lesions were analyzed, and five pathways were identified using differentially expressed genes. The "Crosstalk between DCs and NK cells" pathway was notable due to a high number of modulated genes. The molecules significantly involved in this pathway were identified, and our findings were validated in newly obtained CL biopsies. We found increased expression of TLR4, TNFRSF1B, IL-15, IL-6, CD40, CCR7, TNF and IFNG, confirming the analysis of publicly available datasets. These findings reveal the "crosstalk between DCs and NK cells" as a potential pathway to be further explored in the pathogenesis of CL, especially the expression of CCR7, which is correlated with lesion development.

Aims: Pre-existing conditions, such as age, hypertension, obesity, and diabetes, constitute known risk factors for severe COVID-19. However, the impact of prediabetes mellitus (PDM) on COVID-19 severity is less clear. This study aimed to evaluate the influence of PDM in the acute and long-term phases of COVID-19., Materials and Methods: We compared inflammatory mediators, laboratory and clinical parameters and symptoms in COVID-19 patients with prediabetes (PDM) and without diabetes (NDM) during the acute phase of infection and at three months post-hospitalization., Results: Patients with PDM had longer hospital stays and required intensive care unit admission more frequently than NDM. Upon hospitalization, PDM patients exhibited higher serum levels of interleukin 6 (IL-6), which is related to reduced partial pressure of oxygen (PaO 2 ) in arterial blood, oxygen saturation (SpO 2 ) and increased COVID-19 severity. However, at three months after discharge, those with PDM did not exhibit significant alterations in laboratory parameters or residual symptoms; however, PDM was observed to influence the profile of reported symptoms., Conclusions: PDM seems to be associated with increased risk of severe COVID-19, as well as higher serum levels of IL-6, which may constitute a potential biomarker of severe COVID-19 risk in affected patients. Furthermore, while PDM correlated with more severe acute-phase COVID-19, no long-term worsening of sequelae was observed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bonyek-Silva, Cerqueira-Silva, Nunes, Machado, Cruz, Pereira, Estrela, Silva, Isis, Barral, Oliveira, Khouri, Serezani, Brodskyn, Caldas, Barral-Netto, Boaventura and Tavares.)

Human cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a pronounced inflammatory response associated with ulcer development. Monocytes/macrophages, the main cells harboring parasites, are largely responsible for parasite control. Toll-like receptor (TLR) signaling leads to the transcription of inflammatory mediators, such as IL-1β and TNF during innate immune response. TLR antagonists have been used in the treatment of inflammatory disease. The neutralization of these receptors may attenuate an exacerbated inflammatory response. We evaluated the ability of TLR2 and TLR4 antagonists to modulate host immune response in L. braziliensis -infected monocytes and cells from CL patient skin lesions. Following TLR2 and TLR4 neutralization, decreased numbers of infected cells and internalized parasites were detected in CL patient monocytes. In addition, reductions in oxidative burst, IL-1β, TNF and CXCL9 production were observed. TNF production by cells from CL lesions also decreased after TLR2 and TLR4 neutralization. The attenuation of host inflammatory response after neutralizing these receptors suggests the potential of TLR antagonists as immunomodulators in association with antimonial therapy in human cutaneous leishmaniasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Carneiro, Dórea, Oliveira, Guimarães, Brodskyn, Carvalho and Bacellar.)

Diabetes is a known risk factor for severe coronavirus disease 2019 (COVID-19), the disease caused by the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there is a lack of knowledge about the mechanisms involved in the evolution of COVID-19 in individuals with diabetes. We aimed to evaluate whether the chronic low-grade inflammation of diabetes could play a role in the development of severe COVID-19. We collected clinical data and blood samples of patients with and without diabetes hospitalized for COVID-19. Plasma samples were used to measure inflammatory mediators and peripheral blood mononuclear cells, for gene expression analysis of the SARS-CoV-2 main receptor system ( ACE2 / TMPRSS2 ), and for the main molecule of the leukotriene B 4 (LTB 4 ) pathway ( ALOX5 ). We found that diabetes activates the LTB 4 pathway and that during COVID-19 it increases ACE2 / TMPRSS2 as well as ALOX5 expression. Diabetes was also associated with COVID-19-related disorders, such as reduced oxygen saturation as measured by pulse oximetry/fraction of inspired oxygen (FiO 2 ) and arterial partial pressure of oxygen/FiO 2 levels, and increased disease duration. In addition, the expressions of ACE2 and ALOX5 are positively correlated, with increased expression in patients with diabetes and COVID-19 requiring intensive care assistance. We confirmed these molecular results at the protein level, where plasma LTB 4 is significantly increased in individuals with diabetes. In addition, IL-6 serum levels are increased only in individuals with diabetes requiring intensive care assistance. Together, these results indicate that LTB 4 and IL-6 systemic levels, as well as ACE2 / ALOX5 blood expression, could be early markers of severe COVID-19 in individuals with diabetes., (© 2021 by the American Diabetes Association.)

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 10 6  cells per well on 24-well plates in order to obtain 2 x 10 5  macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

The initial production of inflammatory mediators dictates host defense as well as tissue injury. Inflammasome activation is a constituent of the inflammatory response by recognizing pathogen and host-derived products and eliciting the production of IL-1β and IL-18 in addition to inducing a type of inflammatory cell death termed "pyroptosis." Leukotriene B 4 (LTB 4 ) is a lipid mediator produced quickly (seconds to minutes) by phagocytes and induces chemotaxis, increases cytokine/chemokine production, and enhances antimicrobial effector functions. Whether LTB 4 directly activates the inflammasome remains to be determined. Our data show that endogenously produced LTB 4 is required for the expression of pro-IL-1β and enhances inflammasome assembly in vivo and in vitro. Furthermore, LTB 4 -mediated Bruton's tyrosine kinase (BTK) activation is required for inflammasome assembly in vivo as well for IL-1β-enhanced skin host defense. Together, these data unveil a new role for LTB 4 in enhancing the expression and assembly of inflammasome components and suggest that while blocking LTB 4 actions could be a promising therapeutic strategy to prevent inflammasome-mediated diseases, exogenous LTB 4 can be used as an adjuvant to boost inflammasome-dependent host defense., Competing Interests: The authors declare no competing interest.

Background: Skin lesions from patients infected with Leishmania braziliensis has been associated with inflammation induced by cytotoxic CD8+ T cells. In addition, CD8+ T cell-mediated cytotoxicity has not been linked to parasite killing. Meanwhile, the cytotoxic role played by natural killer (NK) cells in cutaneous leishmaniasis (CL) remains poorly understood., Methods: In this study, we observed higher frequencies of NK cells in the peripheral blood of CL patients compared with healthy subjects, and that NK cells expressed more interferon-γ, tumor necrosis factor (TNF), granzyme B, and perforin than CD8+ T cells., Results: We also found that most of the cytotoxic activity in CL lesions was triggered by NK cells, and that the high levels of granzyme B produced in CL lesions was associated with larger lesion size. Furthermore, an in vitro blockade of granzyme B was observed to decrease TNF production., Concclusions: Our data, taken together, suggest an important role by NK cells in inducing inflammation in CL, thereby contributing to disease immunopathology., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The cell wall has a critical role in the host immune response to fungal pathogens. In this study, we investigated the influence of two cell wall fractions of the dimorphic fungi Paracoccidioides brasiliensis (Pb) in the in vitro generation of monocyte-derived dendritic cells (MoDCs). Monocytes were purified from the peripheral blood of healthy donors and cultivated for 7 days in medium supplemented with IL-4 and GM-CSF in the presence of Pb cell wall fractions: the alkali-insoluble F1, constituted by β-1,3-glucans, chitin and proteins, and the alkali-soluble F2, mainly constituted by α-glucan. MoDCs phenotypes were evaluated regarding cell surface expression of CD1a, DC-SIGN, HLA-DR, CD80, and CD83 and production of cytokines. The α-glucan-rich cell wall fraction downregulated the differentiation of CD1a + MoDCs, a dendritic cell subset that stimulate Th1 responses. The presence of both cell fractions inhibited DC-SIGN and HLA-DR expression, while the expression of maturation markers was differentially induced in CD1a - MoDCs. Differentiation upon F1 and F2 stimulation induced mixed profile of inflammatory cytokines. Altogether, these data demonstrate that Pb cell wall fractions differentially induce a dysregulation in DCs differentiation. Moreover, our results suggest that cell wall α-glucan promote the differentiation of CD1a - DCs, potentially favoring Th2 polarization and contributing to pathogen persistence., (Copyright © 2019 Souza, Favali, Soares, Tavares, Jerônimo, Veloso Junior, Marina, Santos, Brodskyn and Bocca.)

Dendritic cells (DC) are a diverse group of leukocytes responsible for bridging innate and adaptive immunity. Despite their functional versatility, DCs exist primarily in two basic functional states: immature and mature. A large body of evidence suggests that upon interactions with pathogens, DCs undergo intricate cellular processes that culminate in their activation, which is paramount to the orchestration of effective immune responses against Leishmani a parasites. Herein we offer a concise review of the emerging hallmarks of DCs activation in leishmaniasis as well as a comprehensive discussion of the following underlying molecular events: DC- Leishmania interaction, antigen uptake, costimulatory molecule expression, parasite ability to affect DC migration, antigen presentation, metabolic reprogramming, and epigenetic alterations.

Blood-sucking phlebotomine sand flies (Diptera: Psychodidae) transmit leishmaniasis as well as arboviral diseases and bartonellosis. Sand fly females become infected with Leishmania parasites and transmit them while imbibing vertebrates' blood, required as a source of protein for maturation of eggs. In addition, both females and males consume plant-derived sugar meals as a source of energy. Plant meals may comprise sugary solutions such as nectar or honeydew (secreted by plant-sucking homopteran insects), as well as phloem sap that sand flies obtain by piercing leaves and stems with their needle-like mouthparts. Hence, the structure of plant communities can influence the distribution and epidemiology of leishmaniasis. We designed a next-generation sequencing (NGS)-based assay for determining the source of sand fly plant meals, based upon the chloroplast DNA gene ribulose bisphosphate carboxylase large chain ( rbcL ). Here, we report on the predilection of several sand fly species, vectors of leishmaniasis in different parts of the world, for feeding on Cannabis sativa We infer this preference based on the substantial percentage of sand flies that had fed on C. sativa plants despite the apparent "absence" of these plants from most of the field sites. We discuss the conceivable implications of the affinity of sand flies for C. sativa on their vectorial capacity for Leishmania and the putative exploitation of their attraction to C. sativa for the control of sand fly-borne diseases., Competing Interests: The authors declare no conflict of interest.

Localized cutaneous leishmaniasis (LCL) is a chronic disease characterized by ulcerated skin lesion(s) and uncontrolled inflammation. The mechanisms underlying the pathogenesis of LCL are not completely understood, and little is known about posttranscriptional regulation during LCL. MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression and can be implicated in the pathogenesis of LCL. We investigated the involvement of miRNAs and their targets genes in human LCL using publicly available transcriptome data sets followed by ex vivo validation. Initial analysis highlighted that miRNA expression is altered during LCL, as patients clustered separately from controls. Joint analysis identified eight high confidence miRNAs that had altered expression (-1.5 ≤ fold change ≥ 1.5; p  < 0.05) between cutaneous ulcers and uninfected skin. We found that the expression of miR-193b and miR-671 are greatly associated with their target genes, CD40 and TNFR, indicating the important role of these miRNAs in the expression of genes related to the inflammatory response observed in LCL. In addition, network analysis revealed that miR-193b, miR-671, and TREM1 correlate only in patients who show faster wound healing (up to 59 days) and not in patients who require longer cure times (more than 60 days). Given that these miRNAs are associated with control of inflammation and healing time, our findings reveal that they might influence the pathogenesis and prognosis of LCL.

[This corrects the article DOI: 10.1155/2016/3967436.].

Neutrophils mediate early responses against pathogens, and they become activated during endothelial transmigration toward the inflammatory site. In the current study, human neutrophils were activated in vitro with immobilized extracellular matrix proteins, such as fibronectin (FN), collagen, and laminin. Neutrophil activation by FN, but not other extracellular matrix proteins, induces the release of the granules' contents, measured as matrix metalloproteinase 9 and neutrophil elastase activity in culture supernatant, as well as reactive oxygen species production. Upon contact with Leishmania amazonensis-infected macrophages, these FN-activated neutrophils reduce the parasite burden through a mechanism independent of cell contact. The release of granule proteases, such as myeloperoxidase, neutrophil elastase, and matrix metalloproteinase 9, activates macrophages through TLRs, leading to the production of inflammatory mediators, TNF-α and leukotriene B4 (LTB4), which are involved in parasite killing by infected macrophages. The pharmacological inhibition of degranulation reverted this effect, abolishing LTB4 and TNF production. Together, these results suggest that FN-driven degranulation of neutrophils induces the production of LTB4 and TNF by infected macrophages, leading to the control of Leishmania infection., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Leishmaniasis is a group of neglected diseases whose clinical manifestations depend on factors from the host and the pathogen. It is an important public health problem worldwide caused by the protozoan parasite from the Leishmania genus. Cutaneous Leishmaniasis (CL) is the most frequent form of this disease transmitted by the bite of an infected sandfly into the host skin. The parasites can be uptook and/or recognized by macrophages, neutrophils, and/or dendritic cells (DCs). Initially, DCs were described to play a protective role in activating the immune response against Leishmania parasites. However, several reports showed a dichotomic role of DCs in modulating the host immune response to susceptibility or resistance in CL. In this review, we discuss (1) the interactions between DCs and parasites from different species of Leishmania and (2) the crosstalk of DCs and other cells during CL infection. The complexity of these interactions profoundly affects the adaptive immune response and, consequently, the disease outcome, especially from Leishmania species of the New World.

Neutrophils are rapidly recruited to the site of Leishmania infection and play an active role in capturing and killing parasites. They are the main source of leukotriene B4 (LTB4), a potent proinflammatory lipid mediator. However, the role of LTB4 in neutrophil infection by Leishmania amazonensis is not clear. In this study, we show that L. amazonensis or its lipophosphoglycan can induce neutrophil activation, degranulation, and LTB4 production. Using pharmacological inhibitors of leukotriene synthesis, our findings reveal an LTB4-driven autocrine/paracrine regulatory effect. In particular, neutrophil-derived LTB4 controls L. amazonensis killing, degranulation, and reactive oxygen species production. In addition, L. amazonensis infection induces an early increase in Toll-like receptor 2 expression, which facilitates parasite internalization. Nuclear factor kappa B (NFkB) pathway activation represents a required upstream event for L. amazonensis-induced LTB4 synthesis. These leishmanicidal mechanisms mediated by neutrophil-derived LTB4 act through activation of its receptor, B leukotriene receptor 1 (BLT1)., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

We recently demonstrated that immunization with polyester poly(lactide-co-glycolide acid) (PLGA) nanoparticles loaded with the 11-kDa Leishmania vaccine candidate kinetoplastid membrane protein 11 (KMP-11) significantly reduced parasite load in vivo. Presently, we explored the ability of the recombinant PLGA nanoparticles to stimulate innate responses in macrophages and the outcome of infection with Leishmania braziliensis in vitro. Incubation of macrophages with KMP-11-loaded PLGA nanoparticles significantly decreased parasite load. In parallel, we observed the augmented production of nitric oxide, superoxide, TNF-α and IL-6. An increased release of CCL2/MCP-1 and CXCL1/KC was also observed, resulting in macrophage and neutrophil recruitment in vitro. Lastly, the incubation of macrophages with KMP-11-loaded PLGA nanoparticles triggered the activation of caspase-1 and the secretion of IL-1β and IL-18, suggesting inflammasome participation. Inhibition of caspase-1 significantly increased the parasite load. We conclude that KMP-11-loaded PLGA nanoparticles promote the killing of intracellular Leishmania parasites through the induction of potent innate responses., From the Clinical Editor: In this novel study, KMP-11-loaded PLGA nanoparticles are demonstrated to promote the killing of intracellular Leishmania parasites through enhanced innate immune responses by multiple mechanisms. Future clinical applications would have a major effect on our efforts to address parasitic infections., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects salivary components that change the environment at the feeding site. Mice immunized with Phlebotomus papatasi salivary gland (SG) homogenate are protected against Leishmania major infection, while immunity to Lutzomyia intermedia SG homogenate exacerbated experimental Leishmania braziliensis infection. In humans, antibodies to Lu. intermedia saliva are associated with risk of acquiring L. braziliensis infection. Despite these important findings, there is no information regarding the repertoire of Lu. intermedia salivary proteins., Methods and Findings: A cDNA library from the Salivary Glands (SGs) of wild-caught Lu. intermedia was constructed, sequenced, and complemented by a proteomic approach based on 1D SDS PAGE and mass/mass spectrometry to validate the transcripts present in this cDNA library. We identified the most abundant transcripts and proteins reported in other sand fly species as well as novel proteins such as neurotoxin-like proteins, peptides with ML domain, and three small peptides found so far only in this sand fly species. DNA plasmids coding for ten selected transcripts were constructed and used to immunize BALB/c mice to study their immunogenicity. Plasmid Linb-11--coding for a 4.5-kDa protein--induced a cellular immune response and conferred protection against L. braziliensis infection. This protection correlated with a decreased parasite load and an increased frequency of IFN-γ-producing cells., Conclusions: We identified the most abundant and novel proteins present in the SGs of Lu. intermedia, a vector of cutaneous leishmaniasis in the Americas. We also show for the first time that immunity to a single salivary protein from Lu. intermedia can protect against cutaneous leishmaniasis caused by L. braziliensis.

Background: Leishmania parasites are transmitted to their vertebrate hosts by infected Phlebotomine sand flies during the blood meal of the flies. Sand fly saliva is known to enhance Leishmania spp. infection, while pre-exposure to saliva protects mice against parasitic infections. In this study, we investigated the initial inflammatory leucocyte composition induced by one or three inocula of salivary gland extract (SGE) from Lutzomyia longipalpis in the presence or absence of Leishmania braziliensis., Results: We demonstrated that inoculating SGE once (SGE-1X) or three times (SGE-3X), which represented a co-inoculation or a pre-exposure to saliva, respectively, resulted in different cellular infiltrate profiles. Whereas SGE-1X led to the recruitment of all leucocytes subtypes including CD4(+) T cells, CD4(+)CD25(+) T cells, dendritic cells, macrophages and neutrophils, the immune cell profile in the SGE-3X group differed dramatically, as CD4(+) T cells, CD4(+)CD25(+) T cells, dendritic cells, macrophages and neutrophils were decreased and CD8(+) T cells were increased. The SGE-1X group did not show differences in the ear lesion size; however, the SGE-1X group harbored a higher number of parasites. On the other hand, the SGE-3X group demonstrated a protective effect against parasitic disease, as the parasite burden was lower even in the earlier stages of the infection, a period in which the SGE-1X group presented with larger and more severe lesions. These effects were also reflected in the cytokine profiles of both groups. Whereas the SGE-1X group presented with a substantial increase in IL-10 production, the SGE-3X group showed an increase in IFN-γ production in the draining lymph nodes. Analysis of the inflammatory cell populations present within the ear lesions, the SGE-1X group showed an increase in CD4(+)FOXP3(+) cells, whereas the CD4(+)FOXP3(+) population was reduced in the SGE-3X group. Moreover, CD4(+) T cells and CD8(+) T cells producing IFN-γ were highly detected in the ears of the SGE-3X mice prior to infection. In addition, upon treatment of SGE-3X mice with anti-IFN-γ monoclonal antibody, we observed a decrease in the protective effect of SGE-3X against L. braziliensis infection., Conclusions: These results indicate that different inocula of Lutzomyia longipalpis salivary gland extract can markedly modify the cellular immune response, which is reflected in the pattern of susceptibility or resistance to Leishmania braziliensis infection.

Background: Canine Visceral Leishmaniasis (CVL) is a zoonotic disease caused by Leishmania infantum, transmitted by the bite of Lutzomyia longipalpis sand flies. Dogs are the main domestic reservoir of the parasite. The establishment of an experimental model that partially reproduces natural infection in dogs is very important to test vaccine candidates, mainly regarding those that use salivary proteins from the vector and new therapeutical approaches., Methodology/principal Findings: In this report, we describe an experimental infection in dogs, using intradermal injection of Leishmania infantum plus salivary gland homogenate (SGH) of Lutzomyia longipalpis. Thirty-five dogs were infected with 1×10(7) parasites combined with five pairs of Lutzomyia longipalpis salivary glands and followed for 450 days after infection and clinical, immunological and parasitological parameters were evaluated. Two hundred and ten days after infection we observed that 31,4% of dogs did not display detectable levels of anti-Leishmania antibodies but all presented different numbers of parasites in the lymph nodes. Animals with a positive xenodiagnosis had at least 3,35×10(5) parasites in their lymph nodes. An increase of IFN-γ and IL-10 levels was detected during infection. Twenty two percent of dogs developed symptoms of CVL during infection., Conclusion: The infection model described here shows some degree of similarity when compared with naturally infected dogs opening new perspectives for the study of CVL using an experimental model that employs the combination of parasites and sand fly saliva both present during natural transmission.

Leishmania spp. cause a broad spectrum of diseases collectively known as leishmaniasis. Leishmania braziliensis is the main etiological agent of American cutaneous leishmaniasis and mucocutaneous leishmaniasis. During experimental infection with L. braziliensis, BALB/c mice develop an adaptive immune response that is associated with lesion healing and, in parallel, parasite persistence within draining lymph nodes (dLNs). In the Leishmania major model of cutaneous leishmaniasis, regulatory T cells (Tregs) play an important role in immune regulation, preventing pathological immune responses but at the same time precluding sterile cure. In this study we investigated the role of Tregs during experimental infection with L. braziliensis. CD4(+)CD25(+) T cells were detected throughout the duration of clinical disease both at the ear and in dLNs of infected mice. These cells expressed Treg markers such as glucocorticoid-induced TNF-receptor-related protein (GITR), the α chain of the αεβ7 integrin (CD103), and the forkhead/winged helix transcription factor, Foxp3, and were able to suppress the proliferation of CD4(+)CD25(-) cells. Importantly, a high frequency of Foxp3(+) cells accumulated at the site of infection and in dLNs. We next investigated the outcome of a reinfection with L. braziliensis in terms of Treg distribution and disease reactivation. Interestingly, a secondary inoculation with L. braziliensis did not preclude an efficient recall response to L. braziliensis at a distal site, despite the presence of Tregs. Within dLNs, reinfection did not promote parasite dissemination or a differential recruitment of either CD4(+)CD25(+)Foxp3(+) or CD4(+)IL-10(+) T cells. On the contrary, parasites were mainly detected in the LN draining the primary infection site where a high frequency of CD4(+)IFN-γ(+) T cells was also present. Collectively these data show that during experimental infection, Tregs are present in healed mice but this population does not compromise an effective immune response upon reinfection with L. braziliensis., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

When an haematophagous sand fly vector insect bites a vertebrate host, it introduces its mouthparts into the skin and lacerates blood vessels, forming a hemorrhagic pool which constitutes an intricate environment of cell interactions. In this scenario, the initial performance of host, parasite, and vector "authors" will heavily influence the course of Leishmania infection. Recent advances in vector-parasite-host interaction have elucidated "co-authors" and "new roles" not yet described. We review here the stimulatory role of Lutzomyia longipalpis saliva leading to inflammation and try to connect them in an early context of Leishmania infection.

Background: Nucleosomal histones are intracellular proteins that are highly conserved among Leishmania species. After parasite destruction or spontaneous lysis, exposure to these proteins elicits a strong host immune response. In the present study, we analyzed the protective capability of Leishmania infantum chagasi nucleosomal histones against L. braziliensis infection using different immunization strategies., Methodology/principal Findings: BALB/c mice were immunized with either a plasmid DNA cocktail (DNA) containing four Leishmania nucleosomal histones or with the DNA cocktail followed by the corresponding recombinant proteins plus CpG (DNA/Protein). Mice were later challenged with L. braziliensis, in the presence of sand fly saliva. Lesion development, parasite load and the cellular immune response were analyzed five weeks after challenge. Immunization with either DNA alone or with DNA/Protein was able to inhibit lesion development. This finding was highlighted by the absence of infected macrophages in tissue sections. Further, parasite load at the infection site and in the draining lymph nodes was also significantly lower in vaccinated animals. This outcome was associated with increased expression of IFN-γ and down regulation of IL-4 at the infection site., Conclusion: The data presented here demonstrate the potential use of L. infantum chagasi nucleosomal histones as targets for the development of vaccines against infection with L. braziliensis, as shown by the significant inhibition of disease development following a live challenge.

It was recently shown that immunization of hamsters with DNA plasmids coding LJM19, a sand fly salivary protein, partially protected against a challenge with Leishmania chagasi, whereas immunization with KMP11 DNA plasmid, a Leishmania antigen, induced protection against L. donovani infection. In the present study, we evaluated the protective effect of immunization with both LJM19 and KMP11 DNA plasmid together. Concerning the protection against an infection by L. chagasi, immunization with DNA plasmids coding LJM19 or KMP11, as well as with both plasmids combined, induced IFN-γ production in draining lymph nodes at 7, 14 and 21 days post-immunization. Immunized hamsters challenged with L. chagasi plus Salivary Gland Sonicate (SGS) from Lutzomyia longipalpis showed an enhancement of IFN-γ/IL-10 and IFN-γ/TGF-β in draining lymph nodes after 7 and 14 days of infection. Two and five months after challenge, immunized animals showed reduced parasite load in the liver and spleen, as well as increased IFN-γ/IL-10 and IFN-γ/TGF-β ratios in the spleen. Furthermore, immunized animals remained with a normal hematological profile even five months after the challenge, whereas L. chagasi in unimmunized hamsters lead to a significant anemia. The protection observed with LJM19 or KMP11 DNA plasmids used alone was very similar to the protection obtained by the combination of both plasmids., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Neutrophils are considered the host's first line of defense against infections and have been implicated in the immunopathogenesis of Leishmaniasis. Leishmania parasites are inoculated alongside vectors' saliva, which is a rich source of pharmacologically active substances that interfere with host immune response. In the present study, we tested the hypothesis that salivary components from Lutzomyia longipalpis, an important vector of visceral Leishmaniasis, enhance neutrophil apoptosis. Murine inflammatory peritoneal neutrophils cultured in the presence of SGS presented increased surface expression of FasL and underwent caspase-dependent and FasL-mediated apoptosis. This proapoptosis effect of SGS on neutrophils was abrogated by pretreatment with protease as well as preincubation with antisaliva antibodies. Furthermore, in the presence of Leishmania chagasi, SGS also increased apoptosis on neutrophils and increased PGE(2) release and decreased ROS production by neutrophils, while enhancing parasite viability inside these cells. The increased parasite burden was abrogated by treatment with z-VAD, a pan caspase inhibitor, and NS-398, a COX-2 inhibitor. In the presence of SGS, Leishmania-infected neutrophils produced higher levels of MCP-1 and attracted a high number of macrophages by chemotaxis in vitro assays. Both of these events were abrogated by pretreatment of neutrophils with bindarit, an inhibitor of CCL2/MCP-1 expression. Taken together, our data support the hypothesis that vector salivary proteins trigger caspase-dependent and FasL-mediated apoptosis, thereby favoring Leishmania survival inside neutrophils, which may represent an important mechanism for the establishment of Leishmania infection.

Background: Leishmania transmission occurs in the presence of insect saliva. Immunity to Phlebotomus papatasi or Lutzomyia longipalpis saliva or salivary components confers protection against an infection by Leishmania in the presence of the homologous saliva. However, immunization with Lutzomyia intermedia saliva did not protect mice against Leishmania braziliensis plus Lu. intermedia saliva. In the present study, we have studied whether the immunization with Lu. longipalpis saliva or a DNA plasmid coding for LJM19 salivary protein would be protective against L. braziliensis infection in the presence of Lu. intermedia saliva, the natural vector for L. braziliensis., Methodology/principal Findings: Immunization with Lu. longipalpis saliva or with LJM19 DNA plasmid induced a Delayed-Type Hypersensitivity (DTH) response against Lu. longipalpis as well as against a Lu. intermedia saliva challenge. Immunized and unimmunized control hamsters were then intradermally infected in the ears with L. braziliensis in the presence of Lu. longipalpis or Lu. intermedia saliva. Animals immunized with Lu. longipalpis saliva exhibited smaller lesion sizes as well as reduced disease burdens both at lesion site and in the draining lymph nodes. These alterations were associated with a significant decrease in the expression levels of IL-10 and TGF-β. Animals immunized with LJM19 DNA plasmid presented similar findings in protection and immune response and additionally increased IFN-γ expression., Conclusions/significance: Immunization with Lu. longipalpis saliva or with a DNA plasmid coding LJM19 salivary protein induced protection in hamsters challenged with L. braziliensis plus Lu. intermedia saliva. These findings point out an important role of immune response against saliva components, suggesting the possibility to develop a vaccine using a single component of Lu. longipalpis saliva to generate protection against different species of Leishmania, even those transmitted by a different vector.

Background: During blood feeding, sand flies inject Leishmania parasites in the presence of saliva. The types and functions of cells present at the first host-parasite contact are critical to the outcome on infection and sand fly saliva has been shown to play an important role in this setting. Herein, we investigated the in vivo chemotactic effects of Lutzomyia intermedia saliva, the vector of Leishmania braziliensis, combined or not with the parasite., Methods and Findings: We tested the initial response induced by Lutzomyia intermedia salivary gland sonicate (SGS) in BALB/c mice employing the air pouch model of inflammation. L. intermedia SGS induced a rapid influx of macrophages and neutrophils. In mice that were pre-sensitized with L. intermedia saliva, injection of SGS was associated with increased neutrophil recruitment and a significant up-regulation of CXCL1, CCL2, CCL4 and TNF-alpha expression. Surprisingly, in mice that were pre-exposed to SGS, a combination of SGS and L. braziliensis induced a significant migration of neutrophils and an important modulation in cytokine and chemokine expression as shown by decreased CXCL10 expression and increased IL-10 expression., Conclusion: These results confirm that sand fly saliva modulates the initial host response. More importantly, pre-exposure to L. intermedia saliva significantly modifies the host's response to L. braziliensis, in terms of cellular recruitment and expression of cytokines and chemokines. This particular immune modulation may, in turn, favor parasite multiplication.

Neutrophils play an active role in the control of infections caused by intracellular pathogens such as Leishmania. In the present study, we investigated the effect of neutrophil depletion at the time of Leishmania braziliensis infection of BALB/c mice and how neutrophils interact with the infected macrophage to promote parasite elimination. The in vivo depletion of neutrophils led to a significant increase in parasite load and enhanced the Th1-Th2 immune response in this experimental model of infection. BALB/c mice coinoculated with both parasites and live neutrophils displayed lower parasite burdens at the site of infection and in the draining lymph nodes. In vitro, we observed that live neutrophils significantly reduced the parasite load in L. braziliensis-infected murine macrophages, an effect not observed with Leishmania major. L. braziliensis elimination was dependent on the interaction between neutrophils and macrophages and was associated with TNF-alpha as well as superoxide production. Furthermore, cooperation between neutrophils and macrophages toward parasite elimination was also observed in experiments performed with L. braziliensis-infected human cells and, importantly, with two other New World Leishmania species. These results indicate that neutrophils play an important and previously unappreciated role in L. braziliensis infection, favoring the induction of a protective immune response.

The miniexon gene has a central role in the processing of polycistronic pre-mRNA of kinetoplastids. It is added to the 5' extremity of each mRNA, supplying the 5'-capped structure to the molecule. Previous studies in Leishmania (Leishmania) major showed that the overexpression of the miniexon array attenuates the virulence of the parasite in in vivo assays. The results presented here extend those findings to Viannia subgenus. Leishmania (Viannia) braziliensis was transfected with a cosmid harboring a tandem array of one hundred miniexon gene copies and then characterized by Northern blot analysis. The overexpression of the exogenous gene was confirmed and its effect on the virulence of L. (V.) braziliensis was investigated in hamsters. In BALB/c mice we could not detect parasites during the course of 15 weeks of infection. In addition, hamsters infected with transfectants overexpressing the miniexon gene exhibited only a minor footpad swelling of late onset and failed to develop progressive lesion, these attenuated parasites could be recovered from the inoculation site 1 year after infection. The persistence of parasites in the host indicates that a stable line overexpressing the miniexon may be tested as live vaccine against leishmaniasis.