Protein disulfide isomerase (PDI) associates with NADPH oxidase and is required for phagocytosis of Leishmania chagasi promastigotes by macrophages

PDI, a redox chaperone, is involved in host cell uptake of bacteria/viruses, phagosome formation, and vascular NADPH oxidase regulation. PDI involvement in phagocyte infection by parasites has been poorly explored. Here, we investigated the role of PDI in in vitro infection of J774 macrophages by amastigote and promastigote forms of the protozoan Leishmania chagasi and assessed whether PDI associates with the macrophage NADPH oxidase complex. Promastigote but not amastigote phagocytosis was inhibited significantly by macrophage incubation with thiol/PDI inhibitors DTNB, bacitracin, phenylarsine oxide, and neutralizing PDI antibody in a parasite redox-dependent way. Binding assays indicate that PDI preferentially mediates parasite internalization. Bref-A, an ER-Golgi-disrupting agent, prevented PDI concentration in an enriched macrophage membrane fraction and promoted a significant decrease in infection. Promastigote phagocytosis was increased further by macrophage overexpression of wild-type PDI and decreased upon transfection with an antisense PDI plasmid or PDI siRNA. At later stages of infection, PDI physically interacted with L. chagasi, as revealed by immunoprecipitation data. Promastigote uptake was inhibited consistently by macrophage preincubation with catalase. Additionally, loss-or gain-of-function experiments indicated that PMA-driven NADPH oxidase activation correlated directly with PDI expression levels. Close association between PDI and the p22phox NADPH oxidase subunit was shown by confocal colocalization and coimmunoprecipitation. These results provide evidence that PDI not only associates with phagocyte NADPH oxidase but also that PDI is crucial for efficient macrophage infection by L. chagasi. J. Leukoc. Biol. 86: 989-998; 2009.

Uptake and antileishmanial activity of meglumine antimoniate-containing liposomes in Leishmania (Leishmania) major-infected macrophages

Leishmaniasis is a parasitic disease caused by the intramacrophage protozoa Leishmania spp. and may be fatal if left untreated. Although pentavalent antimonials are toxic and their mechanism of action is unclear, they remain the first-line drugs for treatment of leishmaniasis. An effective therapy could be achieved by delivering antileishmanial drugs to the site of infection. Compared with free drugs, antileishmanial agent-containing liposomes are more effective, less toxic and have fewer adverse side effects. The aim of this study was to develop novel meglumine antimoniate (MA)-containing liposome formulations and to analyse their antileishmanial activity and uptake by macrophages. Determination of the 50% inhibitory concentration (IC(50)) values showed that MA-containing liposomes were >= 10-fold more effective than the free drug, with a 5-fold increase in selectivity index, higher activity and reduced macrophage toxicity. The concentration required to kill 100% of intracellular amastigotes was >= 40-fold lower when MA was encapsulated in liposomes containing phosphatidylserine compared with the free drug. Fluorescence microscopy analysis revealed increased uptake of fluorescent liposomes in infected macrophages after short incubation times compared with non-infected macrophages. In conclusion, these data suggest that MA encapsulated in liposome formulations is more effective against Leishmania-infected macrophages than the non-liposomal drug. Development of liposome formulations is a valuable approach to the treatment of infectious diseases involving the mononuclear phagocyte system. (C) 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

In vitro effect of Aloe vera, Coriandrum sativum and Ricinus communis fractions on Leishmania infantum and on murine monocytic cells

In South America, visceral leishmaniasis is a zoonosis caused by the protozoan species Leishmania infantum (syn. L. chagasi) and is primarily transmitted through the bite of the female Lutzomyia longipalpis. Its main reservoir in urban areas is the dog. The application of control measures recommended by health agencies have not achieved significant results in reducing the incidence of human cases, and the lack of effective drugs to treat dogs resulted in the prohibition of this course of action in Brazil. Therefore, it is necessary to search new alternatives for the treatment of canine and human visceral leishmaniasis. The objectives of this study were to evaluate the in vitro effect of fractions from Aloe vera (aloe), Coriandrum sativum (coriander), and Ricinus communis (castor) on promastigotes and amastigotes of L. infantum and to analyze the toxicity against the murine monocytic cells RAW 264.7. To determine the viability of these substances on 50% parasites (IC50), we used a tetrazolium dye (MU) colorimetric assay (bromide 3-4.5-dimethylthiazol-2-yl-2,5-dephenyltetrazolium), and on amastigotes we performed an in situ ELISA. All fractions were effective against L. infantum promastigotes and did not differ from the positive control pentamidine (p > 0.05). However, the R. communis ethyl acetate and chloroform fractions, as well as the C. sativum methanol fraction, were the most effective against amastigotes and did not differ from the positive control amphotericin B (p > 0.05). The R. communis ethyl acetate fraction was the least toxic, presenting 83.5% viability of RAW 264.7 cells, which was similar to the results obtained with amphotericin B (p > 0.05). Based on these results, we intend to undertake in vivo studies with R. communis ethyl acetate fractions due the high effectiveness against amastigotes and promastigotes of L. infantum and the low cytotoxicity towards murine monocytic cells. (C) 2011 Elsevier B.V. All rights reserved.

A longitudinal study on the transmission dynamics of human Leishmania (Leishmania) infantum chagasi infection in Amazonian Brazil, with special reference to its prevalence and incidence

This was a longitudinal study carried out during a period over 2 years with a cohort of 946 individuals of both sexes, aged 1 year and older, from an endemic area of American visceral leishmaniasis (AVL) in Para State, Brazil. The object was to analyze the transmission dynamics of human Leishmania (Leishmania) infantum chagasi infection based principally on the prevalence and incidence. For diagnosis of the infection, the indirect fluorescent antibody test (IFAT) and leishmanin skin test (LST) were performed with amastigote and promastigote antigens of the parasite, respectively. The prevalence by LST (11.2%) was higher (p < 0.0001) than that (3.4%) by IFAT, and the combined prevalence by both tests was 12.6%. The incidences by LST were also higher (p < 0.05) than those by IFAT at 6 (4.7% A- 0.6%), 12 (4.7% A- 2.7%), and 24 months (2.9% A- 0.3%). Moreover, there were no differences (p > 0.05) between the combined incidences by both tests on the same point surveys, 5.2%, 6.3%, and 3.6%. During the study, 12 infected persons showed high IFAT IgG titers with no LST reactions: five children and two adults developed AVL (2,560-10,120), and two children and three adults developed subclinical oligosymptomatic infection (1,280-2,560). The combined tests diagnosed a total of 231 cases of infection leading to an accumulated prevalence of 24.4%.

Leishmania chagasi infection in cats with dermatologic lesions from an endemic area of visceral leishmaniosis in Brazil

Although dogs are considered the main domestic reservoirs for Visceral Leishmaniosis (VL), which is caused in the Americas by Leishmania chagasi, infected cats have also been recently found in endemic areas of several countries and became a public health concern. Accordingly, the purpose of this study was to evaluate cats with dermatologic lesions from an endemic area of VL and the natural infection of L. chagasi. A total of 55 cats were selected between April 2008 and November 2009 from two major animal shelters of Aracatuba, Southeastern Brazil. All cats underwent general and dermatologic examinations, followed by direct parasitological examination of lymphoid organs, immunosorbent assay (ELISA) and indirect immunofluorescence (IFAT). In addition, detection of amastigotes was performed by immunohistochemistry (IHC) in skin lesions of all cats. VL was diagnosed in 27/55 (49.1%) cats with dermatological problems. Amastigotes were found in lymphoid organs of 10/27 (37.0%) cats; serology of 14/27 (51.9%), 6/27 (22.2%) and 5/27 (18.5%) cats was positive for ELISA, IFAT and both, respectively. The IHC identified 9/27 (33.3%) cats; 5/27 (18.5%) were positive only for IHC and therefore increased the overall sensitivity. Specific FIV antibodies were found in 6/55(10.9%) cats, of which 5/6 (83.3%) had leishmaniosis. Real time PCR followed by amplicon sequencing successfully confirmed L chagasi infection. In conclusion, dermatological lesions in cats from endemic areas was highly associated to visceral leishmaniosis, and therefore skin IHC and differential diagnosis of LV should be always conducted in dermatological patients in such areas. (c) 2011 Elsevier B.V. All rights reserved.

A prospective study on the dynamics of the clinical and immunological evolution of human Leishmania (L.) infantum chagasi infection in the Brazilian Amazon region

This prospective study was carried out from October 2003 to December 2005 and involved a cohort of 946 individuals of both genders, aged 1-89 years, from an endemic area for American visceral leishmaniasis (AVL), in Para State, Brazil. The aim of the study was to analyze the dynamics of the clinical and immunological evolution of human Leishmania ( L.) infantum chagasi infection represented by the following clinical-immunological profiles: asymptomatic infection (AI); symptomatic infection (SI = AVL); subclinical oligosymptomatic infection (SOI); subclinical resistant infection (SRI); and indeterminate initial infection (III). Infection diagnosis was determined by the indirect fluorescent antibody test and leishmanin skin test. In total, 231 cases of infection were diagnosed: the AI profile was the most frequent (73.2%), followed by SRI (12.1%), III (9.9%), SI (2.6%) and SOI (2.2%). The major conclusion regarding evolution dynamics was that the III profile plays a pivotal role from which the cases evolve to either the resistant, SRI and AI, or susceptible, SOI and SI, profiles; only one of the 23 III cases evolved to SI, while most evolved to either SRI (nine cases) or SOI (five cases) and eight cases remained as III. (C) 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Further evidences on a new diagnostic approach for monitoring human Leishmania (L.) infantum chagasi infection in Amazonian Brazil

This was a prospective study carried out during a period over 2 years (May/2006-September/2008) with a cohort of 1,099 individuals of both genders, aged 1 year old and older, from an endemic area of American visceral leishmaniasis (AVL) in Para state, Brazil. The object was to analyze the prevalence and incidence of human Leishmania (L.) infantum chagasi infection as well as the dynamics evolution of its clinical-immunological profiles prior identified: (1) asymptomatic infection (AI); (2) symptomatic infection (SI = AVL); (3) sub-clinical oligosymptomatic infection (SOI); (4) sub-clinical resistant infection (SRI) and; (5) indeterminate initial infection (III). The infection diagnosis was performed by using both the indirect fluorescent antibody test and leishmanin skin test with amastigotes and promastigotes antigens of L. (L.) i. chagasi, respectively. A total of 187 cases of infection were recorded in the prevalence (17%), 117 in the final incidence (6.9%), and 304 in the accumulated prevalence (26.7%), which provided the following distribution into the clinical-immunological profiles: AI, 51.6%; III, 22.4%; SRI, 20.1%; SOI, 4.3%; and SI (=AVL), 1.6%. The major finding regarding the dynamics evolution of infection was concerned to III profile, from which the cases of infection evolved to either the resistant profiles, SRI (21 cases, 30.8%) and AI (30 cases, 44.1%), or the susceptible SI (=AVL; 1 case, 1.5%); the latter 16 cases remained as III till the end of the study. These results provided the conclusion that this diagnostic approach may be useful for monitoring human L. (L.) i. chagasi infection in endemic area and preventing the high morbidity of severe AVL cases.

A cross-sectional study on the clinical and immunological spectrum of human Leishmania (L.) infantum chagasi infection in the Brazilian Amazon region

The objectives of this study were to identify individuals with symptomatic and/or asymptomatic infection due to Leishmania (L.) infantum chagasi; to study the two types of infection, both clinically and immunologically, and to determine the prevalence rate of infection at the beginning of the study. This was a cross-sectional study with a cohort of 946 individuals, of both genders, from the age of 1 year, living in the municipality of Barcarena, PA, Brazil, an area endemic for American visceral leishmaniasis (AVL). The leishmanin skin test (LST) and the indirect fluorescent test (IFAT), were used for the diagnosis of infection. One hundred and twenty cases of infection were diagnosed, with a prevalence rate of 12.6%; eight cases showed high seroreactivity (1280-10240, IgG) in IFAT and no LST reaction; four of these cases were typical AVL and four had subclinical oligosymptomatic infection. Using two immunological methods with a clinical examination of the infected individuals enabled the identification of five clinical-immunological profiles which may promote a better understanding of the interaction between L. (L.) i. chagasi and the human immune response: asymptomatic infection (AI) 73.4%; subclinical resistant infection (SRI) 15%; subclinicat oligosymptomatic infection (SOI) 3%; symptomatic infection (AVL) 3% and indeterminate initial infection (III) 5%. (C) 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host. (c) 2010 Elsevier Ltd. All rights reserved.

The Hus1 homologue of Leishmania major encodes a nuclear protein that participates in DNA damage response

The protozoan parasite Leishmania presents a dynamic and plastic genome in which gene amplification and chromosome translocations are common phenomena. Such plasticity hints at the necessity of dependable genome maintenance pathways. Eukaryotic cells have evolved checkpoint control systems that recognize altered DNA structures and halt cell cycle progression allowing DNA repair to take place. In these cells, the PCNA-related heterotrimeric complex formed by the proteins Hus1, Rad9, and Rad1 is known to participate in the early steps of replicative stress sensing and signaling. Here we show that the Hus1 homolog of Leishmania major is a nuclear protein that improves the cell capability to cope with replicative stress. Overexpression of LmHus1 confers resistance to the genotoxic drugs hydroxyurea (HU) and methyl methanesulfonate (MMS) and resistance to HU correlates to reduced net DNA damage upon LmHus1 expression. (C) 2011 Elsevier B.V. All rights reserved.

Hypoxia modulates phenotype, inflammatory response, and leishmanial infection of human dendritic cells

Development of hypoxic areas occurs during infectious and inflammatory processes and dendritic cells (DCs) are involved in both innate and adaptive immunity in diseased tissues. Our group previously reported that macrophages exposed to hypoxia were infected with the intracellular parasite Leishmania amazonensis, but showed reduced susceptibility to the parasite. This study shows that although hypoxia did not alter human DC viability, it significantly altered phenotypic and functional characteristics. The expression of CD1a, CD80, and CD86 was significantly reduced in DCs exposed to hypoxia, whereas CD11c, CD14, CD123, CD49 and HLA-DR expression remained unaltered in DCs cultured in hypoxia or normoxia. DC secretion of IL-12p70, the bioactive interleukin-12 (IL-12), a cytokine produced in response to inflammatory mediators, was enhanced under hypoxia. In addition, phagocytic activity (Leishmania uptake) was not impaired under hypoxia, although this microenviroment induced infected DCs to reduce parasite survival, consequently controlling the infection rate. All these data support the notion that a hypoxic microenvironment promotes selective pressure on DCs to assume a phenotype characterized by pro-inflammatory and microbial activities in injured or inflamed tissues and contribute to the innate immune response.

The influence of glutathione modulators on the course of Leishmania major infection in susceptible and resistant mice

Glutathione (GSH) has an important dual role in parasite-host relationship in Leishmania major infection. Our previous studies showed that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in the protection of Leishmania against the toxic effect of nitrogen-derived reactive species. On the other hand, GSH also is very important to the modulation of the effective immune response, inducting NO production and leishmanicidal activity of macrophages. In the present study, we investigated the role of host GSH during the course of L. major infection, analysing the size of footpad lesions and parasite load from mice treated with two GSH modulators, N-acethyl-L-cysteine (NAC) and buthionine sulphoximine (BSO). Resistant mice treated with BSO, which depletes GSH develop exacerbated lesions, but only harbour higher parasite load in their lesions 2 weeks post-infection. Although the NAC treatment does not affect the footpad lesions development in susceptible BALB/c mice, it significantly reduced the tissue parasitism in the lesions throughout the course of infection. Interestingly, the treatment with BSO did not change the course of L. major infection on susceptible mice when compared with nontreated mice. These results suggest that GSH is an important antioxidant modulator during anti-Leishmania immune response in vivo.

Occurrence of antibodies anti-Neospora caninum, anti-Toxoplasma gondii, and anti-Leishmania chagasi in serum of dogs from Para State, Amazon, Brazil

A cross-sectional study was conducted to determine the occurrence of anti-Toxoplasma gondii, anti-Neospora caninum, and anti- Leishmania chagasi antibodies in dogs of the state of Para, Brazil. For this purpose, 129 blood samples were collected from dogs of different ages and gender. Samples of 72 dogs were collected from 39 rural properties from 19 municipalities, and 57 samples were from stray dogs, collected after captivity by the Center of Zoonosis Control from the municipality of Santar,m. The sera were analyzed for anti-T. gondii and anti-N. caninum antibodies by indirect fluorescent antibody tests with cutoff values of 1:16 and 1:50, respectively. For the presence of L. chagasi antibodies, enzyme-linked immunosorbent assay was used and positive results were confirmed by immunochromatographic method using the recombinant antigen K39. Of the total of 129 dogs, 90 (69.8%) were positive for T. gondii, 16 (12.4%) for N. caninum, and 30 (23.3%) for L. chagasi. Antibodies for all three parasites were found simultaneously in seven dogs (5.4%), mostly in urban dogs (six of seven). No association was observed related to gender and location (urban or rural) of dogs and occurrence of N. caninum and T. gondii antibodies although, regarding L. chagasi, higher prevalence was found in females (P < 0.02) and in dogs from urban location (P < 0.001). From the 39 farms, in 30 (76.9%) at least one dog was positive for T. gondii or N. caninum or both. Higher occurrence of Leishmania antibodies was observed in N. caninum-negative dogs (P < 0.05).

Transovarial passage of Leishmania infantum kDNA in artificially infected Rhipicephalus sanguineus

Phlebotomine sand flies are the only proven biological vectors of Leishmania parasites. However, Rhipicephalus sanguineus ticks have long been suspected to transmit Leishmania infantum in studies carried out in laboratory and natural conditions. In the present study, 5 mu l of L. infantum promastigotes (1 x 10(6) cells per ml) was injected into the hemocel through the coxa 1 of four engorged females (F1, F2, F3 and F4). Control ticks (F5 and F6) were injected with sterile phosphate-buffered saline (PBS) using the same procedure. Then, these females, their eggs, and the originated larvae were tested by real time polymerase chain reaction (real-time PCR) for the presence of L. infantum kinetoplast DNA (kDNA). Females and eggs were tested after the end of the oviposition period (about 5 weeks post-inoculation) whereas larvae were tested about 4 months after the inoculation of females. All artificially infected females were positive for L. infantum kDNA. In addition, two pools of eggs (one from F2 and other from F4) and four pools of larvae (one from each F1 and F4 and two from F2) were positive for L infantum kDNA. These results showed, for the first time, the transovarial passage of L. infantum kDNA in R. sanguineus ticks, thus suggesting that the transovarial transmission of L. infantum protozoa in ticks is worth to be investigated. (C) 2010 Elsevier Inc. All rights reserved.