Peel of araticum fruit (Annona crassiflora Mart.) as a source of antioxidant compounds with α-amylase, α-glucosidase and glycation inhibitory activities.

Annona crassiflora Mart., whose fruit is popularly known as araticum, is a member of the Annonaceae family found in the Brazilian Cerrado. Although this plant has several medicinal uses, its bioactive molecules are not fully understood. A bioguided assay was performed to identify the main bioactive compounds of A. crassiflora fruit peel from the ethanol extract fractions with antioxidant capacity and α-amylase, α-glucosidase and glycation inhibitory activities. Ethyl acetate and n-butanol fractions showed, respectively, higher antioxidant capacity (DPPH IC50 1.5±0.1 and 0.8±0.1µgmL-1, ORAC 3355±164 and 2714±79µmoltroloxeq/g, and FRAP 888±16 and 921±9µmoltroloxeq/g) and inhibitory activities against α-amylase (IC50 4.5±0.8 and 1.7±0.3µgmL-1), α-glucosidase (IC50 554.5±158.6 and 787.8±140.6µgmL-1) and glycation (IC50 14.3±3.3 and 16.0±4.2µgmL-1), and lower cytotoxicity, compared to the other fractions and crude ethanol extract. The HPLC-ESI-MS/MS analysis identified various biomolecules known as potent antioxidants, such as chlorogenic acid, (epi)catechin, procyanidins, caffeoyl-hexosides, quercetin-glucosides and kaempferol. The fruit peel of A. crassiflora, a specie from Cerrado, the Brazilian Savanna, provided a source of antioxidant compounds with properties to block carbohydrate digestive enzymes and formation of glycation products. Thus, there is potential to use the by-products of araticum in order to identify and isolate phytochemicals for application in nutraceutical supplements, food additives and pharmaceuticals products.

Comparison of parasitological, immunological and molecular methods for evaluation of fecal samples of immunosuppressed rats experimentally infected with Strongyloides venezuelensis.

Definitive diagnosis of strongyloidiasis in humans is typically achieved by detection of larvae in fecal samples. However, limitations on sensitivity of parasitological methods emphasize the need for more robust diagnostic methods. The aim of this study was to compare the diagnostic value of three methods: eggs per gram of feces (EPG), coproantigen detection by enzyme linked immunosorbent assay (ELISA), and DNA detection by conventional polymerase chain reaction (PCR). The assays were performed at 0 and 5, 8, 13, 21 and 39 days post-infection (dpi) using fecal samples from experimentally infected immunocompetent and immunosuppressed rats. In immunocompetent rats, eggs were detected in feces on days 5, 8 and 13 dpi; coproantigen detection and PCR amplification were successful at all post-infection time points (5, 8, 13, 21 and 39 dpi). In immunosuppressed rats, eggs were detected at 5, 8, 13 and 21; coproantigen detection and PCR amplification were successful at all post-infection time points. In conclusion, these results suggest that coproantigen detection and PCR may be more sensitive alternatives to traditional methods such as EPG for diagnosis of Strongyloides venezuelensis infection.

Azithromycin is able to control Toxoplasma gondii infection in human villous explants.

BACKGROUND: Although Toxoplasma gondii infection is normally asymptomatic, severe cases of toxoplasmosis may occur in immunosuppressed patients or congenitally infected newborns. When a fetal infection is established, the recommended treatment is a combination of pyrimethamine, sulfadiazine and folinic acid (PSA). The aim of the present study was to evaluate the efficacy of azithromycin to control T. gondii infection in human villous explants. METHODS: Cultures of third trimester human villous explants were infected with T. gondii and simultaneously treated with either PSA or azithromycin. Proliferation of T. gondii, as well as production of cytokines and hormones by chorionic villous explants, was analyzed. RESULTS: Treatment with either azithromycin or PSA was able to control T. gondii infection in villous explants. After azithromycin or PSA treatment, TNF-α, IL-17A or TGF-ß1 levels secreted by infected villous explants did not present significant differences. However, PSA-treated villous explants had decreased levels of IL-10 and increased IL-12 levels, while treatment with azithromycin increased production of IL-6. Additionally, T. gondii-infected villous explants increased secretion of estradiol, progesterone and HCG+ß, while treatments with azithromycin or PSA reduced secretion of these hormones concurrently with decrease of parasite load. CONCLUSIONS: In conclusion, these results suggest that azithromycin may be defined as an effective alternative drug to control T. gondii infection at the fetal-maternal interface.

Heme oxygenase-1 activity is involved in the control of Toxoplasma gondii infection in the lung of BALB/c and C57BL/6 and in the small intestine of C57BL/6 mice.

Heme oxygenase-1 (HO-1) is an enzyme that catabolizes free heme, which induces an intense inflammatory response. The expression of HO-1 is induced by different stimuli, triggering an anti-inflammatory response during biological stress. It was previously verified that HO-1 is able to induce indoleamine 2,3-dioxygenase (IDO), an enzyme that is induced by IFN-γ in Toxoplasma gondii infection. To verify the role of HO-1 during in vivo T. gondii infection, BALB/c and C57BL/6 mice were infected with the ME49 strain and treated with zinc protoporphyrin IX (ZnPPIX) or hemin, which inhibit or induce HO-1 activity, respectively. The results show that T. gondii infection induced high levels of HO-1 expression in the lung of BALB/c and C57BL6 mice. The animals treated with ZnPPIX presented higher parasitism in the lungs of both lineages of mice, whereas hemin treatment decreased the parasite replication in this organ and in the small intestine of infected C57BL/6 mice. Furthermore, C57BL/6 mice infected with T. gondii and treated with hemin showed higher levels of IDO expression in the lungs and small intestine than uninfected mice. In conclusion, our data suggest that HO-1 activity is involved in the control of T. gondii in the lungs of both mouse lineages, whereas the hemin, a HO-1 inducer, seems to be involved in the control of parasitism in the small intestine of C57BL/6 mice.

Adjuvant and immunostimulatory effects of a D-galactose-binding lectin from Synadenium carinatum latex (ScLL) in the mouse model of vaccination against neosporosis.

Vaccination is an important control measure for neosporosis that is caused by a coccidian parasite, Neospora caninum, leading to abortion and reproductive disorders in cattle and serious economic impacts worldwide. A D-galactose-binding lectin from Synadenium carinatum latex (ScLL) was recently described by our group with potential immunostimulatory and adjuvant effects in the leishmaniasis model. In this study, we evaluated the adjuvant effect of ScLL in immunization of mice against neosporosis. First, we investigated in vitro cytokine production by dendritic cells stimulated with Neospora lysate antigen (NLA), ScLL or both. Each treatment induced TNF-α, IL-6, IL-10 and IL-12 production in a dose-dependent manner, with synergistic effect of NLA plus ScLL. Next, four groups of C57BL/6 mice were immunized with NLA + ScLL, NLA, ScLL or PBS. The kinetics of antibody response showed a predominance of IgG and IgG1 for NLA + ScLL group, whereas IgG2a response was similar between NLA + ScLL and NLA groups. Ex vivo cytokine production by mouse spleen cells showed the highest IFN-γ/IL-10 ratio in the presence of NLA stimulation for mice immunized with NLA + ScLL and the lowest for those immunized with ScLL alone. After parasite challenge, mice immunized with NLA + ScLL or ScLL alone presented higher survival rates (70-80%) and lower brain parasite burden as compared to PBS group, but with no significant changes in morbidity and inflammation scores. In conclusion, ScLL combined with NLA was able to change the cytokine profile induced by the antigen or lectin alone for a Th1-biased immune response, resulting in high protection of mice challenged with the parasite, but with low degree of inflammation. Both features may be important to prevent congenital neosporosis, since protection and low inflammatory response are necessary events to guide towards a successful pregnancy.

The biocontrol fungus Trichoderma stromaticum downregulates respiratory burst and nitric oxide in phagocytes and IFN-gamma and IL-10.

Trichoderma stromaticum, a biocontrol agent of the cacao witches' broom pathogen Moniliophthora perniciosa, has been used in Brazil as part of the integrated pest management of cacao. At the present time, little is known about the effects of T. stromaticum on the modulation of in vitro or in vivo immune responses. The present study examined the interaction of T. stromaticum spores with cellular and molecular components of the immune system following intranasal sensitization of mice. Our results showed that T. stromaticum spores prevented the expression and production of inflammatory mediators in macrophages stimulated with interferon (IFN)-γ plus lipopolysaccharide (LPS) and neutrophils stimulated with phorbol myristate 13-acetate (PMA). Quantitative polymerase chain reaction (qPCR) assays revealed that T. stromaticum spores inhibited the expression of dectin-1 and Toll-like-receptor (TLR)2/TLR4. Intranasal injection of BALB/c mice and subsequent challenge with spores of T. stromaticum induced a discrete inflammatory response in the lungs. Interestingly, the spores inhibited local and systemic production of the regulatory IL-10 and proinflammatory IFN-γ cytokines. In addition the spores presented an antiproliferative effect on spleen cells. These findings showed that the biopesticide T. stromaticum may exert immunosuppressive effects in vitro and in vivo.

Transforming growth factor (TGF)-ß1 and interferon (IFN)-γ differentially regulate ICAM-1 expression and adhesion of Toxoplasma gondii to human trophoblast (BeWo) and uterine cervical (HeLa) cells.

Toxoplasma gondii is a parasite able to infect various cell types, including trophoblast cells. Studies have demonstrated that interleukin (IL)-10, transforming growth factor (TGF)-ß1 and interferon (IFN)-γ are involved in the susceptibility of BeWo trophoblast cells to T. gondii infection. Furthermore, T. gondii is able to adhere to the plasma membrane of host cells through intercellular adhesion molecule (ICAM)-1. Thus, the present study aimed to assess the role of IL-10, TGF-ß1 and IFN-γ in the expression of ICAM-1 in BeWo and HeLa cells and to analyze the role of ICAM-1 in the adhesion and invasion of T. gondii to these cells under the influence of these cytokines. For this purpose, BeWo and HeLa cells were treated or not, before and after T. gondii infection, with rIL-10, rTGF-ß1 or rIFN-γ. For the BeWo cells, rIL-10 and rTGF-ß1 favored susceptibility to infection, but only rTGF-ß1 and rIFN-γ increased ICAM-1 expression, and TNF-α release. On the other hand, rIFN-γ downregulated the expression of ICAM-1 triggered by T. gondii in HeLa cells, leading to control of the infection. Moreover, we observed that upregulation of ICAM-1, mediated by cytokine's stimulation, in BeWo and HeLa cells resulted in a high number rate of both parasite adhesion and invasion to these cells, which were strongly reduced after ICAM-1 neutralization. Likewise, the blockage of ICAM-1 molecule also impaired T. gondii infection in human villous explants. Taken together, these findings demonstrate that TGF-ß1 and IFN-γ differentially regulate ICAM-1 expression, which may interfere in the adhesion/invasion of T. gondii to BeWo and HeLa cells for modulating susceptibility to infection.

Major histocompatibility complex (MHC) class II but not MHC class I molecules are required for efficient control of Strongyloides venezuelensis infection in mice.

Strongyloides stercoralis is an intestinal nematode capable of chronic, persistent infection and hyperinfection of the host; this can lead to dissemination, mainly in immunosuppressive states, in which the infection can become severe and result in the death of the host. In this study, we investigated the immune response against Strongyloides venezuelensis infection in major histocompatibility complex (MHC) class I or class II deficient mice. We found that MHC II(-/-) animals were more susceptible to S. venezuelensis infection as a result of the presence of an elevated number of eggs in the faeces and a delay in the elimination of adult worms compared with wild-type (WT) and MHC I(-/-) mice. Histopathological analysis revealed that MHC II(-/-) mice had a mild inflammatory infiltration in the small intestine with a reduction in tissue eosinophilia. These mice also presented a significantly lower frequency of eosinophils and mononuclear cells in the blood, together with reduced T helper type 2 (Th2) cytokines in small intestine homogenates and sera compared with WT and MHC I(-/-) animals. Additionally, levels of parasite-specific immunoglobulin M (IgM), IgA, IgE, total IgG and IgG1 were also significantly reduced in the sera of MHC II(-/-) infected mice, while a non-significant increase in the level of IgG2a was found in comparison to WT or MHC I(-/-) infected mice. Together, these data demonstrate that expression of MHC class II but not class I molecules is required to induce a predominantly Th2 response and to achieve efficient control of S. venezuelensis infection in mice.

Macrophage Migration Inhibitory Factor (MIF) Prevents Maternal Death, but Contributes to Poor Fetal Outcome During Congenital Toxoplasmosis.

Migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays important roles in physiology, pathology, immunology and parasitology, including the control of infection by protozoa parasites such as Toxoplasma gondii. As the MIF function in congenital toxoplasmosis is not fully elucidated yet, the present study brings new insights for T. gondii infection in the absence of MIF based on pregnant C57BL/6MIF-/- mouse models. Pregnant C57BL/6MIF-/- and C57BL/6WT mice were infected with 05 cysts of T. gondii (ME49 strain) on the first day of pregnancy (dop) and were euthanized at 8 dop. Non-pregnant and non-infected females were used as control. Our results demonstrated that MIF-/- mice have more accentuated change in body weight and succumbed to infection first than their WT counterparts. Otherwise, pregnancy outcome was less destructive in MIF-/- mice compared to WT ones, and the former had an increase in the mast cell recruitment and IDO expression and consequently presented less inflammatory cytokine production. Also, MIF receptor (CD74) was upregulated in MIF-/- mice, indicating that a compensatory mechanism may be required in this model of study. The global absence of MIF was associated with attenuation of pathology in congenital toxoplasmosis, but resulted in female death probably because of uncontrolled infection. Altogether, ours results demonstrated that part of the immune response that protects a pregnant female from T. gondii infection, favors fetal damage.

Recombinant TgHSP70 Immunization Protects against Toxoplasma gondii Brain Cyst Formation by Enhancing Inducible Nitric Oxide Expression.

Toxoplasma gondii is known to cause congenital infection in humans and animals and severe disease in immunocompromised individuals; consequently development of vaccines against the parasite is highly necessary. Under stress conditions, T. gondii expresses the highly immunogenic heat shock protein 70 (TgHSP70). Here, we assessed the protective efficacy of rTgHSP70 immunization combined with Alum in oral ME-49 T. gondii infection and the mechanisms involved on it. It was observed that immunized mice with rTgHSP70 or rTgHSP70 adsorbed in Alum presented a significantly reduced number of cysts in the brain that was associated with increased iNOS+ cell numbers in the organ, irrespective the use of the adjuvant. Indeed, ex vivo experiments showed that peritoneal macrophages pre-stimulated with rTgHSP70 presented increased NO production and enhanced parasite killing, and the protein was able to directly stimulate B cells toward antibody producing profile. In addition, rTgHSP70 immunization leads to high specific antibody titters systemically and a mixed IgG1/IgG2a response, with predominance of IgG1 production. Nonetheless, it was observed that the pretreatment of the parasite with rTgHSP70 immune sera was not able to control T. gondii internalization and replication by NIH fibroblast neither peritoneal murine macrophages, nor anti-rTgHSP70 antibodies were able to kill T. gondii by complement-mediated lysis, suggesting that these mechanisms are not crucial to resistance. Interestingly, when in combination with Alum, rTgHSP70 immunization was able to reduce inflammation in the brain of infected mice and in parallel anti-rTgHSP70 immune complexes in the serum. In conclusion, immunization with rTgHSP70 induces massive amounts of iNOS expression and reduced brain parasitism, suggesting that iNOS expression and consequently NO production in the brain is a protective mechanism induced by TgHSP70 immunization, therefore rTgHSP70 can be a good candidate for vaccine development against toxoplasmosis.

Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy.

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure.

Immunization with MIC1 and MIC4 induces protective immunity against Toxoplasma gondii.

Host cell invasion by Toxoplasma gondii is tightly coupled to the apical release of micronemal proteins (MIC). In this work, we evaluated the protective effect encountered in C57BL/6 mice immunized with MIC1 and MIC4 purified from soluble tachyzoite antigens by affinity to immobilized lactose. The immunized mice presented high serum levels of IgG1 and IgG2b specific antibodies. MIC1/4-stimulated spleen cells from immunized mice produced IL-2, IL-12, IFN-gamma, IL-10, but not IL-4, suggesting the induction of a polarized Th1 type immune response. When orally challenged with 40 cysts of the ME49 strain, the immunized mice had 68% fewer brain cysts than the control mice. Immunization was associated with 80% survival of the mice challenged with 80 cysts, contrasting with 100% mortality of the non-immunized mice in the acute phase. In this phase, there was much lower parasitism in the lungs and small intestine of the immunized mice, and they did not exhibit the early-stage signs of intestinal necrosis, which was clearly detected in the control mice. Our data demonstrate that MIC1 and MIC4 triggered a protective response against toxoplasmosis, and that these antigens are targets for the further development of a vaccine.

Strength and Aerobic Physical Exercises Are Able to Increase Survival of Toxoplasma gondii-Infected C57BL/6 Mice by Interfering in the IFN-γ Expression.

Physical exercise has been implicated in several immunophysiological improvements, particularly during the aging process, when an immunocompromised status could be established. Toxoplasma gondii is a protozoan parasite that causes a widespread opportunistic infection, which may present severe consequences, mainly to the fetus and immunocompromised patients. It is estimated that one-third of the human population worldwide has been infected by this parasite, being the reactivation during immunesenescence an unexplored public health issue. The major purpose of the present study was to observe the immunophysiological differences between exercised vs. sedentary C57BL/6 male mice that have been experimentally infected by T. gondii. In the first set of experiments, the animals were infected after exercising and three groups were set up: experimental groups-infected sedentary (IS, n = 6); infected exercised (IEx, n = 6) and control group-non-infected sedentary (NIS, n = 6). When stimulated in vitro by T. gondii-soluble tachyzoite antigen, it was found that splenocytes from exercised group produced higher levels of IFN-γ, as well as of IFN-γ/IL-10 ratios in comparison with splenocytes from sedentary animals (P < 0.001). However, it was not found significant differences concerning quantification of T. gondii genomic DNA by qRT-PCR and immunohistochemistry analysis in brain cysts from both group of animals (P > 0.05). In order to further investigate the consequences of these data for the host, a second set of experiments was performed, when the animals were infected before exercising and four groups of animals were established for comparison purpose, as follows: experimental groups-infected sedentary (IS, n = 7); infected exercised (IEx, n = 6) and control groups-non-infected sedentary (NIS, n = 6) and non-infected exercised (NIEx, n = 6). It was found significant differences in the survival rates of the exercised group the animals, as they survived longer than sedentary groups (P = 0.0005). In both sets of experiments, mice have been submitted to moderate exercises: aerobic (14 m/min; 3 x/week) and strength (60-80% of one maximum repetition; 2 x/week). Overall, our findings are showing that the aerobic and strength exercises are able to modulate immune response against T. gondii infection, being these immunological features beneficial to the host.

Calomys callosus chronically infected by Toxoplasma gondii clonal type II strain and reinfected by Brazilian strains is not able to prevent vertical transmission.

Considering that Toxoplasma gondii has shown high genetic diversity in Brazil, the aim of this study was to determine whether Calomys callosus chronically infected by the ME-49 strain might be susceptible to reinfection by these Brazilian strains, including vertical transmission of the parasite. Survival curves were analyzed in non-pregnant females chronically infected with ME-49 and reinfected with the TgChBrUD1 or TgChBrUD2 strain, and vertical transmission was analyzed after reinfection of pregnant females with these same strains. On the 19th day of pregnancy (dop), placentas, uteri, fetuses, liver, spleen, and lung were processed for detection of the parasite. Blood samples were collected for humoral and cellular immune response analyses. All non-pregnant females survived after reinfection and no changes were observed in body weight and morbidity scores. In pregnant females, parasites were detected in the placentas of ME-49 chronically infected females and reinfected females, but were only detected in the fetuses of reinfected females. TgChBrUD2 reinfected females showed more impaired pregnancy outcomes, presenting higher numbers of animals with fetal loss and a higher resorption rate, in parallel with higher levels of pro-inflammatory cytokines and IgG2a subclass antibodies. Vertical transmission resulting from chronic infection of immunocompetent C. callosus is considered a rare event, being attributed instead to either reactivation or reinfection. That is, the pregnancy may be responsible for reactivation of the latent infection or the reinfection may promote T. gondii vertical transmission. Our results clearly demonstrate that, during pregnancy, protection against T. gondii can be breached after reinfection with parasites belonging to different genotypes, particularly when non-clonal strains are involved in this process and in this case the reinfection promoted vertical transmission of both type II and Brazilian T. gondii strains.

Toxoplasma gondii: in vivo expression of BAG-5 and cyst formation is independent of TNF p55 receptor and inducible nitric oxide synthase functions.

Wild type, TNFRp55(-/-), iNOS(-/-) and IFN-gamma(-/-) mice were infected with Toxoplasma gondii strain ME-49, and the central nervous system (CNS), lungs, liver, spleen, heart and kidneys were examined for the presence of parasites expressing tachyzoite-specific (SAG-1) and bradyzoite-specific (BAG-5) antigens. During the acute phase of infection, the peripheral organs, but not the CNS, of the IFN-gamma(-/-) mice are heavily parasitized by tachyzoites and there are no signs of parasites expressing BAG-5. In contrast, the tissues from TNFRp55(-/-) and inducible nitric oxide synthase (iNOS)(-/-) mice, mainly the CNS, presented high numbers of parasites expressing SAG-1 and/or BAG-5. Tachyzoite transformation into bradyzoite and cyst development was shown to be normal in the tissues from TNFRp55(-/-) and iNOS(-/-) mice, as indicated by the high numbers of BAG-5/PAS positive cysts. Consistently, reactivation of infection in IFN-gamma(-/-) mice was rapid and characterized by a dramatic increase in SAG-1, contrasting with slow course in the TNFRp55(-/-) or iNOS(-/-) mice associated with a relatively small increase in SAG-1- and/or BAG-5-positive parasites. In conclusion, our results suggest that the control of multiplication of tachyzoites is largely dependent on endogenous IFN-gamma with partial involvement of TNFRp55 and iNOS. In contrast, induction of BAG-5 expression and cyst formation during toxoplasmosis seems to be dependent on IFN-gamma, but independent of TNFRp55 and iNOS functions.

Heterologous antibodies to evaluate the kinetics of the humoral immune response in dogs experimentally infected with Toxoplasma gondii RH strain.

An IgM capture ELISA using heterologous antibodies was developed to evaluate the kinetics of the humoral immune response in dogs experimentally infected with Toxoplasma gondii RH strain. Detection of parasite in tissues from inoculated dogs was evaluated by mouse bioassay and immunohistochemical techniques. Serum samples were obtained at regular intervals up to 62 days post-inoculation (p.i.), when the animals were necropsied and their tissues examined. Antibody levels were measured by IgM capture ELISA (McELISA), indirect hemagglutination (IHA), indirect fluorescent antibody test (IgG-IFAT) and indirect immunoenzymatic assay (IgG-ELISA). All dogs seroconverted but only one exhibited severe clinical signs of infection. IgM antibodies were detected by McELISA from the seventh day on, with decreasing IgM levels around the 27th day. Similar results were obtained from IHA, although McELISA showed earlier and longer detection of IgM antibodies. IgG antibodies were detected from the seventh day on, and throughout the period of observation. Immunohistochemical findings and mouse bioassay revealed the presence of free tachyzoites in tissues of the clinically affected dog only. These results suggest that T. gondii acute infection in dogs shows a remarkably transient IgM synthesis, and this feature may constitute an important marker of active infection. Furthermore, McELISA was shown to be a potential tool to diagnose canine toxoplasmosis.

Toxoplasma gondii soluble tachyzoite antigen triggers protective mechanisms against fatal intestinal pathology in oral infection of C57BL/6 mice.

Toxoplasma gondii induces a potent IL-12 response early in infection that results in IFN-γ-dependent control of parasite growth. It was previously shown that T. gondii soluble tachyzoite antigen (STAg) injected 48 hr before intraperitoneal infection reduces lipoxin A4 and 5-lipoxygenase (5-LO)-dependent systemic IL-12 and IFN-γ production as well as hepatic immunopathology. This study investigated the ability of STAg-pretreatment to control the fatal intestinal pathology that develops in C57BL/6 mice orally infected with 100 T. gondii cysts. STAg-pretreatment prolonged the animals' survival by decreasing tissue parasitism and pathology, mainly in the ilea. Protection was associated with decreases in the systemic IFN-γ levels and IFN-γ and TNF message levels in the ilea and with increased TGF-ß production in this tissue, but protection was independent of 5-LO and IL-4. STAg-pretreatment decreased CD4(+) T cell, NK cell, CD11b(+) monocyte and CD11b(+)CD11c(+) dendritic cell numbers in the lamina propria and increased CD8(+) T cells in the intestinal epithelial compartment. In parallel, decreases were observed in iNOS and IL-17 expression in this organ. These results demonstrate that pretreatment with STAg can induce the recruitment of protective CD8(+) T cells to the intraepithelial compartment and decrease proinflammatory immune mechanisms that promote intestinal pathology in T. gondii infection.

The impaired pregnancy outcome in murine congenital toxoplasmosis is associated with a pro-inflammatory immune response, but not correlated with decidual inducible nitric oxide synthase expression.

Congenital toxoplasmosis is associated with adverse pregnancy outcome. Despite the type 1 immune response, C57BL/6 mice are more susceptible than BALB/c mice to Toxoplasma gondii infection. Additionally, successful pregnancy appears to be correlated with type 2 T helper maternal immunity and regulatory T cells. In order to investigate the mechanisms of susceptibility/resistance to congenital toxoplasmosis in mice with different genetic backgrounds and the influence of inducible nitric oxide synthase in pregnancy outcome, groups of C57BL/6, BALB/c and C57BL/6 iNOS(-/-) females were orally infected with T. gondii ME-49 strain on day 1 of pregnancy and were sacrificed on day 8 p.i. and day 19 p.i. The uterus and placenta were evaluated for the foetal resorption rate, parasite load, immunological and histological changes. C57BL/6 mice presented inflammatory foci in the decidua (endometrium) of the uterus at a higher frequency than BALB/c mice on day 8 p.i., and a large number of pregnant C57BL/6 mice presented necrotic implantation sites. The parasite was seldom found in the uterus or placenta of either lineage of mice. Interestingly, there was no observed difference in inducible nitric oxide synthase expression in the uterus and placenta of infected mice. In addition, higher levels of TNF-α were detected in serum samples from C57BL/6 mice compared with BALB/c mice. Accordingly, C57BL/6 mice presented with levels of 90% abortion compared with 50% in BALB/c mice on day 19 p.i. C57BL/6 iNOS(-/-) mice showed low placental parasite counts and high absorption rates, similar to wild type mice. The data suggest that the impaired pregnancy outcome due to T. gondii infection in C57BL/6 mice could be associated with a higher inflammatory response leading to cell apoptosis and necrosis of implantation sites compared with BALB/c mice, and this phenomenon was not due to inducible nitric oxide synthase expression in the decidua.

ArtinM, a D-mannose-binding lectin from Artocarpus integrifolia, plays a potent adjuvant and immunostimulatory role in immunization against Neospora caninum.

ArtinM and Jacalin (JAC) are lectins from the jackfruit (Artocarpus integrifolia) that have important role in modulation of immune responses to pathogens. Neospora caninum is an Apicomplexa parasite that causes neuromuscular disease in dogs and reproductive disorders in cattle, with economic impact on the livestock industry. Hence, we evaluated the adjuvant effect of ArtinM and JAC in immunization of mice against neosporosis. Six C57BL/6 mouse groups were subcutaneously immunized three times at 2-week intervals with Neospora lysate antigen (NLA) associated with lectins (NLA+ArtinM and NLA+JAC), NLA, ArtinM and JAC alone, and PBS (infection control). Animals were challenged with lethal dose of Nc-1 isolate and evaluated for morbidity, mortality, specific antibody response, cytokine production by spleen cells, brain parasite burden and inflammation. Our results demonstrated that ArtinM was able to increase NLA immunogenicity, inducing the highest levels of specific total IgG and IgG2a/IgG1 ratio, ex vivo Th1 cytokine production, increased survival, the lowest brain parasite burden, along with the highest inflammation scores. In contrast, NLA+JAC immunized group showed intermediate survival, the highest brain parasite burden and the lowest inflammation scores. In conclusion, ArtinM presents stronger immunostimulatory and adjuvant effect than Jacalin in immunization of mice against neosporosis, by inducing a protective Th1-biased pro-inflammatory immune response and higher protection after parasite challenge.

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-γ, TNF-α, IL-10 and TGF-ß 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.