BALB/c Mice Infected with Antimony Treatment Refractory Isolate of Leishmania braziliensis Present Severe Lesions due to IL-4 Production

Background: Leishmania braziliensis is the main causative agent of cutaneous leishmaniasis in Brazil. Protection against infection is related to development of Th1 responses, but the mechanisms that mediate susceptibility are still poorly understood. Murine models have been the most important tools in understanding the immunopathogenesis of L. major infection and have shown that Th2 responses favor parasite survival. In contrast, L. braziliensis-infected mice develop strong Th1 responses and easily resolve the infection, thus making the study of factors affecting susceptibility to this parasite difficult. Methodology/Principal Findings: Here, we describe an experimental model for the evaluation of the mechanisms mediating susceptibility to L. braziliensis infection. BALB/c mice were inoculated with stationary phase promastigotes of L. braziliensis, isolates LTCP393(R) and LTCP15171(S), which are resistant and susceptible to antimony and nitric oxide (NO), respectively. Mice inoculated with LTCP393(R) presented larger lesions that healed more slowly and contained higher parasite loads than lesions caused by LTCP15171(S). Inflammatory infiltrates in the lesions and production of IFN-gamma, TNF-alpha, IL-10 and TGF-beta were similar in mice inoculated with either isolate, indicating that these factors did not contribute to the different disease manifestations observed. In contrast, IL-4 production was strongly increased in LTCP393(R)-inoculated animals and also arginase I (Arg I) expression. Moreover, anti-IL-4 monoclonal antibody (mAb) treatment resulted in decreased lesion thickness and parasite burden in animals inoculated with LTCP393(R), but not in those inoculated with LTCP15171(S). Conclusion/Significance: We conclude that the ability of L. braziliensis isolates to induce Th2 responses affects the susceptibility to infection with these isolates and contributes to the increased virulence and severity of disease associated with them. Since these data reflect what happens in human infection, this model could be useful to study the pathogenesis of the L. braziliensis infection, as well as to design new strategies of therapeutic intervention.

A Method for Generation of Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells

The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM) as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L.) amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells.

PREVALENCE OF ANTIBODIES TO TRYPANOSOMA CRUZI, LEISHMANIA INFANTUM, ENCEPHALITOZOON CUNICULI, SARCOCYSTIS NEURONA, AND NEOSPORA CANINUM IN CAPYBARA, HYDROCHOERUS HYDROCHAERIS, FROM SAO PAULO STATE, BRAZIL

Little is known about the importance of capybara. Hydrochoerus hydrochaeris, as reservoirs for parasites of zoonotic or veterinary importance. Sera from 63 capybaras, from 6 counties in the state of Sao Paulo, Brazil, were examined for antibodies to Trypanosoma cruel, Leishmania infantum, Encephalitozoon cuniculi. Sarcacystis neurona, and Neospora caninum using an indirect immunofluorescent antibody test. Five (8%) of the 63 capybaras had antibodies to T cruzi epimastigotes. None of the samples from capybara reacted positively with L. infantum promastigotes or with spores of E. cuniculi. Two (3%) of the serum samples were positive for antibodies to S. neurona merozoites, and 2 (3%) of the serum samples were positive for antibodies to N. caninum tachyzoites. A serum sample from 1 capybara was positive for antibodies to both T cruzi and N. caninum. None of the remaining 62 samples reacted with more than 1 parasite.

Leishmania (Viannia) braziliensis transfectants overexpressing the miniexon gene lose virulence in vivo

The miniexon gene has a central role in the processing of polycistronic pre-mRNA of kinetoplastids. It is added to the 5` extremity of each mRNA, supplying the 5`-capped structure to the molecule. Previous studies in Leishmania (Leishmania) major showed that the overexpression of the miniexon array attenuates the Virulence of the parasite in in vivo assays. The results presented here extend those findings to Vionnia subgenus. Leishmania (Vionnia) braziliensis was transfected with a cosmid harboring a tandem array of one hundred miniexon gene copies and then characterized by Northern blot analysis. The overexpression of the exogenous gene was confirmed and its effect on the virulence of L (V.) braziliensis was investigated in hamsters. In BALB/c mice we could not detect parasites during the course of 15 weeks of infection. In addition, hamsters infected with transfectants overexpressing the miniexon gene exhibited only a minor footpad swelling of late onset and failed to develop progressive lesion, these attenuated parasites could be recovered from the inoculation site 1 year after infection. The persistence of parasites in the host indicates that a stable line overexpressing the miniexon may be tested as live vaccine against leishmaniasis. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

A quantitative and morphometric study of mast cells in cutaneous leishmaniasis

Background Mast cells (MCs) are related with healing process in chronic inflammatory diseases, although in cutaneous leishmaniasis (CL) its importance is unknown. The aim of this study was to determine the correlation of MC with clinical findings in patients with the localized form of CL. Methods A cohort of 85 patients with CL was evaluated. MCs count was performed in pre-treatment biopsies and correlation with clinical findings and Leishmania species determined by PCR were performed. Results The MCs count in patients with CL caused by Leishmania (V.) braziliensis was 14.3 +/- 9.8 cells/mm(2), and 7.0 +/- 6.5 cells/mm(2) in patients with L. (L.) amazonensis (P < 0.05). The linear regression of MCs count with the age showed a tendency of cell number decreasing, according to ageing of the patient (r(2) = 0.05; P < 0.05). The association of disease`s duration and MCs count was positive (r(2) = 0.11; P < 0.05). There was not any association of MCs count with number of lesions neither with Leishmania antigen expression. The MCs count was higher in patients with earlier healing after treatment (P < 0.05). Conclusion MC can be important in CL and related with healing lesion.

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.

Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs

The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination.

Species-specific identification of Leishmania in naturally infected sand flies captured in Mato Grosso do Sul State, Brazil

An increase in cutaneous and visceral leishmaniasis cases has been reported in recent years in the state of Mato Grosso do Sul, Brazil, and little is known to date about their etiological agents. An investigation into natural Leishmania infection of sand flies captured in this state between December 2003 and August 2004 was carried out. Mini-exon sequences were used as targets to identify Leishmania, and an RFLP technique was employed for those identified as belonging to the Viannia subgenus. Calculation of the minimal infection rate (MR) revealed that 1.6% of sand flies captured in the forest, peridomicile and intradomicile were positive. Six species were found to be infected by Leishmania (V.) braziliensis. Interestingly, two of the six species. Lutzomyia longipalpis and Nyssomyia whitmani, were captured in anthropic environments. The findings of this study constitute a useful tool for planning control measures against this disease in the State of Mato Grosso do Sul. (C) 2010 Elsevier B.V. All rights reserved.

Ca(2+)-activated transbilayer movement of plasma membrane phospholipids in Leishmania donovani during ionomycin or thapsigargin stimulation

The protozoan parasite Leishmania causes serious infections in humans all over the world. After being inoculated into the skin through the bite of an infected sandfly, Leishmania promastigotes must gain entry into macrophages to initiate a successful infection. Specific, surface exposed phospholipids have been implicated in Leishmania-macrophage interaction but the mechanisms controlling and regulating the plasma membrane lipid distribution remains to be elucidated. Here, we provide evidence for Ca(2+)-induced phospholipid scrambling in the plasma membrane of Leishmania donovani. Stimulation of parasites with ionomycin increases intracellular Ca(2+) levels and triggers exposure of phosphatidylethanolamine at the cell surface. We found that increasing intracellular Ca(2+) levels with ionomycin or thapsigargin induces rapid transbilayer movement of NBD-labelled phospholipids in the parasite plasma membrane that is bidirectional, independent of cellular ATP and not specific to the polar lipid head group. The findings suggest the presence of a Ca(2+)-dependent lipid scramblase activity in Leishmania parasites. Our studies further show that lipid scrambling is not activated by rapid exposure of promastigotes to higher physiological temperature that increases intracellular Ca(2+) levels. (C) 2011 Elsevier B.V. All rights reserved.

Species diversity of Leishmania (Viannia) parasites circulating in an endemic area for cutaneous leishmaniasis located in the Atlantic rainforest region of northeastern Brazil

OBJECTIVES To identify the aetiological agents of cutaneous leishmaniasis and to investigate the genetic polymorphism of Leishmania (Viannia) parasites circulating in an area with endemic cutaneous leishmaniasis (CL) in the Atlantic rainforest region of northeastern Brazil. METHODS Leishmania spp. isolates came from three sources: (i) patients diagnosed clinically and parasitologically with CL based on primary lesions, secondary lesions, clinical recidiva, mucocutaneous leishmaniasis and scars; (ii) sentinel hamsters, sylvatic or synanthropic small rodents; and (iii) the sand fly species Lutzomyia whitmani. Isolates were characterised using monoclonal antibodies, multilocus enzyme electrophoresis (MLEE) and polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region rDNA locus. RESULTS Seventy-seven isolates were obtained and characterised. All isolates were identified as Leishmania (Viannia) braziliensis serodeme 1 based on reactivity to monoclonal antibodies. MLEE identified 10 zymodemes circulating in the study region. Most isolates were classified as zymodemes closely related to L. (V.) braziliensis, but five isolates were classified as Leishmania (Viannia) shawi. All but three of the identified zymodemes have so far been observed only in the study region. Enzootic transmission and multiclonal infection were observed. CONCLUSIONS Our results confirm that transmission cycle complexity and the co-existence of two or more species in the same area can affect the level of genetic polymorphism in a natural Leishmania population. Although it is not possible to make inferences as to the modes of genetic exchange, one can speculate that some of the zymodemes specific to the region are hybrids of L. (V.) braziliensis and L. (V.) shawi.

In vivo infection by Trypanosoma cruzi: The conserved FLY domain of the gp85/trans-sialidase family potentiates host infection

Trypanosoma cruzi is a protozoan parasite that infects vertebrates, causing in humans a pathological condition known as Chagas` disease. The infection of host cells by T. cruzi involves a vast collection of molecules, including a family of 85 kDa GPI-anchored glycoproteins belonging to the gp85/trans-sialidase superfamily, which contains a conserved cell-binding sequence (VTVXNVFLYNR) known as FLY, for short. Herein, it is shown that BALB/c mice administered with a single dose (1 mu g/animal, intraperitoneally) of FLY-synthetic peptide are more susceptible to infection by T. cruzi, with increased systemic parasitaemia (2-fold) and mortality. Higher tissue parasitism was observed in bladder (7.6-fold), heart (3-fold) and small intestine (3.6-fold). Moreover, an intense inflammatory response and increment of CD4(+) T cells (1.7-fold) were detected in the heart of FLY-primed and infected animals, with a 5-fold relative increase of CD4(+)CD25(+)FoxP3(+) T (Treg) cells. Mice treated with anti-CD25 antibodies prior to infection, showed a decrease in parasitaemia in the FLY model employed. In conclusion, the results suggest that FLY facilitates in vivo infection by T. cruzi and concurs with other factors to improve parasite survival to such an extent that might influence the progression of pathology in Chagas` disease.

Vancomycin use in a brazilian teaching hospital: comparison with the Hospital Infection Controlpractices Advisory Committee Guidelines (HICPAC)

This study describes vancomycin prescribing patterns in an average complexity hospital and compare the guidelines proposed by the Hospital Infection Control Practices Advisory Committee (HICPAC). The study was conducted in a 256-bed secondary-care hospital. Data were collected of all patients given vancomycin from March 2003 to February 2004, using a standardized chart-extraction form designed. Appropriate and inappropriate use was reviewed according to the Hospital Infection Control Practices Advisory Committee (HICPAC) guidelines on prudent vancomycin use. Out of 118 prescriptions, 95 (80.5%) were considered appropriate. Out of these 95 orders, 77 (81.1%) were administered for empiric treatment of suspected Gram-positive infections, 17 (17.9%) were administered for treatment of proven Gram-positive infections (76.5% identified as Staphyloccocus aureus-like agents) and 1 (1.0%) for beta-lactam allergy. The majority of the patients (96.6%) had recently used an antimicrobial medication (3 months). The mean pre-treatment hospitalization period was 11±10 days. Out of the 118 treatments, 67 (56.8%) were for nosocomial infections. The more frequent indications for vancomycin use were pneumonia (48.3%) and primary sepsis (18.6%), accounting for more than 66% of all treatments. No restriction policy was suggested because vancomycin use was considered adequate in the majority of the treatment cases. The broad empiric use of this antimicrobial was greater than expected in the institution and its use should be revised.

Evaluation of the effects of ozone therapy in the treatment of intra-abdominal infection in rats

INTRODUCTION: The antibacterial effect of ozone (O3) has been described in the extant literature, but the role of O3 therapy in the treatment of certain types of infection remains controversial. OBJECTIVES: To evaluate the effect of intraperitoneal (i.p.) O3 application in a cecal ligation/puncture rat model on interleukins (IL-6, IL-10) and cytokine-induced neutrophil chemoattractant (CINC)-1 serum levels, acute lung injury and survival rates. METHODS: Four animal groups were used for the study: a) the SHAM group underwent laparotomy; b) the cecal ligation/puncture group underwent cecal ligation/puncture procedures; and c) the CLP+O2 and CLP+O3 groups underwent CLP+ corresponding gas mixture infusions (i.p.) throughout the observation period. IL-6, CINC-1 and IL-10 concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Acute lung injury was evaluated with the Evans blue dye lung leakage method and by lung histology. P<0.05 was considered significant. RESULTS: CINC-1 was at the lowest level in the SHAM group and was lower for the CLP+O3 group vs. the CLP+O2 group and the cecal ligation/puncture group. IL-10 was lower for the SHAM group vs. the other three groups, which were similar compared to each other. IL-6 was lower for the SHAM group vs. all other groups, was lower for the CLP+O3 or CLP+O2 group vs. the cecal ligation/puncture group, and was similar for the CLP+O3 group vs. the CLP+O2 group. The lung histology score was lower for the SHAM group vs. the other groups. The Evans blue dye result was lower for the CLP+O3 group vs. the CLP+O2 group and the cecal ligation/puncture group but similar to that of the SHAM group. The survival rate for the CLP+O3 group was lower than for the SHAM group and similar to that for the other 2 groups (CLP and CLP+O2). CONCLUSION: Ozone therapy modulated the inflammatory response and acute lung injury in the cecal ligation/puncture infection model in rats, although there was no improvement on survival rates.