Granulomas in parasitic diseases: the good and the bad.

Parasitic diseases affect more than one billion people worldwide, and most of them are chronic conditions in which the treatment and prevention are difficult. The appearance of granulomas, defined as organized and compact structures of macrophages and other immune cells, during various parasitic diseases is frequent, since these structures will only form when individual immune cells do not control the invading agent. Th2-typering various parasitic diseases are frequent, since these structures will only form when individual immune cells do not control the invading agent. The characterization of granulomas in different parasitic diseases, as well as recent findings in this field, is discussed in this review, in order to understand the significance of the granuloma and its modulation in the host-parasite interaction and in the immune, pathological, and parasitological aspects of this interaction. The parasitic granulomatous diseases granulomatous amebic encephalitis, toxoplasmosis, leishmaniasis, neurocysticercosis, and schistosomiasis mansoni are discussed as well as the mechanistic and dynamical aspects of the infectious granulomas.

Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria.

Cerebral malaria (CM) is a multifactorial syndrome involving an exacerbated proinflammatory status, endothelial cell activation, coagulopathy, hypoxia, and accumulation of leukocytes and parasites in the brain microvasculature. Despite significant improvements in malaria control, 15% of mortality is still observed in CM cases, and 25% of survivors develop neurologic sequelae for life-even after appropriate antimalarial therapy. A treatment that ameliorates CM clinical signs, resulting in complete healing, is urgently needed. Previously, we showed a hyperbaric oxygen (HBO)-protective effect against experimental CM. Here, we provide molecular evidence that HBO targets brain endothelial cells by decreasing their activation and inhibits parasite and leukocyte accumulation, thus improving cerebral microcirculatory blood flow. HBO treatment increased the expression of aryl hydrocarbon receptor over hypoxia-inducible factor 1-α (HIF-1α), an oxygen-sensitive cytosolic receptor, along with decreased indoleamine 2,3-dioxygenase 1 expression and kynurenine levels. Moreover, ablation of HIF-1α expression in endothelial cells in mice conferred protection against CM and improved survival. We propose that HBO should be pursued as an adjunctive therapy in CM patients to prolong survival and diminish deleterious proinflammatory reaction. Furthermore, our data support the use of HBO in therapeutic strategies to improve outcomes of non-CM disorders affecting the brain.-Bastos, M. F., Kayano, A. C. A. V., Silva-Filho, J. L., Dos-Santos, J. C. K., Judice, C., Blanco, Y. C., Shryock, N., Sercundes, M. K., Ortolan, L. S., Francelin, C., Leite, J. A., Oliveira, R., Elias, R. M., Câmara, N. O. S., Lopes, S. C. P., Albrecht, L., Farias, A. S., Vicente, C. P., Werneck, C. C., Giorgio, S., Verinaud, L., Epiphanio, S., Marinho, C. R. F., Lalwani, P., Amino, R., Aliberti, J., Costa, F. T. M. Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria.

Hyperbaric oxygen affects endothelial progenitor cells proliferation in vitro.

Hyperbaric oxygen is a clinical treatment that contributes to wound healing by increasing fibroblasts proliferation, collagen synthesis, and production of growth factors, inducing angiogenesis and inhibiting antimicrobial activity. It also has been shown that hyperbaric oxygen treatment (HBO), through the activation of nitric oxide synthase promotes an increase in the nitric oxide levels that may improve endothelial progenitor cells (EPC) mobilization from bone marrow to the peripheral blood and stimulates the vessel healing process. However, cellular mechanisms involved in cell proliferation and activation of EPC after HBO treatment remain unknown. Therefore, the present work aimed to analyze the effect of HBO on the proliferation of pre-treated bone marrow-derived EPC with TNF-alpha. Also, we investigated the expression of ICAM and eNOS by immunochemistry, the production of reactive species of oxygen and performed an in vitro wound healing. Although 1h of HBO treatment did not alter the rate of in vitro wound closure or cell proliferation, it increased eNOS expression and decreased ICAM expression and reactive oxygen species production in cells pre-treated with TNF-alpha. These results indicate that HBO can decrease the inflammatory response in endothelial cells mediated by TNF-alpha, and thus, promote vascular recovery after injury.

Effects of nanoemulsions prepared with essential oils of copaiba- and andiroba against Leishmania infantum and Leishmania amazonensis infections.

Plant products are an important source of bioactive agents against parasitic diseases, including leishmaniasis. Among these products, vegetable oils have gained ground in the pharmaceutical field. Here we report the development of nanoemulsions as a delivery system for copaiba and andiroba oils (nanocopa and nanoandi) in order to test their effects on Leishmania infantum and L. amazonensis. The nanocopa and nanoandi had an average particle size of 76.1 and 88.1, respectively with polydispersity index 0.14 to 0.16 and potential zeta -2.54 to -3.9. The data indicated toxic activity of nanocopa and nanoandi against promastigotes of both Leishmania species ultrastructural analyses by scanning electron microscopy revealed that exposition to nanoemulsions induced oval cell shape and retracted flagella. The treatment with nanocopa and nanoandi led to a reduction in L. infantum and L. amazonensis infection levels in macrophage cultures. The nanoemulsions treatment have significant beneficial effects on all the parameters evaluated in lesions induced by L. amazonensis (lesion size, parasite burden and histopathology) on BALB/c mice. The treatment of L. infantum-infected BALB/c mice with nanoemulsions also showed promising results reducing parasite burden in spleen and liver and improving histopathological features.

Liposomal formulations in the pharmacological treatment of leishmaniasis: a review.

Conventional chemotherapy for leishmaniasis includes considerably toxic drugs and reports of drug-resistance are not uncommon. Liposomal encapsulated drugs appear as an option for the treatment of leishmaniasis, providing greater efficacy for the active and reducing its side effects by promoting superior tissue absorption, favouring drug penetration into the macrophages, and retarding its clearance from the site of action. In this paper, a review on the advances achieved with liposome-based anti-leishmaniasis drug delivery systems is presented. Formulations prepared with either conventional or modified (sugar-coated, cationic, niosomes, peptides- and antibodies-bounded) liposomes for the delivery of pentavalent antimonials, amphotericin B, pentamidine, paromomycyn, and miltefosine were covered. This literature review depicts a scenario of no effective therapeutic agents for the treatment of this neglected disease, where liposomal formulations appear to improve the effectiveness of the available antileishmania agents.

Comparative in vitro toxicity of a graphene oxide-silver nanocomposite and the pristine counterparts toward macrophages.

BACKGROUND: Graphene oxide (GO) is a highly oxidized graphene form with oxygen functional groups on its surface. GO is an excellent platform to support and stabilize silver nanoparticles (AgNP), which gives rise to the graphene oxide-silver nanoparticle (GOAg) nanocomposite. Understanding how this nanocomposite interacts with cells is a toxicological challenge of great importance for future biomedical applications, and macrophage cells can provide information concerning the biocompatibility of these nanomaterials. The cytotoxicity of the GOAg nanocomposite, pristine GO, and pristine AgNP was compared toward two representative murine macrophages: a tumoral lineage (J774) and peritoneal macrophages collected from Balb/c mouse. The production of reactive oxygen species (ROS) by J774 macrophages was also monitored. We investigated the internalization of nanomaterials by transmission electron microscopy (TEM). The quantification of internalized silver was carried out by inductively coupled plasma mass spectrometry (ICP-MS). Nanomaterial stability in the cell media was investigated overtime by visual observation, inductively coupled plasma optical emission spectrometry (ICP OES), and dynamic light scattering (DLS). RESULTS: The GOAg nanocomposite was more toxic than pristine GO and pristine AgNP for both macrophages, and it significantly induced more ROS production compared to pristine AgNP. TEM analysis showed that GOAg was internalized by tumoral J774 macrophages. However, macrophages internalized approximately 60 % less GOAg than did pristine AgNP. The images also showed the degradation of nanocomposite inside cells. CONCLUSIONS: Although the GOAg nanocomposite was less internalized by the macrophage cells, it was more toxic than the pristine counterparts and induced remarkable oxidative stress. Our findings strongly reveal a synergistic toxicity effect of the GOAg nanocomposite. The toxicity and fate of nanocomposites in cells are some of the major concerns in the development of novel biocompatible materials and must be carefully evaluated.

HSP70 of Leishmania amazonensis alters resistance to different stresses and mitochondrial bioenergetics.

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.

Hyperbaric oxygen augments susceptibility to C. difficile infection by impairing gut microbiota ability to stimulate the HIF-1α-IL-22 axis in ILC3.

Hyperbaric oxygen (HBO) therapy is a well-established method for improving tissue oxygenation and is typically used for the treatment of various inflammatory conditions, including infectious diseases. However, its effect on the intestinal mucosa, a microenvironment known to be physiologically hypoxic, remains unclear. Here, we demonstrated that daily treatment with hyperbaric oxygen affects gut microbiome composition, worsening antibiotic-induced dysbiosis. Accordingly, HBO-treated mice were more susceptible to Clostridioides difficile infection (CDI), an enteric pathogen highly associated with antibiotic-induced colitis. These observations were closely linked with a decline in the level of microbiota-derived short-chain fatty acids (SCFAs). Butyrate, a SCFA produced primarily by anaerobic microbial species, mitigated HBO-induced susceptibility to CDI and increased epithelial barrier integrity by improving group 3 innate lymphoid cell (ILC3) responses. Mice displaying tissue-specific deletion of HIF-1 in RORγt-positive cells exhibited no protective effect of butyrate during CDI. In contrast, the reinforcement of HIF-1 signaling in RORγt-positive cells through the conditional deletion of VHL mitigated disease outcome, even after HBO therapy. Taken together, we conclude that HBO induces intestinal dysbiosis and impairs the production of SCFAs affecting the HIF-1α-IL-22 axis in ILC3 and worsening the response of mice to subsequent C. difficile infection.

Macrophages: plastic solutions to environmental heterogeneity.

INTRODUCTION: Macrophages are among the oldest cell types in the animal kingdom, and they have a long evolutionary history and experience various evolutionary pressures. It was clear from the earliest studies that variations exist in macrophage populations. Macrophages are known to adapt to their microenvironment. Although the paradigm for macrophage plasticity is their flexible program driven by environmental signals, the most common working hypothesis is that of a dichotomy between two major macrophage phenotypes, M1 and M2. METHODS: A PubMed and Web of Science databases search was performed providing evidences that numerous authors have expanded the concept of plasticity and conducted experimental studies focusing on the complex program of phenotypes. RESULTS AND CONCLUSIONS: This review evaluated a number of issues relating to macrophage plasticity, environmental heterogeneity and the potential for changes to be reversal or non reversal in an ecological context. The ecological principles of phenotypic plasticity which can assist in evaluating and interpreting macrophage experimental data are discussed as well.

Leishmania and its relationships with bacteria.

Leishmaniasis is a zoonotic and neglected disease, which represents an important public health problem worldwide. Different species of Leishmania are associated with different manifestations, and a practical problem that can worsen the condition of hosts infected with Leishmania is the secondary infection caused by bacteria. This review aims to examine the importance and prevalence of bacteria co-infection during leishmaniasis and the nature of this ecological relationship. In the cases discussed in this review, the facilitation phenomenon, defined as any interaction where the action of one organism has a beneficial effect on an organism of another species, was considered in the Leishmania-bacteria interaction, as well as the effects on one another and their consequences for the host.

Improved efficacy of meglumine antimoniate incorporated in anionic liposomes against Leishmania infantum infecting canine macrophages.

OBJECTIVES: Leishmaniasis is a zoonotic disease and several drugs have been used in the treatment, including meglumine antimoniate (AME). The chemotherapy reaches clinical cure but does not eliminate parasites, contributing to drug resistance. To improve AME efficacy we incorporated it in anionic liposomes. The antiparasitic activity and intracellular localization were investigated in canine macrophages infected with Leishmania infantum. METHODS: Liposomes (L-AME) is composed of egg phosphatidylcholine, cholesterol, palmitoyl oleoyl phosphatidyl serine and α-tocopherol (4 : 3 : 0.4 : 0.07 mol%) plus AME. L-AME size, polydispersity, zeta potential and morphology were analysed as well as antileishmanial activity and intracellular localization in DH82 macrophages. KEY FINDINGS: Liposomes (360 nm) zeta potential range from -40 to -65 mV, had 23% encapsulation efficiency and were stable for 180 days at 4°C. Free AME was cytotoxic towards L. infantum infected macrophages (ID50 = 0.012 M) while L-AME did not reduce cell viability. L-AME colocalized with parasites inside macrophages in a time-dependent manner, and reduced the percentage of infected cells and the number of intracellular parasites, decreasing the infection index (75-80%) twice as compared with AME treatment. CONCLUSIONS: Liposomal AME is a promising delivery system for treating visceral leishmaniasis, improving meglumine efficacy against L. infantum and minimizing its cytotoxicity towards canine macrophages.

Evaluation of prime and prime-boost immunization strategies in BALB/c mice inoculated with Leishmania infantum transfected with toxic plasmids.

The etiologic agents of visceral leishmaniasis are Leishmania infantum and Leishmania donovani. Despite the variety of drugs available to treat leishmaniasis, most lead to serious adverse effects, and resistance to these drugs has been reported. Currently, no leishmaniasis vaccine is available for humans. That is why the group developed transgenic L. infantum promastigote lines, which express toxic proteins after differentiation into amastigotes. That is why group developed the pFL-AMA plasmid and transfected it into L. Infantum promastigotes. This plasmid was expressed only in the amastigote form of the parasite. Sequences encoding toxic proteins (active bovine trypsin and egg avidin) were inserted in this plasmid, and the transfected parasites died after the differentiation process. In this study, two immunization protocols were performed in BALB/c mice: prime and prime-boost immunization prior to challenge with the wild-type L. infantum (WT). The parasite burdens in the spleen, liver, and bone marrow were evaluated to verify immunological protection. Histopathological analysis of the spleen and liver and the humoral immune response were also performed. The data showed that the parasite burden was reduced in prime-boosted mice in the spleen, liver, and bone marrow, indicating that mice immunized with two doses of the transfected parasites were satisfactorily protected. High levels of IgG, IgG1, and IgG2a antibodies were observed, as well as the presence of anti-inflammatory cytokine Interleukine-10 and pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF-α) and Interferon-γ (IFN - γ) suggesting a Th1/Th2 mix response, in addition to the presence of multinucleated giant cells in the spleen and lymphocyte infiltration in the liver. Therefore, L. infantum transfected with a toxic plasmid is an excellent vaccine candidate against visceral leishmaniasis and the application of a boost before the challenge promotes greater protection against WT L. infantum infection.

Leishmania (Viannia) braziliensis replicates in mouse bone marrow.

Leishmaniasis is a neglected disease caused by species of the protozoan Leishmania. Leishmania (Viannia) braziliensis causes the cutaneous and mucocutaneous forms of the disease. Experimental cutaneous infection of mice is one of the most important preclinical research models of leishmaniasis. Here, we investigated the course of infection in mice inoculated with two reference strains of L. (V.) braziliensis (MHOM/BR/00/BA788 strain [BA] and MHOM/BR/94/H-3227 strain [CE]). Although both parasite strains induced detectable footpad lesions, BA-infected mice developed small non-ulcerated lesions that self-healed, whereas CE-infected mice developed small non-ulcerated lesions that did not regress. The parasites were detected in the footpad lesions, lymph nodes draining the site of inoculation, spleen, and bone marrow of mice infected with BA or CE parasites at 6 and 25 weeks post-inoculation. These data indicate that L (V.) braziliensis-infected mice harbor parasites that spread, even when these animals do not display overt lesions. In addition, this is the first report of the presence of the parasite in the bone marrow of mice inoculated with L. (V.) braziliensis.

Monitoring Leishmania infantum Infections in Female Lutzomyia longipalpis by Using DNA Extraction on Cation Exchange Paper and PCR Pool Testing

Visceral leishmaniasis remains a serious public health issue, and Brazil was among the seven countries with the highest prevalence of this disease worldwide. The measures to control this disease are not easily developed, and the improvement of its diagnosis, surveillance, and control is still needed. This study aimed to carry out the polymerase chain reaction (PCR) diagnosis of Leishmania infantum in vector samples in some municipalities of the State of São Paulo, which included two municipalities with human disease transmission and two with dog transmission only. Vectors were collected in traps with luminous bait. Next, they were killed at -4 °C and kept in 70% alcohol. Groups of ten female insects (pools) were mashed on cation exchange paper (fine cellulose phosphate with 18 µEq/cm² ionic exchange capacity) for DNA extraction. The PCR was carried out to identify the natural infection of the Leishmania genus in female Lutzomyia longipalpis (Lu. Longipalpis). Out of the 3,880 Lu. longipalpis phlebotomines, 1060 were female and 2820 were male (3:1). The method used to extract the DNA in pools of ten phlebotomines and the PCR resulted in sensitivity, specificity, practicality, and faster analyses when compared to the individual analysis method. The procedure described can be used on a large scale in the leishmaniasis epidemiological surveillance, enabling a higher number of analyses and the optimization of human resources because the traditional diagnostic method is carried out via desiccation of the insect digestive system and microscopic examination, which is time-demanding and there is the need of manual skills.

Virulence and DNA sequence analysis of Cronobacter spp. isolated from infant cereals.

Cronobacter spp. is an opportunistic pathogen that causes severe infections, affecting newborns and infants, and is also an emerging cause of hospital-acquired infection in elderly populations. These infections are mainly associated with the consumption of infant formulas, even though these bacteria have been isolated from other foods as well. Cronobacter spp. invades epithelial cells and escapes the immune response mechanisms, multiplying inside macrophages. However, the pathogenesis and virulence factors of these bacteria have not been fully elucidated and need to be further studied. Therefore, this study aimed to evaluate the ability of Cronobacter spp. strains isolated from infant cereals to invade and survive within macrophages, investigate the virulence phenotype using the Galleria mellonella model, and identify possible genes involved in bacterial pathogenesis through pan-genome analysis. All the isolates were able to invade macrophages and the survival of bacteria decreased over a 72 h period, with bacterial cell counts reaching up to 106 CFU/ml. Cronobacter sakazakii isolate 112 exhibited a similar mortality rate (40-70%) to the ATCC BAA 894 strain (Cronobacter sakazakii) in G. mellonella assay. In addition, some unique virulence genes (isolate 7, ada_2, tcmA_1, acrB_3; isolate 78, ampC_2, rihC_1 and isolate 112, fimH, ylpA, gtrA) were identified within isolates with the invasive profile in the in vivo and in vitro assays. Furthermore, isolates from different species were grouped into seven distinct clusters in the pan-genome analysis. The most virulent isolates (7, 78, and 112) were grouped in distinct subclusters in the cladogram. This work revealed potential Cronobacter spp. pathogenic strains recovered from infant cereals.

Use of in vivo and in vitro systems to select Leishmania amazonensis expressing green fluorescent protein.

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.

Exploring TERRA during Leishmania major developmental cycle and continuous in vitro passages.

Telomeres from different eukaryotes, including trypanosomatids, are transcribed into TERRA noncoding RNAs, crucial in regulating chromatin deposition and telomere length. TERRA is transcribed from the C-rich subtelomeric strand towards the 3'-ends of the telomeric array. Using bioinformatics, we confirmed the presence of subtelomeric splice acceptor sites at all L. major chromosome ends. Splice leader sequences positioned 5' upstream of L. major chromosomes subtelomeres were then mapped using SL-RNA-Seq libraries constructed from three independent parasite life stages and helped confirm TERRA expression from several chromosomes ends. Northern blots and RT-qPCR validated the results showing that L. major TERRA is processed by trans-splicing and polyadenylation coupled reactions. The number of transcripts varied with the parasite's life stage and continuous passages, being more abundant in the infective forms. However, no putative subtelomeric promoters involved in TERRA's transcriptional regulation were detected. In contrast, the observed changes in parasite's telomere length during development, suggest that differences in telomeric base J levels may control TERRA transcription in L. major. Also, TERRA-R loops' detection, mainly in the infective forms, was suggestive of TERRA's involvement in telomere protection. Therefore, Leishmania TERRA shares conserved features with other eukaryotes and advances new telomere specific functions in a Public Health-impacting parasite.

Hypoxia, hypoxia-inducible factor-1α and vascular endothelial growth factor in a murine model of Schistosoma mansoni infection.

Schistosomiasis mansoni is a chronic parasitic disease where much of the symptomatology is attributed to granuloma formation, an immunopathological reaction against Schistosoma eggs. To more clearly understand the immunopathology of schistosomiasis, the tissue microenvironment generated by S. mansoni infected mice was investigated. Using the hypoxia marker pimonidazole, we provide immunohistochemical evidence that hypoxia occurred in inflammatory cells infiltrated around the eggs and cells surrounding granulomas in the liver, intestine, spleen and lungs of infected mice. Hypoxia-inducible factor-1α (HIF-1α) was mainly expressed in inflammatory cells surrounding the eggs and in hepatocytes surrounding cellular and fibrocellular granulomas in infected mouse liver. HIF-1α expression was also verified in granulomas in the other tissues tested (intestine, spleen and lungs). Vascular endothelial growth factor (VEGF) expression was observed in the extracellular space surrounding inflammatory cells in liver granuloma. The VEGF expression pattern verified in infected mouse liver was very similar to that observed in the other tissues tested. A strong positive correlation occurred between pimonidazole binding and HIF-1α and VEGF expression in the tissues tested, except for lung. This work is the first evidence that infection by a helminth parasite, S. mansoni, produces a hypoxic tissue microenvironment and induces HIF-1α and VEGF expression.

Phenotype evaluation of human and canine isolates of Leishmania infantum.

Human visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in countries of South and Central America are caused by Leishmania infantum and has been endemic in Brazil for several years. The parasite biodiversity as well as the pharmacologic properties of drugs and the host species, are involved in the efficacy or inefficacy of leishmaniasis treatments. Although there are substantial number of reports describing the genetic characterization of the clinical field isolates of L. infantum,the phenotypic parameters have been less studied. In this study isolates from human and canine leishmaniasis (Hum1 and Can1) obtained in Campinas, São Paulo state, Brazil were identified as L. infantum. The Hum1 and Can1 isolates exhibited typical promastigote growth pattern. Regarding morphological features Can1 isolate differed in cell size. The infectivity in vitro of both isolatesis lower compared to the reference strain of L. infantum. Moreover, the in vivo infectivity of the three parasites is similar in Balb/c mice. The Hum1 isolate is more sensitive to leishmanial drugs (amphotericin B, miltefosine and glucantime) than the Can1 isolate when inside human macrophages, but not when inside canine macrophages. These findings indicated that L. infantum isolates differs in some phenotypic characteristics.

Leishmania infantum transfected with toxic plasmid induces protection in mice infected with wild type L. infantum or L. amazonensis.

Leishmania infantum infection may cause visceral leishmaniasis (VL), a fatal disease having worldwide distribution, that may be silent or asymptomatic. The latter indicates that immunity is naturally developed in some individuals, and, therefore, a vaccine against VL would be possible. Molecular mechanisms of gene expression are being understood in Leishmania, and this knowledge may be useful for vaccine development. The aim of this study was developing an attenuated strain by regulating the expression of toxic proteins in a stage specific manner. For that purpose, the 3' UTR of an amastin gene, known by its increased expression in the amastigote phase, was selected for direct the expression of exogenous proteins. This construct (pFL-AMA), firstly, was proved effective for the expression of mCherry specifically in the intracellular form of L. infantum, as demonstrated by fluorescence microscopy, flow cytometry and Western blotting. Afterwards, mCherry coding sequence was replaced, in the pFL-AMA plasmid, by either egg avidin or the active form of bovine trypsin. Viability of transfected parasites was evaluated in promastigote axenic cultures and in in vitro infection of macrophages. Both lines of transfected parasites showed a limited capacity to multiply inside macrophages. BALB/c mice were inoculated intraperitoneally (i.p.) with a single dose consisting of 2 × 106L. infantum promastigotes transfected with plasmids bearing the toxic genes. After 10 weeks post-inoculation, no parasites were recovered by limiting dilution in either liver or spleen, but a specific immunological response was detected. The immunization with transfected parasites induced cellular and humoral immune responses with activation of TCD4+, TCD8+ and B cells, having a TH1-type response with increased levels of pro-inflammatory cytokines such as IFN-γ, TNF-α and IL-6. In parallel groups of mice, a challenge consisting on 1 × 106 virulent parasites of either L. infantum (inoculated i.p.) or L. amazonensis subcutaneously (s.c.) was performed. Vaccinated mice, challenged with L. infantum, showed lower parasite burdens in liver, spleen and bone marrow than infected mice with WT L. infantum (non-vaccinated); similarly, vaccinated mice developed smaller footpad inflammation than control group. These data support this strategy as an efficient immunization system aimed to the development of vaccines against different forms of leishmaniasis.