Transcriptome profiling of Paracoccidioides brasiliensis yeast-phase cells recovered from infected mice brings new insights into fungal response upon host interaction

Paracoccidioides brasiliensis is a fungal human pathogen with a wide distribution in Latin America. It causes paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. Although gene expression in P. brasiliensis had been studied, little is known about the genome sequences expressed by this species during the infection process. To better understand the infection process, 4934 expressed sequence tags (ESTs) derived from a non-normalized cDNA library from P. brasiliensis (isolate Pb01) yeast-phase cells recovered from the livers of infected mice were annotated and clustered to a UniGene (clusters containing sequences that represent a unique gene) set with 1602 members. A large-scale comparative analysis was performed between the UniGene sequences of P. brasiliensis yeast-phase cells recovered from infected mice and a database constructed with sequences of the yeast-phase and mycelium transcriptome (isolate Pb01) (https://dna.biomol.unb.br/Pb/), as well as with all public ESTs available at GenBank, including sequences of the P. brasiliensis yeast-phase transcriptome (isolate Pb18) (http:// www.ncbi.nlm.nih.gov/). The focus was on the overexpressed and novel genes. From the total, 3184 ESTs (64.53%) were also present in the previously described transcriptome of yeast-form and mycelium cells obtained from in vitro cultures (https://dna.biomol.unb.br/Pb/) and of those, 1172 ESTs (23.75% of the described sequences) represented transcripts overexpressed during the infection process. Comparative analysis identified 1750 ESTs (35.47% of the total), comprising 649 UniGene sequences representing novel transcripts of P. brasiliensis, not previously described for this isolate or for other isolates in public databases. KEGG pathway mapping showed that the novel and overexpressed transcripts represented standard metabolic pathways, including glycolysis, amino acid biosynthesis, lipid and sterol metabolism. The unique and divergent representation of transcripts in the cDNA library of yeast cells recovered from infected mice suggests differential gene expression in response to the host milieu.

Developmental and reproductive patterns of Triatoma brasiliensis infected with Trypanosoma cruzi under laboratory conditions

The aim of this work was to study the interaction between Trypanosoma cruzi-1 and Triatoma brasiliensis. A group of 1st instar nymphs was initially fed on T. cruzi-infected mice and a control group was fed on uninfected mice. From the second feeding onwards, both groups were otherwise fed on non-infected mice. The resulting adults were grouped in pairs: infected male/uninfected female, uninfected male/infected female, infected male and female and uninfected male/uninfected female. The infection affected only the 1st instar nymphs, which took significantly more time to reach the 2nd instar than uninfected nymphs. The differences in the molting time between the infected and uninfected nymphs from the 2nd to the 5th instars were not statistically significant. Both groups presented similar rates of nymphal mortality and reproductive performance was not significantly affected by infection in any of the treatments.

The Liver Plays a Major Role in Clearance and Destruction of Blood Trypomastigotes in Trypanosoma cruzi Chronically Infected Mice

Intravenous challenge with Trypanosoma cruzi can be used to investigate the process and consequences of blood parasite clearance in experimental Chagas disease. One hour after intravenous challenge of chronically infected mice with 5610 6 trypomastigotes, the liver constituted a major site of parasite accumulation, as revealed by PCR. Intact parasites and/or parasite remnants were visualized at this time point scattered in the liver parenchyma. Moreover, at this time, many of liver-cleared parasites were viable, as estimated by the frequency of positive cultures, which considerably diminished after 48 h. Following clearance, the number of infiltrating cells in the hepatic tissue notably increased: initially (at 24 h) as diffuse infiltrates affecting the whole parenchyma, and at 48 h, in the form of large focal infiltrates in both the parenchyma and perivascular spaces. Phenotypic characterization of liver-infiltrating cells 24 h after challenge revealed an increase in Mac1(+), CD8(+) and CD4(+) cells, followed by natural killer (NK) cells. As evidence that liver-infiltrating CD4(+) and CD8(+) cells were activated, increased frequencies of CD69(+) CD8(+), CD69(+) CD4(+) and CD25(+) CD122(+) CD4(+) cells were observed at 24 and 48 h after challenge, and of CD25(-)CD122(+)CD4(+) cells at 48 h. The major role of CD4(+) cells in liver protection was suggested by data showing a very high frequency of interferon (IFN)-gamma-producing CD4(+) cells 24 h after challenge. In contrast, liver CD8(+) cells produced little IFN-gamma, even though they showed an enhanced potential for secreting this cytokine, as revealed by in vitro T cell receptor (TCR) stimulation. Confirming the effectiveness of the liver immune response in blood parasite control during the chronic phase of infection, no live parasites were detected in this organ 7 days after challenge.

Leukotriene B(4)-loaded microspheres as a new approach to enhance antimicrobial responses in Histoplasma capsulatum-infected mice

Histoplasmosis is a pulmonary disease characterised by chronic granulomatous and suppurative inflammatory reactions caused by Histoplasma capsulatum. Regarding new therapies to control fungal infections, the aim of this study was to investigate whether pulmonary administration of leukotriene B(4) (LTB(4))-loaded microspheres (MS) could confer protection to 5-lipoxygenase knockout (5-LO(-/-)) mice infected by H. capsulatum. In this study, MS containing LTB4 were administered intranasally to mice infected by H. capsulatum. On Day 14 after the infection, fungal recovery from the lungs and histology were evaluated and inflammatory cytokines were measured. Pulmonary administration of LTB(4)-loaded MS was able to reduce fungal recovery from infected lungs. Production of important inflammatory cytokines related to host defence was augmented following MS administration to the lungs. Lung histology also showed that infected mice presented a clear reduction in the fungal burden following the pulmonary release of LTB4 from MS. Our study provides evidence that the proposed biodegradable microparticulate system, which can release LTB4 to the lungs, can be employed as therapy, enhancing the antimicrobial activity of host cells during histoplasmosis. (C) 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS(-/-)) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS(-/-) mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS(-/-) mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS(-/-) mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS(-/-) mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-gamma and TNF-alpha) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis. (c) 2008 Elsevier Masson SAS. All rights reserved.

Deficiency of IL-12p40 subunit determines severe paracoccidioidomycosis in mice

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis. To investigate the role of interleukin (IL)-12 in this disease, IL-12p40(-/-) deficient mice (IL-12p40(-/-)) and wild type mice (WT) were infected intravenously with viable yeast cells of P. brasiliensis 18 isolate. We found that, unlike WT mice, IL-12p40(-/-) mice did not control fungal proliferation and dissemination and succumbed to infection by day 21 after inoculation. Additionally, IL-12p40(-/-) mice presented a higher number of granulomas/mm(2) in lung tissue than WT mice, and showed unorganized granulomas containing high numbers of yeast cells. Moreover, IL-12p40(-/-) mice did not release detectable levels of IFN-gamma, but they produced high levels of IL-10, as well as IgG1 antibody. Additionally, splenocytes from both infected IL-12p40(-/-) and WT mice exhibited a suppressed Con-A-induced T cell proliferative response. Our findings suggest that the IL-12p40 subunit mediates resistance in paracoccidioidomycosis by inducting IFN-gamma production and a Th1 immune response

Activity of praziquantel against Hymenolepis nana, at different development stages, in experimentally infected mice

Single doses of praziquantel were administered by oral route, at various time intervals, following the experimental infection of mice with Hymenolepis nana eggs (2000 per animal), to investigate the drug action against different development stages of the parasite. It was shown that either 25 or 50 mg/kg given on the 4th day after inoculation had just a partial effect against the cysticercoids. Moreover, 25 mg/kg given on the 7th day was not able to kill all juvenile forms as well. However, this dose administered on the 10th day, when the parasites had reached maturity taut oviposition was not yet initiated was 100% efficacious. The same degree of efficacy was achieved with the administration of 25 mg/kg on the 14th day when the fully mature worms already lay eggs. These animal findings indicate that in the treatment of human hymenolepiasis praziquantel, 25 mg/kg, should be taken twice, 10 days apart, so that the second dose kills the larval and juvenile forms which have survived the first one. This should be particularly recommended for treating H. nana infection in close communities.

Anaphylaxis with Schistosoma mansoni extracts in normal and infected mice

Methods generally utilized for studies on anaphylaxis to protein antigens such as determination of histamine release to the blood, hemoconcentration, histamine release from peritoneal mast cells and passive cutaneous anaphylaxis (PCA) were used to investigate some aspects of the anaphylaxis to parasite antigens in Schistosoma mansoni infected mice. The release of histamine to the blood and significant rates of hemoconcentration were induced by intravenous injection of schistosomula or cercarial extracts into 10-13 weeks infected mice. Cercarial, schistosomula, worm tegument and soluble egg antigens were able to trigger histamine release from peritoneal mast cells from chronically infected mice. In spite of the PCA reaction beeing detected within 2 hours of sensitization (IgG1antibodies) in 6 of 8 tested sera from chronically infected mice, no detectable reactions were obtained after 48 hours sensitization (IgE antibodies). Although IgE was not detected in the circulation, by the PCA technique, the results indicate that the infected mice contained IgE antibodies bound to their mast cells.

Evolution of subpatent parasitaemia in Trypanosoma cruzi chronically infected mice with the help of a cyclophosphamide amplification transfer assay

We have evaluated the sensitivity of the classical blood subinoculation method, modified through cyclophosphamide treatment of transferred mice, for the detection of occult parasitaemias in Trypanosoma cruzi chronically infected mice. Besides its simplicity, the method was shown to be highly sensitive for both the "chronic" phase parasites (99% of chronic cases were shown to harbour occult parasitaemias) and for the acute phase parasites (T. cruzi could be detected in 53.8% of animals transferred with one Y strain parasite and in 20% of animals transferred with one CL strain parasite). Using acute phase bloodforms, the assay proved to be more sensitive than conventional subinoculation when dealing with the CL, but not the Y strain of the parasite. With the help of this parasite detection tool, we have studied during a one year period, the evolution of subpatent parasitaemias in a group of mice which survived through chemotherapy from lethal acute phase of T. cruzi infection. Cyclophosphamide transfer assay revealed occult parasitaemias in 100% of the chronic animals, nevertheless, continuous and discontinuous patterns of positivity were observed.

Effect of wide spectrum anti-helminthic drugs upon Schistosoma mansoni experimentally infected mice

Mebendazole, albendazole, levamisole and thiabendazole are well known as active drugs against several nematode species, and against cestodes as well, when the first two drugs are considered. None of the drugs have proven activity, however, against trematodes. We tested the effect of these drugs on the fecal shedding of schistosome eggs and the recovering of adult schistosomes, after portal perfusion in Schistosoma mansoni experimentally infected mice. Balb/c mice infected with 80 S. mansoni cercariae were divided into three groups, each in turn subdivided into four other groups, for each tested drug. The first group was treated with each one of the studied drugs 25 days after S. mansoni infection; the second group was submitted to treatment with each one of the drugs 60 days after infection. Finally, the third group, considered as control, received no treatment. No effect upon fecal shedding of S. mansoni eggs and recovering of schistosomes after portal perfusion was observed when mice were treated with either mebendazole or albendazole. Mice treated with either levamisole or thiabendazole, on the other hand, showed a significant reduction in the recovering of adult schistosomes after portal perfusion, mainly when both drugs were given during the schistosomula evolution period, i.e., 25 days after cercariae penetration, probably due to unspecific immunomodulation

Effects of vitamin C supplementation on acute phase Chagas disease in experimentally infected mice with Trypanosoma cruzi QM1 strain

The tissue changes that occur in Chagas disease are related to the degree of oxidative stress and antioxidant capacity of affected tissue. Studies with vitamin C supplementation did not develop oxidative damage caused by Chagas disease in the host, but other studies cite the use of peroxiredoxins ascorbate - dependent on T. cruzi to offer protection against immune reaction. Based on these propositions, thirty "Swiss" mice were infected with T. cruzi QM1 strain and treated with two different vitamin C doses in order to study the parasitemia evolution, histopathological changes and lipid peroxidation biomarkers during the acute phase of Chagas disease. The results showed that the parasite clearance was greater in animals fed with vitamin C overdose. There were no significant differences regarding the biomarkers of lipid peroxidation and inflammatory process or the increase of myocardium in animals treated with the recommended dosage. The largest amount of parasite growth towards the end of the acute phase suggests the benefit of high doses of vitamin C for trypomastigotes. The supplementation doesn't influence the production of free radicals or the number of amastigote nests in the acute phase of Chagas disease.

EFFICACY OF NITAZOXANIDE AGAINST Toxocara canis: LARVAL RECOVERY AND HUMORAL IMMUNE RESPONSE IN EXPERIMENTALLY INFECTED MICE

SUMMARY The efficacy of nitazoxanide (NTZ) against toxocariasis was investigated in an experimental murine model and results were compared to those obtained using mebendazole. Sixty male BALB/c mice, aged six to eight weeks-old, were divided into groups of 10 each; fifty were orally infected with 300 larvaed eggs of T. canisand grouped as follows, G I: infected untreated mice; G II: infected mice treated with MBZ (15 mg/kg/day) 10 days postinfection (dpi); G III: infected mice treated with NTZ (20 mg/kg/day) 10 dpi; G IV: infected mice treated with MBZ 60 dpi; G V: infected mice treated with NTZ 60 dpi; GVI: control group comprising uninfected mice. Mice were bled via retro-orbital plexus on four occasions between 30 and 120 dpi. Sera were processed using the ELISA technique to detect IgG anti- Toxocaraantibodies. At 120 dpi, mice were sacrificed for larval recovery in the CNS, liver, lungs, kidneys, eyes and carcass. Results showed similar levels of anti- ToxocaraIgG antibodies among mice infected but not submitted to treatment and groups treated with MBZ or NTZ, 10 and 60 dpi. Larval recovery showed similar values in groups treated with NTZ and MBZ 10 dpi. MBZ showed better efficacy 60 dpi, with a 72.6% reduction in the parasite load compared with NTZ, which showed only 46.5% reduction. We conclude that administration of these anthelmintics did not modify the humoral response in experimental infection by T. canis. No parasitological cure was observed with either drug; however, a greater reduction in parasite load was achieved following treatment with MBZ.

EVALUATION OF THE THERAPEUTIC EFFICACY OF LEVAMISOLE HYDROCHLORIDE ON THIRD-STAGE LARVAE OF Lagochilascaris minor IN EXPERIMENTALLY INFECTED MICE

Lagochilascariosis, a disease caused by Lagochilascaris minor, affects the neck, sinuses, tonsils, lungs, the sacral region, dental alveoli, eyeballs and the central nervous system of humans. A cycle of autoinfection may occur in human host tissues characterized by the presence of eggs, larvae and adult worms. This peculiarity of the cycle hinders therapy, since there are no drugs that exhibit ovicidal, larvicidal and vermicidal activity. Given these facts, we studied the action of levamisole hydrochloride on third-stage larvae in the migration phase (G1) and on encysted larvae (G3) of L. minor. To this end, 87 inbred mice of the C57BL/6 strain were divided into test groups comprising 67 animals (G1-37; G3-30) and a control group (G2-10; G4-10) with 20 animals. Each animal was inoculated orally with 2,000 infective eggs of the parasite. The animals of the test groups were treated individually with a single oral dose of levamisole hydrochloride at a concentration of 0.075 mg. The drug was administered either 30 minutes prior to the parasite inoculation (G1 animals) or 120 days after the inoculation (G3 animals). The mice in the control groups were not treated with the drug. After the time required for the migration and the encysting of L. minor larvae, all the animals were euthanized and their tissues examined. The data were analyzed using the Student's unpaired t-test and the Levene test. The groups showed no statistically significant difference. Levamisole hydrochloride was ineffective on third-stage larvae of L. minor. These findings explain the massive expulsion of live adult worms, as well as the use of long treatment schemes, owing to the persistence of larvae and eggs in human parasitic lesions.

Hepatic pathology in Capillaria hepatica infected mice

Septal fibrosis of the liver regularly develops in rats infected with Capillaria hepatica. To find out whether such fibrosis also occurs in mice, 20 animals were submitted to infection with either 100 or 300 embryonated eggs and histologically examined after several periods of time, from 30 to 110 days afterwards. Results showed that mice developed acute, severe, diffuse and focal hepatic lesions that were soon modulated to focal areas of fibrosis containing eggs and worm remnants, despite the fact that a few worms remained alive, at least up to 110 days after inoculation. Areas of perisinusoidal fibrosis appeared in the proximity and around focal parasitic lesions, but clear-cut septal fibrosis was not observed. Why septal fibrosis forms in rats, but not in mice during C. hepatica infection, only further studies can clarify. Mice seem to show better host/parasite relationship than rats in regard to C. hepatica infection.