RESUMEN
A role of IL-10 is down-regulating T-cell responses to schistosome antigens. Since SmATPDases can be correlated to modulation of the immune response, we evaluated the expression of enzymes in S. mansoni eggs. Faecal samples were collected from 40 infected individuals to detect coding regions of the SmATPDases. The cytokines were measured in supernatants of PBMC. The analysis was performed by the global median determination and set up high producers (HP) of cytokines. Six individuals expressed SmATPDase1, six expressed SmATPDase2 and six expressed both enzymes. The group who expressed only SmATPDase1 showed a high frequency of IFN-γ, TNF IL-4 HP; individuals who expressed only SmATPDase2 showed a high frequency of IFN-γ, IL-6 and IL-4 HP; and individuals who expressed both enzymes showed a high frequency of IL-10 HP. The comparison of the IFN-γ/IL-10 ratio presented higher indices in the group who had SmATPDase 2 expression than those who had the expression of both enzymes. The positive correlation between infection intensity and IL-10 levels remained only in the positive SmATPDase group. The IL-10 is the only cytokine induced by the expression of both enzymes. Our data suggest that the expression of both enzymes seems to be a factor that modulates the host immune response by inducing high IL-10 production.
Asunto(s)
Schistosoma mansoni , Esquistosomiasis mansoni , Animales , Humanos , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Leucocitos Mononucleares , Citocinas/metabolismoRESUMEN
Extracellular adenosine plays important roles in modulating the immune responses. We have previously demonstrated that infection of dendritic cells (DC) by Leishmania amazonensis leads to increased expression of CD39 and CD73 and to the selective activation of the low affinity A2B receptors (A2B R), which contributes to DC inhibition, without involvement of the high affinity A2A R. To understand this apparent paradox, we now characterized the alterations of both adenosine receptors in infected cells. With this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Fluorescence microscopy revealed that L. amazonensis infection stimulates the recruitment of A2B R, but not of A2A R, to the surface of infected DC, without altering the amount of mRNA or the total A2B R density, an effect dependent on lipophosphoglycan (LPG). Log-phase promastigotes or axenic amastigotes of L. amazonensis do not stimulate A2B R recruitment. A2B R clusters are localized in caveolin-rich lipid rafts and the disruption of these membrane domains impairs A2B R recruitment and activation. More importantly, our results show that A2B R co-localize with CD39 and CD73 forming a "purinergic cluster" that allows for the production of extracellular adenosine in close proximity with these receptors. We conclude that A2B R activation by locally produced adenosine constitutes an elegant and powerful evasion mechanism used by L. amazonensis to down-modulate the DC activation.
Asunto(s)
5'-Nucleotidasa/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Caveolina 1/metabolismo , Células Dendríticas/inmunología , Leishmaniasis/inmunología , Microdominios de Membrana/inmunología , Receptor de Adenosina A2B/metabolismo , Animales , Células Dendríticas/metabolismo , Células Dendríticas/parasitología , Células Dendríticas/patología , Inmunidad , Inmunomodulación , Leishmania/inmunología , Leishmaniasis/metabolismo , Leishmaniasis/parasitología , Leishmaniasis/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/parasitología , Macrófagos/patología , Masculino , Microdominios de Membrana/parasitología , Microdominios de Membrana/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
Distinct populations of Trypanosoma cruzi interact with mammalian cardiac muscle cells causing different inflammation patterns and low heart functionality. During T. cruzi infection, the extracellular ATP is hydrolyzed to tri- and/or diphosphate nucleotides, based on the infectivity, virulence, and regulation of the inflammatory response. T. cruzi carries out this hydrolysis through the T. cruzi ectonucleotidase, NTPDase-1 (TcNTPDase-1). This study aimed to evaluate the role of TcNTPDase-1 in culture rich in metacyclic trypomastigote forms (MT) and cell culture-derived trypomastigote forms (CT) from Colombiana (discrete typing unit - DTU I), VL-10 (DTU II), and CL (DTU VI) strains of T. cruzi. For this, we measured TcNTPDase-1 activity in suramin-treated and untreated parasites and infected J774 cells and C57BL/6 mice with suramin pre-treated parasites to assess parasitic and inflammatory cardiac profile in the acute phase of infection. Our data indicated a higher TcNTPDase-1 activity for ATP in culture rich in metacyclic trypomastigote forms from Colombiana strain in comparison to those from VL-10 and CL strains. The cell culture-derived trypomastigote forms from CL strain presented higher capacity to hydrolyze ATP than those from Colombiana and VL-10 strains. Suramin inhibited ATP hydrolysis in all studied parasite forms and strains. Suramin pre-treated parasites reduced J774 cell infection and increased nitrite production in vitro. In vivo studies showed a reduction of inflammatory infiltrate in the cardiac tissues of animals infected with cell culture-derived trypomastigote forms from suramin pre-treated Colombiana strain. In conclusion, TcNTPDase-1 activity in trypomastigotes forms drives part of the biological characteristics observed in distinct DTUs and may induce cardiac pathogenesis during T. cruzi infection.
Asunto(s)
Antígenos CD , Apirasa , Enfermedad de Chagas , Proteínas Protozoarias , Trypanosoma cruzi , Factores de Virulencia , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Apirasa/genética , Apirasa/metabolismo , Línea Celular Tumoral , Enfermedad de Chagas/enzimología , Enfermedad de Chagas/genética , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Especificidad de la Especie , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidad , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The development of hybrids has been an effective approach to generate novel yeast strains with optimal technological profile for use in beer production. This study describes the generation of a new yeast strain for lager beer production by direct mating between two Saccharomyces cerevisiae strains isolated from cachaça distilleries: one that was strongly flocculent, and the other with higher production of acetate esters. The first step in this procedure was to analyze the sporulation ability and reproductive cycle of strains belonging to a specific collection of yeasts isolated from cachaça fermentation vats. Most strains showed high rates of sporulation, spore viability, and homothallic behavior. In order to obtain new yeast strains with desirable properties useful for lager beer production, we compare haploid-to-haploid and diploid-to-diploid mating procedures. Moreover, an assessment of parental phenotype traits showed that the segregant diploid C2-1d generated from a diploid-to-diploid mating experiment showed good fermentation performance at low temperature, high flocculation capacity, and desirable production of acetate esters that was significantly better than that of one type lager strain. Therefore, strain C2-1d might be an important candidate for the production of lager beer, with distinct fruit traces and originating using a non-genetically modified organism (GMO) approach.IMPORTANCE Recent work has suggested the utilization of hybridization techniques for the generation of novel non-genetically modified brewing yeast strains with combined properties not commonly found in a unique yeast strain. We have observed remarkable traits, especially low temperature tolerance, maltotriose utilization, flocculation ability, and production of volatile aroma compounds, among a collection of Saccharomyces cerevisiae strains isolated from cachaça distilleries, which allow their utilization in the production of beer. The significance of our research is in the use of breeding/hybridization techniques to generate yeast strains that would be appropriate for producing new lager beers by exploring the capacity of cachaça yeast strains to flocculate and to ferment maltose at low temperature, with the concomitant production of flavoring compounds.
RESUMEN
Endogenous nucleotides produced by various group of cells under inflammatory conditions act as potential danger signals in vivo. Extracellularly released nucleotides such as ATP are rapidly hydrolyzed to adenosine by the coordinated ectonucleotidase activities of CD39 and CD73. Leishmania is an obligate intracellular parasite of macrophages and capable of modulating host immune response in order to survive and multiply within host cells. In this study, the activity of CD73 induced by Leishmania amazonensis in infected macrophages has been investigated and correlated with parasite survival and infection in vitro. For this, the expression of CD39 and CD73, by flow cytometry, in murine peritoneal macrophages infected with metacyclic promastigotes of L. amazonensis has been analyzed. Our results showed that L. amazonensis-infected macrophages, unlike LPS-treated macrophages, increased CD73 expression. It was also noted that when CD73 enzymatic activity was blocked by α, ß-methyleneadenosine 5'-diphosphate sodium salt (APCP), macrophage parasitism was significantly decreased. Interestingly, these effects were not associated with the production of TNF-α, IL-10, or nitric oxide (NO). Together, these data demonstrate that L. amazonensis induces a regulatory phenotype in macrophages, which by activating the CD39/CD73 pathway allows parasite survival through the action of immunomodulatory adenosine receptors.
Asunto(s)
5'-Nucleotidasa/metabolismo , Adenosina Trifosfatasas/metabolismo , Leishmania mexicana/enzimología , Macrófagos Peritoneales/parasitología , Animales , Antígenos CD/metabolismo , Apirasa/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
ENTPDases are enzymes known for hydrolyzing extracellular nucleotides and playing an essential role in controlling the nucleotide signaling via nucleotide/purinergic receptors P2. Moreover, ENTPDases, together with Ecto-5´-nucleotidase activity, affect the adenosine signaling via P1 receptors. These signals control many biological processes, including the immune system. In this context, ATP is considered as a trigger to inflammatory signaling, while adenosine (Ado) induces anti-inflammatory response. The trypanosomatids Leishmania and Trypanosoma cruzi, pathogenic agents of Leishmaniasis and Chagas Disease, respectively, have their own ENTPDases named "TpENTPDases," which can affect the nucleotide signaling, adhesion and infection, in order to favor the parasite. Besides, TpENTPDases are essential for the parasite nutrition, since the Purine De Novo synthesis pathway is absent in them, which makes these pathogens dependent on the intake of purines and nucleopurines for the Salvage Pathway, in which TpENTPDases also take place. Here, we review information regarding TpNTPDases, including their known biological roles and their effect on the purinergic signaling. We also highlight the roles of these enzymes in parasite infection and their biotechnological applications, while pointing to future developments.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Biotecnología , Receptores Purinérgicos/metabolismo , Trypanosomatina/enzimología , Transducción de SeñalRESUMEN
A model of skin infection with Leishmania amazonensis with low doses of parasites is compared to infection with high doses of L. amazonensis and low and high doses of Leishmania major. C57BL/6 mice were infected with 10³ or 10(6) parasites in the ear and the outcome of infection was assessed. The appearance of lesions in mice infected with 10³ parasites was delayed compared to mice infected with 10(6) Leishmania and parasites were detectable at the infection site before lesions became apparent. Mice infected with L. amazonensis displayed persistent lesions, whereas infection with L. major spontaneously healed in all groups, although lymphocytes persisted at the site of infection after healing. Macrophages persisted only in L. amazonensis-infected mice. High-dose L. amazonensis-infected mice produced lower levels of IFN-γ and TNF than mice infected with L. major. No correlation between the persistence of parasites and IL-10 levels and the production of nitric oxide or urea by macrophages was found. We conclude that infection with low doses of L. amazonensis in the dermis changes the course of infection by delaying the appearance of lesions. However, low-dose infection does not change the outcomes of susceptibility and cytokine production described for subcutaneous infection with high numbers of parasites.
Asunto(s)
Citocinas/inmunología , Leishmania major , Leishmania mexicana , Leishmaniasis Cutánea/parasitología , Linfocitos/parasitología , Macrófagos/parasitología , Animales , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Inmunohistoquímica , Leishmania major/inmunología , Leishmania mexicana/inmunología , Leishmaniasis Cutánea/inmunología , Linfocitos/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Factores de TiempoRESUMEN
We describe the relationship between lesion outcome and histopathological hallmarks in susceptible (BALB/c) and resistant (C57BL/6 and IL-4-deficient BALB/c) mouse strains over the course of a 12-week-infection with Leishmania major in the ear. The infiltration of mononuclear cells and polymorphonuclear cells occurred within 6 h and mononuclear cells predominated one week post-infection. Permissive intracellular growth of the pathogen was associated with non-healing lesions. In contrast, tissue damage and clearance of the parasite was observed in healing lesions and was associated with inducible nitric oxide synthase expression. The identification of the structural components of tissue reaction to the parasite in this study furthers our understanding of subjacent immune effector mechanisms.
Asunto(s)
Oído Externo/parasitología , Interleucina-4/inmunología , Leishmania major , Leishmaniasis Cutánea/patología , Óxido Nítrico Sintasa de Tipo II/inmunología , Animales , Susceptibilidad a Enfermedades , Oído Externo/patología , Femenino , Interleucina-4/deficiencia , Leishmaniasis Cutánea/inmunología , Masculino , Mastocitos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Schistosomiasis is a neglected parasitic disease that affects millions of people worldwide and is caused by helminth parasites from the genus Schistosoma. When caused by S. mansoni, it is associated with the development of a hepatosplenic disease caused by an intense immune response to the important antigenic contribution of adult worms and to the presence of eggs trapped in liver tissue. Although the importance of the spleen for the establishment of immune pathology is widely accepted, it has received little attention in terms of the molecular mechanisms operating in response to the infection. Here, we interrogated the spleen proteome using a label-free shotgun approach for the potential discovery of molecular mechanisms associated to the peak of the acute phase of inflammation and the development of splenomegaly in the murine model. Over fifteen hundred proteins were identified in both infected and control individuals and 325 of those proteins were differentially expressed. Two hundred and forty-two proteins were found upregulated in infected individuals while 83 were downregulated. Functional enrichment analyses for differentially expressed proteins showed that most of them were categorized within pathways of innate and adaptive immunity, DNA replication, vesicle transport and catabolic metabolism. There was an important contribution of granulocyte proteins and antigen processing and presentation pathways were augmented, with the increased expression of MHC class II molecules but the negative regulation of cysteine and serine proteases. Several proteins related to RNA processing were upregulated, including splicing factors. We also found indications of metabolic reprogramming in spleen cells with downregulation of proteins related to mitochondrial metabolism. Ex-vivo imunophenotyping of spleen cells allowed us to attribute the higher abundance of MHC II detected by mass spectrometry to increased number of macrophages (F4/80+/MHC II+ cells) in the infected condition. We believe these findings add novel insights for the understanding of the immune mechanisms associated with the establishment of schistosomiasis and the processes of immune modulation implied in the host-parasite interactions.
Asunto(s)
Proteoma , Proteómica , Schistosoma , Esquistosomiasis/diagnóstico , Esquistosomiasis/metabolismo , Esplenomegalia/metabolismo , Animales , Cromatografía Liquida , Modelos Animales de Enfermedad , Femenino , Inmunofenotipificación , Espectrometría de Masas , Ratones , Proteómica/métodos , Esquistosomiasis/parasitología , Bazo/citología , Bazo/metabolismo , Esplenomegalia/parasitologíaRESUMEN
Vaccine-induced protection against leishmaniasis is largely dependent on cell-mediated type 1 response and IL-12-driven IFN-gamma production. Surprisingly, our previous data showed that IL-12/23p40(-/-) mice could be vaccinated against L. amazonensis and were able to produce limited amounts of IFN-gamma. Since the role of CD8+ T in immunization against L. amazonensis is obscure, the aim of this study was to evaluate the effects of CD8+ cells in protection against L. amazonensis in IL-12/23p40(-/-) mice. In order to deplete CD8+ cells, one group of vaccinated animals was treated with anti-CD8 mAb. Infection was followed for 8 weeks. The vaccinated CD8+ -depleted group developed smaller lesions than the non-depleted group. CD8 depletion did not affect tissue parasitism or antibody response against the parasite, and treated animals displayed milder inflammation and better tissue integrity. IFN-gamma production in spleen and draining lymph node was impaired in the depleted group, suggesting that CD8+ cells produced this cytokine in IL-12-independent vaccination. Such results suggest that this T cell subset contributes to augmented pathology in IL12/23p40(-/-) mice vaccinated and challenged with L. amazonensis. Although these cells could produce some IFN-gamma the in the absence of IL-12, they do not affect the parasite tissue load.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Subunidad p40 de la Interleucina-12/deficiencia , Leishmania/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis/inmunología , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/inmunología , Citocinas/biosíntesis , Citocinas/deficiencia , Citocinas/inmunología , Femenino , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Subunidad p40 de la Interleucina-12/inmunología , Leishmaniasis/parasitología , Leishmaniasis/prevención & control , Vacunas contra la Leishmaniasis/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos AnimalesRESUMEN
We evaluated the ability of Lactobacillus delbrueckii UFV H2b20, a probiotic candidate, to stimulate the production of inflammatory cytokines and to induce macrophage activation and Th1 differentiation in peripheral blood mononuclear cells (PBMC) from healthy volunteers. Our results show that PBMC stimulated with heat-killed Lact. delbrueckii produced elevated levels of IL-12, IFN-gamma and TNF-alpha but no IL-10. IFN-gamma production was IL-12 dependent with NK cells as the main source. Furthermore, PBMC infected with Leishmania amazonensis presented elevated microbicidal activity when co-incubated with Lact. delbrueckii. Finally, Lact. delbrueckii was capable of inducing in vitro differentiation of L. amazonensis-specific Th1 cells. These findings suggest that this probiotic may be used as an adjuvant in vaccination protocols.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Citocinas/biosíntesis , Lactobacillus delbrueckii/inmunología , Células TH1/inmunología , Adulto , Diferenciación Celular , Células Cultivadas , Femenino , Humanos , Leucocitos Mononucleares/inmunología , Activación de Macrófagos , Masculino , Persona de Mediana Edad , Linfocitos T/citología , Linfocitos T/inmunología , VacunaciónRESUMEN
This study evaluated the potential of a Leishmania antigen vaccine in protecting BALB/c mice against Leishmania chagasi. Mice received two subcutaneous doses of L. amazonensis vaccine with Corynebacterium parvum and subsequent boost was done without adjuvant. One week later, mice were challenged with L. chagasi. We observed that this vaccine caused a significant reduction in parasite load in liver and spleen and induced a high production of IFN-gamma and IL-4 by spleen cells from vaccinated mice in response to Leishmania antigen. Together, our data show that this vaccine is capable of inducing a Th1/Th2 response that is important to control parasite replication.
Asunto(s)
Antígenos de Protozoos/inmunología , Leishmania/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis/inmunología , Propionibacterium acnes/inmunología , Animales , Femenino , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Leishmaniasis/parasitología , Leishmaniasis/prevención & control , Vacunas contra la Leishmaniasis/administración & dosificación , Vacunas contra la Leishmaniasis/uso terapéutico , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/parasitología , Ratones , Ratones Endogámicos BALB C , Bazo/efectos de los fármacos , Bazo/metabolismo , Bazo/parasitología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th2/efectos de los fármacos , Células Th2/inmunologíaRESUMEN
Differently from others Leishmania species, infection by the protozoan parasite L. amazonensis is associated with a lack of antigen-specific T-cell responses. Dendritic cells (DC) are essential for the innate immune response and for directing the differentiation of T-helper lymphocytes. Previously, we showed that L. amazonensis infection impairs DC activation through the activation of adenosine A2B receptor, and here, we evaluated the intracellular events triggered by this receptor in infected cells. To this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Our results show, for the first time, that L. amazonensis increases the production of cAMP and the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in infected DC by a mechanism dependent on the A2B receptor. Furthermore, L. amazonensis impairs CD40 expression and IL-12 production by DC, and the inhibition of adenylate cyclase, phosphoinositide 3-kinase (PI3K), and ERK1/2 prevent these effects. The increase of ERK1/2 phosphorylation and the inhibition of DC activation by L. amazonensis are independent of protein kinase A (PKA). In addition, C57BL/6J mice were inoculated in the ears with metacyclic promastigotes, in the presence of PSB1115, an A2B receptor antagonist. PSB1115 treatment increases the percentage of CD40+ DC on ears and draining lymph nodes. Furthermore, this treatment reduces lesion size and tissue parasitism. Lymph node cells from treated mice produce higher levels of IFN-γ than control mice, without altering the production of IL-10. In conclusion, we suggest a new pathway used by the parasite (A2B receptor â cAMP â PI3K â ERK1/2) to suppress DC activation, which may contribute to the decrease of IFN-γ production following by the deficiency in immune response characteristic of L. amazonensis infection.
RESUMEN
Protozoa of the genus Leishmania are intracellular parasites of macrophages and may cause diverse clinical forms of leishmaniasis, including cutaneous, diffuse cutaneous, mucocutaneous and visceral leishmaniasis. Infection with L. major in mice indicates that a protective immune response is achieved when Th1 cells are developed. Thus, adoptive or vaccine-induced protection against leishmaniasis is largely dependent on cell-mediated immunity and IFN-gamma production. Induction of a Th1 response is dependent on the presence of IL-12 whilst lymphocytes are activated. This study was aimed at evaluating the role of IL-12 during infection with L. amazonensis and after vaccination with Leishvacin (killed Leishmania amazonensis promastigotes), since the role of this cytokine in vaccine-induced immunity with this preparation in experimental models or in humans is not yet elucidated. Hence, C57BL/6 interleukin-12-deficient mice (IL-12p40(-/-)) and wild-type controls (wt) were infected with L. amazonensis and the course of infection, parasite burden and cytokine production were compared. IL-12p40(-/-) mice were more susceptible to L. amazonensis than wt: lesions and parasite burden were larger in IL-12p40(-/-) when compared to wt. Interestingly, IL-4 was not produced in the absence of IL-12 in response to infection with L. amazonensis. To evaluate the role of IL-12 in the vaccine-induced immunity against L. amazonensis infection, IL-12p40(-/-) wt mice were vaccinated in the base of the tail and subsequently challenged with L. amazonensis in the footpads. Surprisingly, vaccinated IL-12p40(-/-) mice developed smaller lesions and had fewer parasites in footpads than non-vaccinated controls. Lymph node and spleen cells from vaccinated IL-12p40(-/-) mice did not produce high levels of IFN-gamma in response do in vitro stimulus with antigen. Hence, partial protection against infection with L. amazonensis could be obtained in the absence of functional IL-12 and a typical Th1 response.
Asunto(s)
Interleucina-12/deficiencia , Leishmania mexicana/inmunología , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/prevención & control , Subunidades de Proteína/deficiencia , Vacunas Antiprotozoos/farmacología , Animales , Femenino , Interferón gamma/biosíntesis , Interleucina-12/genética , Subunidad p40 de la Interleucina-12 , Interleucina-4/biosíntesis , Leishmaniasis Cutánea/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Subunidades de Proteína/genéticaRESUMEN
Infection by protozoan parasites is part of the most common Tropical Neglected Diseases. In the case of leishmaniasis, several millions of people are at risk of contracting the disease. In spite of innumerous studies that elucidated the immune response capable of killing the parasite, the understanding of the evasion mechanisms utilized by the parasite to survive within the very cell responsible for its destruction is still incomplete. In this review, we offer a new approach to the control of the immune response against the parasite. The ability of the parasite to modulate the levels of extracellular ATP and adenosine either by directly acting on the levels of these molecules or by inducing the expression of CD39 and CD73 on the infected cell may influence the magnitude of the immune response against the parasite contributing to its growth and survival.
Asunto(s)
Leishmania/inmunología , Leishmaniasis/inmunología , Humanos , Transducción de SeñalRESUMEN
This work aimed to develop nanoemulsions (NE) containing cholesterol and Amphotericin B (AmB) evaluating the influence of a lipophilic amine (stearylamine; STE) on drug encapsulation efficiency (EE), cytotoxicity on macrophages and in vitro antileishmanial activity. The EE of AmB in NE was nearly 100% regardless of STE concentration. Stability studies showed that AmB-loaded NE with or without STE were stable revealing that AmB content and EE remained constant after 180days. In significant contrast, the EE for AmB in NE without cholesterol drastically decreased showing that this co-surfactant significantly improved the retention of drug in NE. The electronic absorption and circular dichroism (CD) data revealed that the signal characteristic of self-associated free AmB, the most toxic form to the host cells, was virtually absent in the spectra of AmB-loaded NE. In agreement, NE-induced toxicity toward macrophages was significantly lower than that observed for the conventional AmB. STE enhanced both cytotoxicity and the activity against intracellular amastigotes of AmB-loaded NE. However, selectivity index values for AmB-loaded NE were considerably higher than that observed for conventional AmB. AmB-loaded and cholesterol-stabilized NE constitutes an attractive alternative for the treatment of leishmaniasis.
Asunto(s)
Anfotericina B/química , Antiprotozoarios/química , Leishmania/efectos de los fármacos , Leishmaniasis , Nanopartículas/química , Anfotericina B/administración & dosificación , Animales , Antiprotozoarios/administración & dosificación , Línea Celular , Colesterol/administración & dosificación , Colesterol/química , Emulsiones , Leishmania/fisiología , Leishmaniasis/tratamiento farmacológico , RatonesRESUMEN
Leishmania amazonensis, the causal agent of diffuse cutaneous leishmaniasis, is known for its ability to modulate the host immune response. Because a relationship between ectonucleotidase activity and the ability of Leishmania to generate injury in C57BL/6 mice has been demonstrated, in this study we evaluated the involvement of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of L. amazonensis in the process of infection of J774-macrophages. Our results show that high-activity parasites show increased survival rate in LPS/IFN-γ-activated cells, by inhibiting the host-cell NO production. Conversely, inhibition of E-NTPDase activity reduces the parasite survival rates, an effect associated with increased macrophage NO production. E-NTPDase activity generates substrate for the production of extracellular adenosine, which binds to A2B receptors and reduces IL-12 and TNF-α produced by activated macrophages, thus inhibiting NO production. These results indicate that E-NTPDase activity is important for survival of L. amazonensis within macrophages, showing the role of the enzyme in modulating macrophage response and lower NO production, which ultimately favors infection. Our results point to a new mechanism of L. amazonensis infection that may pave the way for the development of new treatments for this neglected disease.
Asunto(s)
Adenosina Trifosfatasas/inmunología , Leishmaniasis/enzimología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Adenosina Trifosfatasas/efectos adversos , Animales , Modelos Animales de Enfermedad , Leishmaniasis/inmunología , Leishmaniasis/parasitología , Macrófagos/citología , Ratones , Ratones Endogámicos C57BLRESUMEN
To date, there is no vaccine available against human leishmaniasis. Although some vaccination protocols can induce immunity in murine models, they fail to induce protection in humans. The reasons for that remain unclear. The aim of the present study was to characterize the changes in the pattern of the immune response during subcutaneous vaccination with Leishvacin® in mice. We also investigated whether IFN-γ and nitric oxide synthase are indispensable for the protection elicited by the vaccine. C57BL/6 WT vaccinated mice showed smaller lesions and fewer numbers of parasites in footpads until 8 weeks post-infection. Up to this time, they produced higher levels of IFN-γ, IL-2, IL-4, IL-17A and IL-10 and higher specific antibody response than control non-vaccinated mice. Moreover, we showed that IFN-γ, most likely by induction of iNOS expression, is essential for immunity. However, after 12 weeks of infection, we observed loss of difference in lesion size and parasite burden between the groups. Loss of resistance was associated with the disappearance of differences in cytokine patterns between vaccinated and control mice, but not of antibody response, which remained different until a later time of infection. The reversal of resistance to L. amazonensis could not be explained by upregulation of regulatory cytokines. Our data point to a subversion of the host immune response by L. amazonensis even when a protective response was previously induced.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Citocinas/inmunología , Leishmania mexicana/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Cutánea/prevención & control , Vacunación , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunoglobulina G/inmunología , Interferón gamma/inmunología , Interleucina-10/inmunología , Interleucina-17/inmunología , Interleucina-4/inmunología , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/metabolismo , Propionibacterium acnes/inmunologíaRESUMEN
BACKGROUND: Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum) is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (â¼70 kDa and â¼45 kDa) have been found in the L. infantum genome. Here, we studied the â¼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection. METHODOLOGY/PRINCIPAL FINDINGS: We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP â=â UDP> ADP> UTP â=â ATP) in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis. CONCLUSIONS/SIGNIFICANCE: In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of NTPDases led to lower levels of in vitro adhesion and infection, suggesting that these proteins are possible targets for rational drug design.
Asunto(s)
Apirasa/metabolismo , Leishmania infantum/enzimología , Leishmaniasis Visceral/parasitología , Macrófagos/parasitología , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Apirasa/química , Apirasa/genética , Línea Celular , Perros , Femenino , Leishmania infantum/química , Leishmania infantum/citología , Leishmania infantum/metabolismo , Ganglios Linfáticos/parasitología , Ratones , Datos de Secuencia Molecular , Filogenia , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that "stressful conditions" will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.