Toxicological and toxicogenetic effects of plants used in popular medicine and in cattle food

Toxicological and toxicogenetic effects of aqueous (tea) and hexanica fruit extract of Indigofera suffruticosa Mill, and hydroalcoholic root extract od Solanum agrarium Stendt. Were evaluated in Balb C male mice intraperitoneally exposed. A hepatotoxic effect was observed just for animals treated with aqueous fruit extract of I. suffruticosa. In relation to the toxicogenetic effect, just the group trreated with 12.5% of toxic dose of aqueous fruit extract of I. suffruticosa showed a statistically significant increase in the frequency of cells with chromosome aberrations (cytogenetic effect), although a slight increase was also observed for the highest dose (25% of LF50_ of hydroalcoholic root extract of S. agrarium. The results obtanied show that before S. agrarium is used as medicine and before the wide use of I. suffruticosa in cattle food, careful evaluation must be done.

Schizotrypanids: the occurence of dermatitis in immunodeficiency animals infected with Trypanosoma cruzi

Congenitally athymic nude Balb/c (nu/nu) and their phenothypically normal adult and neonate littermates (nu/+), the C3H/HeN as well, were intraperitoneally infected with two strains (Y or CL) of Trypanossoma cruzi. The nude mice and the neonates developed a severe parasitaemia, the susceptible C3H/HeN also presented high level and adult Balb/c mice presented parasitaemia similar to that observed in outbred mice. Erythematous skin lesions were observed initially in infected athymic nude and neonates, being charactherized by nests of amastigotes in the dermis; in C3H/HeN infected mice no nest of parasite was observed but a low-grade inflammatory reaction was seen. In adult Balb/c or OF1 outbred mice nest was found but discreet inflammatory reaction was observed in severe infections.

Involvement of regional lymph nodes after penetration of Schistosoma mansoni cercariae in naive and infected mice

The parotid lymph nodes of naive and previously infected Balb/c mice were studied after, respectively, infection and re-infection with cercariae of Schistosoma mansoni via the ears. Schistosomula were able to pass through the lymph node by following the lymph flow or by penetrating the veins of the medullary cords. The number of nodal mast cells was higher from day 2 to 6 of primary infection; and from day 5 to 11 of re-infection. The amount of degranulating mast cells was significantly higher at day 4 of infection and at day 1 of re-infection. Eosinophils characterized the nodal inflammatory processes observed after day 5 in both primarily-infected and re-infected mice. However, only in the latter the eosinophils were able to adhere to the larval surface. In primarily-infected mice, no intranodal larva presented signs of degeneration. In contrast, in re-infected animals, some degenerating larvae were found inside eosinophilic infiltrates. The eosinophils reached the nodal tissue by migrating through the high endothelial venules and their collecting veins.

Chromoblastomycosis murine model and in vitro test to evaluate the sensitivity of Fonsecaea pedrosoi to ketoconazole, itraconazole and saperconazole

An experimental model of murine chromoblastomycosis and in vitro tests with Fonsecaea pedrosoi were used to test the sensitivity of this fungus to three different antimycotics. The experimental model was standardized in BALB/c mice inoculated intraperitoneally with a 10(6) CFU/ml suspension of a F. pedrosoi isolate. Clinical infection was evident after 5 days of inoculation. Three groups of 27 mice each were used in the experiment. One group was treated with ketoconazole (KTZ), another with itraconazole (ITZ) and the other with saperconazole (SPZ). Antimycotic therapy was continued for 21 days. The control group consisted of 40 mice which were inoculated, but not treated. Infection was documented by macroscopic and microscopic examination of affected tissue in addition to culture of tissue macerates. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) for the F. pedrosoi strain used were done. The in vitro results showed that SPZ was the most active with MIC 0.01 mg/ml and MFC 0.1 mg/ml, followed by ITZ. SPZ was also the most effective in vivo since 63% of the treated animals (p=0.01) showed a curative effect after the observation period. We concluded that SPZ had the best in vitro and in vivo activity against F. pedrosoi.

Induction of protective immunity against Schistosoma mansoni infection by antigens purified from PIII, a fraction of adult worm, associated to the downregulation of granuloma formation

This study was performed in order to define Schistosoma mansoni antigens able to function as modulator agents in BALB/c mice granulomatous hypersensitivity to parasite egg. The antigens P-24, P-35 and P-97 were purified by affinity chromatography from a fraction of S. mansoni adult worm antigenic preparation, denominated PIII, involved in the inhibition of granulomatous response to eggs. Immunization of mice with these antigens, in the presence of Corynebacterium parvum and Al(OH)3 as adjuvant, induced a significant protection degree against challenge infection, as observed by the decrease on worm burden recovered from portal system. In vitro blastogenesis assays revealed that purified antigens were able to induce significant proliferation of spleen cells from S. mansoni-infected mice. This protection was correlated to significant decrease in granuloma size induced by PIII. From these results, we concluded that PIII preparation contains antigens capable of mediating protective anti-parasite immunity and down-regulating granulomatous hypersensitivity to S. mansoni eggs.

A dhfr-ts- Leishmania major Knockout Mutant Cross-protects against Leishmania amazonensis

E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65% smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75% in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57% by SC and 49% by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization.

Vertical toxoplasmosis in a murine model. Protection after immunization with antigens of Toxoplasma gondii incorporated into liposomes

Distinct Toxoplasma gondii antigens were entrapped within liposomes and evaluated for their ability to protect Balb/c mice against congenital transmission: soluble tachyzoite antigen (L/STAg), soluble tissue cyst antigen (L/SCAg), soluble tachyzoite plus tissue cyst (L/STCAg) or purified 32kDa antigen of tachyzoite (L/pTAg). Soluble tachyzoite antigen alone in PBS (STAg) or emulsified in Freund's Complete Adjuvant (FCA/STAg) was also evaluated. Dams were inoculated subcutaneously with these antigens 6, 4 and 2 weeks prior to a challenge with four tissue cysts of the P strain of T. gondii orally between 10 and 14 days of pregnancy. Significant diminution differences were observed between the frequency of infected pups born of the dams immunized with the antigens incorporated into liposomes and that of pups born of the dams immunized with antigen emulsified in FCA or non immunized group (p<0.05). There was a significant decrease in the number of pups born dead in the groups L/STAg, L/SCAg and L/pTAg when compared with pups from all other groups (p <0.05). All dams immunized with or without adjuvant showed an antibody response and a proliferation of T-cells. However, no correlation was found between immune response and protection against the challenge.

Isotype specific immune responses in murine experimental toxocariasis

In this work, a murine experimental model of toxocariasis has been developed in BALB/c, C57BL/10 and C3H murine strains orally inoculated with 4,000 Toxocara canis embryonated eggs, in order to investigate the isotype-specific immune responses against excretory-secretory antigens from larvae. T. canis specific IgG+M, IgM, IgG, IgA, IgG1, IgG2a and IgG3 were tested by ELISA. The dynamics of the specific immunoglobulins (IgG+IgM) production showed a contrasting profile regarding the murine strain. Conversely to the results obtained with the IgM isotype, the IgG antibody class showed similar patterns to those obtained with IgG+IgM antibodies, only in the case of the BALB/c strain, being different and much higher than the obtained with IgG+IgM antibodies, when the C3H murine strain was used. The antibodies IgG+IgM tested in BALB/c and C57BL/10 were both of the IgM and IgG isotypes. Conversely, in the C3H strain only IgG specific antibody levels were detected. The IgG1 subclass responses showed a similar profile in the three murine strains studied, with high values in BALB/c, as in the case of the IgG responses.

In vivo lymphocyte activation and apoptosis by lectins of the Diocleinae subtribe

This paper reports the overall effects of three lectins, extracted from Canavalia brasiliensis, Dioclea violacea, and D. grandiflora, on BALB/c mice popliteal draining lymph nodes. These lectins have presented high stimulatory capacity on lymph node T cells. Additionally, they were able to induce apoptosis and inflammation (frequently associated with high endothelial venule necrosis). The data presented here suggest that the Diocleinae lectins studied can stimulate in vivo T cell activation and apoptosis, as well as present important side effects.

Dissociation between vasodilation and Leishmania infection-enhancing effects of sand fly saliva and maxadilan

In this study, the ability of maxadilan and Lutzomyia longipalpis salivary gland lysate to enhance the infection of CBA mice by Leishmania major and of BALB/c mice by L. braziliensis was tested. No difference was observed between sizes of lesion in CBA mice infected with L. major and treated or not with salivary gland lysate or maxadilan, although they were injected in concentrations that induced cutaneous vasodilation. Although parasites were more frequently observed in foot pads and spleens of animals treated with maxadilan than in the animals treated with salivary gland lysate or saline, the differences were small and not statistically significant. The lesions in BALB/c mice infected with L. braziliensis and treated with maxadilan were slightly larger than in animals that received Leishmania alone. Such differences disappeared 14 weeks after infection, and were statistically significant only in one of two experiments.

The opossum Didelphis virginiana as a synanthropic reservoir of Trypanosoma cruzi in Dzidzilché, Yucatán, México

In México, the role of mammals in the transmission cycle of Trypanosoma cruzi is poorly known. In the State of Yucatán, an endemic area of Chagas disease, both Didelphis virginiana and D. marsupialis occur sympatrically. However, until now, only the former species had been found infected with T. cruzi. To evaluate the role of D. virginiana in a peridomestic transmission, nine periods of capture-recapture were performed around the village of Dzidzilché, Yucatán. The sex, age, reproductive status, location, and presence of infection with T. cruzi were recorded for each opossum. The chromosome morphology was used to identify the opossum species. T. cruzi was identified by the presence of pseudocysts of amastigotes in cardiac muscle fibers of Balb/c mice inoculated with strains isolated from opossums. However, xenodiagnosis was the best diagnostic method. Triatoma dimidiata, the vector, were collected in and around the opossums' nests, and human dwellings; and were checked for T. cruzi. From 102 blood samples of D. virginiana examined 55 (53.9%) were positive to T. cruzi, the only two D. marsupialis captured were negative. Significant differences were found between infection, and both sex and reproductive condition. Eight out of 14 triatomines collected in peridomestic nests (57.1%), and 32 of 197 captured inside houses (16.3%) were found infected, suggesting a peridomestic transmission. The statistically high abundance of infected opossums and triatomines during the dry season (March to May) suggested the existence of a seasonality in the peridomestic transmission of T. cruzi in Dzidzilché.

Protective immunity of single and multi-antigen DNA vaccines against schistosomiasis

We evaluated the usefulness of the combination of three plasmids encoding tegumental (pECL and pSM14) and muscular (pIRV5) antigens of the Schistosoma mansoni on improving the protective immunity over the use of a single antigen as DNA vaccines. Female BALB/c mice were inoculated twice with 25 µg DNA plasmid within two weeks interval. The challenge was performed with 80 cercarias of a regional isolate of S. mansoni (SLM) one week after the last immunization. Six weeks after challenge, all mice were perfused for worm load determination. The following groups were analyzed: saline; empty vector; monovalent formulations of pECL; pSM14 and pIRV5 and also double combinations of pECL/pIRV5 and pIRV5/pSM14 and a triple combination of pECL/pIRV5/pSM14. The protection was expressed as a percentage of worm loads in each group compared with the saline group. The results obtained were 41% (p < 0.05); 52% (p < 0.05); 51% (p < 0.05); 48% (p < 0.05); 55% (p < 0.05); 45% (p < 0.05); 65% (p < 0.05) for each group respectively.

Efficacy of a diarylheptanoid derivative against Leishmania amazonensis

The activity of several diarylheptanoid derivatives (curcuminoids) was previously evaluated against Leishmania amazonensis promastigotes and among them the most active compound was the [1-(4-methoxy-phenyl)-7-(3,4-methoxy-4-hydroxy-phenyl)-1,6-heptadien-3, 5-dione]. This derivative was chosen to be assayed in vivo in a treatment trial. For these experiments, the curcuminoid compound was used in a concentration equivalent to the IC50/24 h, obtained from the previous study. Balb/c mice were inoculated subcutaneously in the footpad with L. amazonensis infective promastigotes and 4 weeks after the inoculation, the animals were treated with different schemes, varying from 1 to 3 doses. In all the experiments, Pentamidine Isethionate was used as reference drug under the same experimental conditions. The results showed that one dose was not enough to heal the lesion, however, with 2 and 3 doses the efficiency of the assayed compound was clear. On the other hand, treatment with Pentamidine Isethionate using the three different schemes was not satisfactory when compared to the curcuminoid derivative.

Morphological studies in a model for dengue-2 virus infection in mice

One of the main difficulties in studying dengue virus infection in humans and in developing a vaccine is the absence of a suitable animal model which develops the full spectrum of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome. It is our proposal to present morphological aspects of an animal model which shows many similarities with the dengue infection in humans. BALB/c mice were intraperitoneally infected with non-neuroadapted dengue virus serotype 2 (DENV-2). Histopathological and morphometrical analyses of liver tissue revealed focal alterations along the infection, reaching wide-ranging portal and centrolobular veins congestion and sinusoidal cell death. Additional ultrastructural observations demonstrated multifocal endothelial injury, platelet recruitment, and alterated hepatocytes. Dengue virus antigen was detected in hepatocytes and in the capillar endothelium of the central lobular vein area. Liver function tests showed high levels of aspartate transaminase and alanine transaminase enzyme activity. Lung tissue showed interstitial pneumonia and mononuclear cells, interseptal oedema, hyperplasia, and hypertrophy of the bronchiolar epithelial cells. DENV-2 led to a transient inflammatory process, but caused focal alterations of the blood-exchange barrier. Viremia was observed from 2nd to 11th day p.i. by isolation of DENV-2 in C6/36 mosquito cell line inoculated with the supernatant of macerated liver, lung, kidney, and cerebellum tissues of the infected mice.

Assessment of a DNA vaccine encoding an anchored-glycosylphosphatidylinositol tegumental antigen complexed to protamine sulphate on immunoprotection against murine schistosomiasis

Protamine sulphate/DNA complexes have been shown to protect DNA from DNase digestion in a lipid system for gene transfer. A DNA-based vaccine complexed to protamine sulphate was used to induce an immune response against Schistosoma mansoni anchored-glycosylphosphatidylinositol tegumental antigen in BALB/c mice. The protection elicited ranged from 33 to 44%. The spectrum of the elicited immune response induced by the vaccine formulation without protamine was characterized by a high level of IgG (IgG1> IgG2a). Protamine sulphate added to the DNA vaccine formulation retained the green fluorescent protein encoding-plasmid longer in muscle and spleen. The experiments in vivo showed that under protamine sulphate effect, the scope of protection remained unchanged, but a modulation in antibody production (IgG1= IgG2a) was observed.