Search

Toxoplasmosis, caused by the mandatory intracellular protozoan Toxoplasma gondii, is a cosmopolitan disease that affects one-third of the world population. The anti-T. gondii in vitro potential of ethanolic extract, ethyl acetate, aqueous fractions, volatile oil, and α-bisabolol isolated from Siparuna guianensis Aubl., Siparunaceae, was evaluated by the β-galactosidase assay in two experimental conditions using T. gondii strain RH-2F1. The ethanolic extract and the ethyl acetate fraction showed cytotoxic activity against fibroblasts only at the highest concentrations (250 and 500 μg/ml) and the volatile oil at a concentration of 500 μg/ml. The tested fractions partially controlled the intracellular proliferation of the parasite, when the treatment was carried out after cell infection. Inhibitory effect of parasitism was most strongly observed in the pre-treatment of parasites before cell infection with the ethanolic extract and the ethyl acetate fraction, showing a direct effect on the parasite.

Congenital toxoplasmosis is represented by the transplacental passage of Toxoplasma gondii from the mother to the fetus. Our studies demonstrated that T. gondii developed mechanisms to evade of the host immune response, such as cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) induction, and these mediators can be produced/stored in lipid droplets (LDs). The aim of this study was to evaluate the role of COX-2 and LDs during T. gondii infection in human trophoblast cells and villous explants. Our data demonstrated that COX-2 inhibitors decreased T. gondii replication in trophoblast cells and villous. In BeWo cells, the COX-2 inhibitors induced an increase of pro-inflammatory cytokines (IL-6 and MIF), and a decrease in anti-inflammatory cytokines (IL-4 and IL-10). In HTR-8/SVneo cells, the COX-2 inhibitors induced an increase of IL-6 and nitrite and decreased IL-4 and TGF-β1. In villous explants, the COX-2 inhibitors increased MIF and decreased TNF-α and IL-10. Furthermore, T. gondii induced an increase in LDs in BeWo and HTR-8/SVneo, but COX-2 inhibitors reduced LDs in both cells type. We highlighted that COX-2 is a key factor to T. gondii proliferation in human trophoblast cells, since its inhibition induced a pro-inflammatory response capable of controlling parasitism and leading to a decrease in the availability of LDs, which are essentials for parasite growth.

Heme oxygenase-1 (HO-1) enzyme exerts beneficial effects at the maternal-fetal interface, especially in trophoblasts, being involved in survival and maturation of these cell phenotypes. Trophoblast cells play essential roles throughout pregnancy, being the gateway for pathogens vertically transmitted, such as Toxoplasma gondii. It was previously shown that HO-1 activity was involved in the control of T. gondii infection in vivo; however, its contribution in trophoblast cells during T. gondii infection, remain undefined. Thus, this study aimed to investigate the influence of HO-1 in T. gondii-infected BeWo and HTR-8/SVneo human trophoblast cells. For this purpose, trophoblast cells were infected and the HO-1 expression was evaluated. T. gondii-infected BeWo cells were treated with hemin or CoPPIX, as inducers of HO-1, or with bilirubin, an end-product of HO-1, and the parasitism was quantified. The involvement of p38 MAPK, a regulator of HO-1, and the cytokine production, were also evaluated. It was found that T. gondii decreased the HO-1 expression in BeWo but not in HTR-8/SVneo cells. When treated with the HO-1 inducers or bilirubin, BeWo cells reduced the parasite proliferation. T. gondii also decreased the p38 MAPK phosphorylation in BeWo cells; on the other hand, HO-1 induction sustained its activation. Finally, the IL-6 production was upregulated by HO-1 induction in T. gondii-infected cells, which was associated with the control of infection.

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)-β1 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-β1 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-β1 or rIFN-γ. For the BeWo cells, rIL-10 and rTGF-β1 favored susceptibility to infection, but only rTGF-β1 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-β1 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.

Background Toxoplasma gondii is a protozoan with worldwide distribution and triggers a strong Th1 immune response in infected susceptible hosts. On the contrary, most helminth infections are characterized by Th2 immune response and the use of helminth-derived antigens to regulate immune response in inflammatory disorders has been broadly investigated. Objectives The aim of this study was to investigate whether treatment with Strongyloides venezuelensis antigen extract (SvAg) would alter immune response against T. gondii. Methods C57BL/6 mice were orally infected with T. gondii and treated with SvAg, and parasitological, histological and immunological parameters were investigated. Results It was observed that SvAg treatment improved survival rates of T. gondii infected mice. At day 7 post-infection, the parasite load was lower in the lung and small intestine of infected SvAg-treated mice than untreated infected mice. Remarkably, SvAg-treated mice infected with T. gondii presented reduced inflammatory lesions in the small intestine than infected untreated mice and decreased intestinal and systemic levels of IFN-γ, TNF-α and IL-6. In contrast, SvAg-treatment increased T. gondii-specific IgA serum levels in infected mice. Conclusions S. venezuelensis antigen extract has anti-parasitic and anti-inflammatory properties during T. gondii infection suggesting as a possible alternative to parasite and inflammation control.

Canine demodicosis is a common inflammatory parasitic skin disease caused by Demodex mites. House dust mites, such as Dermatophagoides spp., play an important role in the pathogenesis of canine atopic dermatitis (AD). The goal of this experimental work was to investigate whether demodectic dogs could be previously exposed/sensitized to house dust mites’ antigens. First the prevalence of demodicosis in a southeastern region of Brazil was investigated by analyzing clinical files of dogs that were admitted to a Veterinary Hospital. Subsequently, the IgG responses to Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df) and IgE to D. pteronyssinus (Dp) were evaluated in two groups, AD or demodicosis dogs. Additionally, the major IgE-binding Dp proteins that are recognized by sera from dogs with demodicosis and AD were evaluated. A total of 2,599 clinical files were analyzed to identify the major parasitic skin diseases in dogs from this region, considering the age, sex and breed of the animals. The epidemiological study identified 111 animals with skin diseases; from these 20.7% presented demodicosis. Afterwards, serum samples were obtained from another groups of demodicosis, AD, and healthy dogs, and analyzed for Dp and Df-specific IgG, and IgE antibody levels, Dp IgG avidity by ELISA and IgE-binding Dp-specific proteins by immunoblot. IgG and IgE antibodies to Dp were detected in sera from additional groups of dogs with AD, demodicosis or healthy, with higher IgE levels to Dp in AD than demodectic or healthy dogs. IgG to Df was detected, despite with smaller levels compared to Dp in sera from demodectic dogs, and also in healthy dogs. Immunoblot showed IgE-binding to Dp proteins in sera of dogs with demodicosis and AD; with strong reactivity for the 72 and 116 kDa antigens detected by sera from demodicosis dogs. However, sera from healthy dogs >12 months old also presented reactivity to these bands. In conclusion, the detection of Dp-IgG and IgE antibodies in sera from demodectic dogs indicates previous exposure and sensitization to the house dust mite, respectively, more than cross-reactivity between demodex mites and Dp antigens detected by canine antibodies. Additionally, higher Dp-specific IgE levels were found in dogs with AD compared with those with demodicosis or healthy, suggesting that Dp-specific IgE could better discriminate dogs with AD from healthy ones or even those with demodicosis. Demodicose canina é uma doença inflamatória comum da pele causada por ácaros do gênero Demodex. Ácaros da poeira doméstica como Dermatophagoides spp. desempenham papel importante na patogênese da dermatite atópica canina (DA). O objetivo desse trabalho experimental foi investigar se cães com demodicose poderiam ser previamente expostos/sensibilizados com antígenos de ácaros da poeira doméstica. A princípio, investigou-se a prevalência de demodicose em uma região sudeste do Brasil, analisando-se prontuários clínicos de cães admitidos em um Hospital Veterinário. Posteriormente, as respostas de IgG a Dermatophagoides pteronyssinus (Dp) e D. farinae (Df) e IgE a D. pteronyssinus (Dp) foram avaliadas em dois grupos, DA ou demodicose. Também foram avaliadas as principais proteínas Dp reconhecidas por anticorpo IgE presente em soros de cães com demodicose e DA. Um total de 2.599 prontuários clínicos foram analisados ​​para identificar as principais doenças parasitárias da pele em cães dessa região, considerando a idade, sexo e raça dos animais. O estudo epidemiológico detectou 111 animais com doenças de pele e destes, 20,7% apresentavam demodicose. Posteriormente, amostras de soro foram obtidas de outros grupos de cães com demodicose, DA ou saudáveis, e analisadas quanto aos níveis de IgG e IgE específicos para Dp e Df, avidez de IgG a Dp por ELISA e proteínas específicas de Dp reconhecidas por IgE por immunoblot. Anticorpos IgG e IgE para Dp foram detectados em soros de grupos adicionais de cães com DA, demodicose ou saudáveis, com níveis mais altos de IgE para Dp na DA do que no soro de animais saudáveis. Níveis de IgG específicos para Df foram detectados, apesar serem menores em comparação com os detectados para Dp em soros de cães demodéticos, e também em cães saudáveis. A análise de immunoblot demonstrou detecção de IgE para proteinas de Dp em soros de cães com demodicose e DA; com forte reatividade para os antígenos de 72 e 116 kDa detectados por soros de cães com demodicose. No entanto, soros de cães saudáveis > 12 meses de idade também apresentaram reatividade a essas bandas. Em conclusão, a detecção de anticorpos Dp-IgG e IgE específicos em soros de cães demodéticos indica exposição prévia e sensibilização aos ácaros, respectivamente, mais do que reatividade cruzada entre ácaros Demodex e antígenos Dp detectados por anticorpos caninos. Além disso, níveis de Dp-IgE específicos mais elevados encontrados em cães com DA, sugerem que esses anticorpos poderiam discriminar melhor cães com DA daqueles saudáveis ou mesmo demodéticos.

Iron is an important constituent of our environment, being necessary for both mammalian and pathogenic protozoa survival. Iron-containing proteins exert a wide range of biological processes such as biodegradation and biosynthesis, as well as immune function, fetal development, and physical and mental well-being. This work aimed to investigate the effect of iron deprivation in Toxoplasma gondii infection outcome. C57BL/6 mice were orally infected with T. gondii and treated with an iron chelator, deferoxamine, or supplemented with iron (ferrous sulfate), and the parasitism as well as immunological and histological parameters were analyzed. It was observed that the infection increased iron accumulation in the organs, as well as systemically, and deferoxamine treatment diminished the iron content in serum samples and intestine. The deferoxamine treatment decreased the parasitism and inflammatory alterations in the small intestine and lung. Additionally, they partially preserved the Paneth cells and decreased the intestinal dysbiosis. The ferrous sulfate supplementation, despite not significantly increasing the parasite load in the organs, increased the inflammatory alterations in the liver. Together, our results suggest that iron chelation, which is commonly used to treat iron overload, could be a promising medicine to control T. gondii proliferation, mainly in the small intestine, and consequently inflammation caused by infection.

Canine distemper is a viral disease that affects several systems on dogs, among them, the cardiovascular system. The aim of this study was to identify canine distemper virus (CDV) in the sinoatrial node (SAN) of dogs serologically positive for distemper by Polymerase Chain Reaction preceded by reverse transcription (RT-PCR), and to analyze gross and microscopic changes of distemper in the heart and other tissues. SAN and tissue fragments were collected from 17 serologically positive dead animals, necropsied from October 2015 to December 2016. In the heart, right heart dilatation was observed in 13 dogs (76.47%) and left concentric hypertrophy in two dogs (11.76%). Microscopically, lymphocytic myocarditis was observed in four (23.53%) dogs and 41.18% presented viral inclusion corpuscles of CDV in the bladder epithelium. Only one (5.88%) dog presented a 319 bp target fragment for distemper virus using primers CDV 1 and CDV 2 at the sinoatrial node. In conclusion, CDV can be located in the sinoatrial node of naturally infected dogs, as demonstrated in this study by the RT-PCR technique, reinforcing the hypothesis that CDV is capable of causing inflammatory lesions in the sinoatrial node of this species. Macroscopic and microscopic cardiac changes are frequently observed in dogs with distemper, mainly cardiac dilatation and myocarditis. Viral inclusions of CDV in bladder epithelial cells are an important microscopic finding for the diagnosis of distemper. A cinomose canina é uma doença viral que afeta vários sistemas, dentre eles o cardiovascular. Objetivou-se identificar o vírus da cinomose canina no nó sinoatrial (NSA) de cães sorologicamente positivos para cinomose, através da reação em cadeia da polimerase, precedida de transcrição reversa (RT-PCR), além de analisar os achados macroscópicos e histológicos da cinomose no coração e outros tecidos. Foram coletados fragmentos de tecidos e do NSA de 17 cães sorologicamente positivos para cinomose que vieram a óbito e foram necropsiados no período de outubro de 2015 a dezembro de 2016. No coração observou-se dilatação cardíaca direita em 76,47% dos cães e hipertrofia concêntrica esquerda em 11,76% dos cães. Microscopicamente observou-se miocardite linfocítica em 23,53% dos cães e 41,18% apresentou corpúsculos de inclusão viral no epitélio vesical. Somente um (5,88%) cão apresentou fragmento alvo de 319 bp para cinomose utilizando os primers VCC1 e VCC2, no nó sinoatrial. Conclui-se que o VCC pode localizar-se no nó sinoatrial de cães naturalmente infectados, como demonstrados neste estudo pela técnica de RT-PCR, reforçando a hipótese de que o VCC é capaz de provocar lesões inflamatórias no nó sinoatrial dessa espécie. Alterações cardíacas macroscópicas e microscópicas, principalmente dilatação cardíaca e miocardite, são frequentemente observadas em cães com cinomose. Inclusões virais nas células epiteliais da bexiga são importantes achados microscópicos para diagnóstico da cinomose.

Annona muricata leaves are used in traditional medicine to manage diabetes mellitus and its complications. The aim of this study was to evaluate the potential in vitro antidiabetic properties of Annona muricata leaf by identifying its main phytochemical constituents and characterizing the phenolic-enriched fractions for their in vitro antioxidant capacity and inhibitory activities against glycoside and lipid hydrolases, advanced glycation end-product formation and lipid peroxidation. Ethanol extract of A. muricata leaf was subjected to a liquid-liquid partitioning and its fractions were used in enzymatic assays to evaluate their inhibitory potential against α-amylase, α-glucosidase and lipase, as well as their antioxidant (DPPH, ORAC, FRAP and Fe2+-ascorbate-induced lipid peroxidation assays) and anti-glycation (BSA-fructose, BSA-methylglyoxal and arginine-methylglyoxal models) capacities. In addition, identification of the main bioactive compounds of A. muricata leaf by HPLC-ESI-MS/MS analysis was carried out. Ethyl acetate (EtOAc) and n-butanol (BuOH) fractions showed, respectively, antioxidant properties (ORAC 3964 ± 53 and 2707 ± 519 μmol trolox eq g−1, FRAP 705 ± 35 and 289 ± 18 μmol trolox eq g−1, and DPPH IC50 4.3 ± 0.7 and 9.3 ± 0.8 μg mL−1) and capacity to reduce liver lipid peroxidation (p

The Apicomplexa protozoanToxoplasma gondiiis a mandatory intracellular parasite and the causative agent of toxoplasmosis. This illness is of medical importance due to its high prevalence worldwide and may cause neurological alterations in immunocompromised persons. In chronically infected immunocompetent individuals, this parasite forms tissue cysts mainly in the brain. In addition,T.gondiiinfection has been related to mental illnesses such as schizophrenia, bipolar disorder, depression, obsessive-compulsive disorder, as well as mood, personality, and other behavioral changes. In the present study, we evaluated the kinetics of behavioral alterations in a model of chronic infection, assessing anxiety, depression and exploratory behavior, and their relationship with neuroinflammation and parasite cysts in brain tissue areas, blood-brain-barrier (BBB) integrity, and cytokine status in the brain and serum. Adult female C57BL/6 mice were infected by gavage with 5 cysts of the ME-49 type IIT.gondiistrain, and analyzed as independent groups at 30, 60 and 90 days postinfection (dpi). Anxiety, depressive-like behavior, and hyperactivity were detected in the early (30 dpi) and long-term (60 and 90 dpi) chronicT.gondiiinfection, in a direct association with the presence of parasite cysts and neuroinflammation, independently of the brain tissue areas, and linked to BBB disruption. These behavioral alterations paralleled the upregulation of expression of tumor necrosis factor (TNF) and CC-chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β and CCL5/RANTES) in the brain tissue. In addition, increased levels of interferon-gamma (IFNγ), TNF and CCL2/MCP-1 were detected in the peripheral blood, at 30 and 60 dpi. Our data suggest that the persistence of parasite cysts induces sustained neuroinflammation, and BBB disruption, thus allowing leakage of cytokines of circulating plasma into the brain tissue. Therefore, all these factors may contribute to behavioral changes (anxiety, depressive-like behavior, and hyperactivity) in chronicT.gondiiinfection.

Vertical transmission of Toxoplasma gondii leads to adverse pregnancy outcomes depending on the time at which the infection occurs and the immunological state of the mother. C57BL/6 and BALB/c mice have been described as susceptible and resistant mouse lineages to congenital T. gondii infection, respectively. This study aimed to elucidate the systemic and local cytokine profile of pregnant mice infected with T. gondii and whether the expression of the transcription factor FOXP3, related to T regulatory cells, is associated with the resistance/susceptibility of these lineages of mice in the context of experimental congenital toxoplasmosis. For this purpose, C57BL/6 and BALB/c females were orally infected with the T. gondii ME-49 strain on the day of vaginal plug detection or day 14 of gestation, examined 7 or 5 days later, respectively, as models of early and late pregnancy. Cytokine levels were measured systemically and in the uterus/placenta. Additionally, the uterus/placenta were evaluated macroscopically for resorption rates and histologically for parasite and FOXP3 immunostaining. The FOXP3 protein expression was also evaluated by western blotting assay. It was found that, during early pregnancy, the infection leads to high IFN-γ, TNF and IL-6 levels systemically, with the TNF levels being higher in C57BL/6 mice. At the maternal-fetal interface, the infection induced high levels of IFN-γ in both mouse lineages; however, higher levels were observed in BALB/c, while high TNF and IL-6 levels were found in C57BL/6, but not in BALB/c mice. In contrast, in late gestation, T. gondii interfered less strongly with the cytokine profile. In early pregnancy, a reduction of FOXP3 expression at the maternal-fetal interface of infected mice was also observed, and the reduction was larger in C57BL/6 compared with BALB/c mice. Additionally, the parasite was seldom found in the uterus/placenta. Thus, the worse pregnancy outcomes observed in C57BL/6 mice were associated with higher TNF systemically, and TNF and IL-6 at the maternal-fetal interface, with lower FOXP3 expression.

Toxoplasma gondii is able to infect a wide range of vertebrates, including humans. Studies show that cyclooxygenase-2 (COX-2) is a modulator of immune response in multiple types of infection, such as Trypanosoma cruzi. However, the role of COX-2 during T. gondii infection is still unclear. The aim of this study was to investigate the role of COX-2 during infection by moderately or highly virulent strains of T. gondii in Calomys callosus rodents and human THP-1 cells. C. callosus were infected with 50 cysts of T. gondii (ME49), treated with COX-2 inhibitors (meloxicam or celecoxib) and evaluated to check body weight and morbidity. After 40 days, brain and serum were collected for detection of T. gondii by real-time PCR and immunohistochemistry or cytokines by CBA. Furthermore, peritoneal macrophages or THP-1 cells, infected with RH strain or uninfected, were treated with meloxicam or celecoxib to evaluate the parasite proliferation by colorimetric assay and cytokine production by ELISA. Finally, in order to verify the role of prostaglandin E2 in COX-2 mechanism, THP-1 cells were infected, treated with meloxicam or celecoxib plus PGE2, and analyzed to parasite proliferation and cytokine production. The data showed that body weight and morbidity of the animals changed after infection by T. gondii, under both treatments. Immunohistochemistry and real-time PCR showed a reduction of T. gondii in brains of animals treated with both COX-2 inhibitors. Additionally, it was observed that both COX-2 inhibitors controlled the T. gondii proliferation in peritoneal macrophages and THP-1 cells, and the treatment with PGE2 restored the parasite growth in THP-1 cells blocked to COX-2. In the serum of Calomys, upregulation of pro-inflammatory cytokines was detected, while the supernatants of peritoneal macrophages and THP-1 cells demonstrated significant production of TNF and nitrite, or TNF, nitrite and MIF, respectively, under both COX-2 inhibitors. Finally, PGE2 treatment in THP-1 cells triggered downmodulation of pro-inflammatory mediators and upregulation of IL-8 and IL-10. Thus, COX-2 is an immune mediator involved in the susceptibility to T. gondii regardless of strain or cell types, since inhibition of this enzyme induced control of infection by upregulating important pro-inflammatory mediators against Toxoplasma.

Ethnopharmacological relevance Sulfadiazine and pyrimethamine are the two drugs used as part of the standard therapy for toxoplasmosis, however; they may cause adverse side effects and fail to prevent relapse in many patients, rendering infected individuals at risk of reactivation upon becoming immunocompromised. Extracts from various parts of Annona muricata have been widely used medicinally for the management, control and/or treatment of several human diseases, acting against parasites that cause diseases in humans. Aim of the study This study was performed to investigate the action of the ethanolic extract of A. muricata (EtOHAm) and its fractions in the control of the apicomplexan parasite Toxoplasma gondii in vitro and in vivo, and the effect of EtOHAm on the inflammatory response and lipid profile alteration induced by in vivo T. gondii infection. Materials and methods The cytotoxicity of EtOHAm and its fractions ethyl acetate (EtOAcAm), n-butanol (BuOHAm), aqueous (H2OAm), hexane (HexAm) and dichloromethane (CH2Cl2Am) was evaluated in NIH/3T3 fibroblasts using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cells were infected with T. gondii, treated with the extracts, and parasite proliferation was analyzed. For the in vivo experiments, C57BL/6 mice were orally infected with T. gondii and, treated with different concentrations of extract fractions that were effective in vitro (EtOHAm, EtOAcAm, HexAm and CH2Cl2Am). Tissue parasitism, histological alterations, systemic cytokine and lipid profile were investigated. Results EtOHAm, EtOAcAm, BuOHAm, H2OAm presented low cytotoxicity until doses of 200 μg/mL, while HexAm and CH2Cl2Am presented toxicity from doses of 100μg/mL. EtOHAm, HexAm and CH2Cl2Am decreased the parasitism in vitro, presenting a therapeutic index of 2.62, 2.44, and 2.96, respectively. In vivo, EtOHAm, HexAm and CH2Cl2Am improved the survival rate of infected animals, however, only EtOHAm was able to decrease the parasitism in the small intestine and lung. Additionally, EtOHAm decreased the systemic interferon (IFN)-γ and tumor necrosis factor (TNF) systemically in infected mice, and was able to maintain the triglycerides and very-low-density lipoprotein (VLDL) lipid fractions at similar levels to uninfected animals. Although treatment with EtOHAm could not control the inflammation induced by oral infection in the tissues analyzed, it was able to preserve the number of goblet cells in the small intestine. Conclusions Ethanolic A. muricata leaf extract could be considered as a good candidate for the development of a complementary/alternative therapy against toxoplasmosis, and also as an anti-inflammatory alternative for decreasing TNF and IFN-γ concentrations and lipid fractions in specific diseases.

Congenital toxoplasmosis is a serious health problem that can lead to miscarriage. HTR-8/SVneo is a first trimester extravillous trophoblast, while BeWo is a choriocarcinoma with properties of villous trophoblast cells. In the placenta, iron is taken up from Fe-transferrin through the transferrin receptor being the ion an important nutrient during pregnancy and also for Toxoplasma gondii proliferation. The aim of this study was to evaluate the role of iron in T. gondii proliferation in BeWo and HTR-8/SVneo cells and in human chorionic villous explants. The cells were infected with T. gondii, iron supplemented or deprived by holo-transferrin or deferoxamine, respectively, and parasite proliferation and genes related to iron balance were analyzed. It was verified that the addition of holo-transferrin increased, and DFO decreased the parasite multiplication in both trophoblastic cells, however, in a more expressive manner in HTR-8/SVneo, indicating that the parasite depends on iron storage in trophoblastic cells for its growth. Also, tachyzoites pretread with DFO proliferate normally in trophoblastic cells demonstrating that DFO itself does not interfere with parasite proliferation. Additionally, T. gondii infection induced enhancement in transferrin receptor mRNA expression levels in trophoblastic cells, and the expression was higher in HTR-8/SVneo compared with BeWo. Finally, DFO-treatment was able to reduce the parasite replication in villous explants. Thus, the iron supplementation can be a double-edged sword; in one hand, it could improve the supplement of an essential ion to embryo/fetus development, and on the other hand, could improve the parasite proliferation enhancing the risk of congenital infection.

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.

Increased resistance to the first-line treatment against P. falciparum malaria, artemisinin-based combination therapies, has been reported. Here, we tested the effect of crude ethanolic extract of the fungus Trichoderma stromaticum (Ext-Ts) on the growth of P. falciparum NF54 in infected human red blood cells (ihRBCs) and its anti-malarial and anti-inflammatory properties in a mouse model of experimental cerebral malaria. For this purpose, ihRBCs were treated with Ext-Ts and analysed for parasitaemia; C57BL/6 mice were infected with P. berghei ANKA (PbA), treated daily with Ext-Ts, and clinical, biochemical, histological and immunological features of the disease were monitored. It was observed that Ext-Ts presented a dose-dependent ability to control P. falciparum in ihRBCs. In addition, it was demonstrated that Ext-Ts treatment of PbA-infected mice was able to increase survival, prevent neurological signs and decrease parasitaemia at the beginning of infection. These effects were associated with systemically decreased levels of lipids and IFN-γ, ICAM-1, VCAM-1 and CCR5 cerebral expression, preserving blood brain barrier integrity and attenuating the inflammatory lesions in the brain, liver and lungs. These results suggest that Ext-Ts could be a source of immunomodulatory and antimalarial compounds that could improve the treatment of cerebral malaria.

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.

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 towards 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.

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.

There is growing evidence in the literature suggesting that caste differentiation in the stingless bee, Melipona scutellaris, and other bees in the genus Melipona, is triggered by environmental signals, particularly a primer pheromone. With the proper amount of food and a chemical stimulus, 25% of females emerge as queens, in agreement with a long-standing “two loci/two alleles model” proposed in the 1950s. We surmised that these larvae must be equipped with an olfactory system for reception of these chemical signals. Here we describe for the first time the diversity of antennal sensilla in adults and the morphology of larvae of M. scutellaris. Having found evidence for putative olfactory sensilla in larvae, we next asked whether olfactory proteins were expressed in larvae. Since the molecular basis of M. scutellaris is still unknown, we cloned olfactory genes encoding chemosensory proteins (CSP) and odorant-binding proteins (OBPs) using M. scutellaris cDNA template and primers designed on the basis CSPs and OBPs previously reported from the European honeybee, Apis mellifera. We cloned two CSP and two OBP genes and then attempted to express the proteins encoded by these genes. With a recombinant OBP, MscuOBP8, and a combinatorial single-chain variable fragment antibody library, we generated anti-MscuOBP8 monoclonal antibody. By immunohistochemistry we demonstrated that the anti-MscuOBP8 binds specifically to the MscuOBP8. Next, we found evidence that MscuOBP8 is expressed in M. scutellaris larvae and it is located in the mandibular region, thus further supporting the hypothesis of olfactory function in immature stages. Lastly, molecular modeling suggests that MscuOBP8 may function as a carrier of primer pheromones or other ligands.

This study aimed to measure the synthesis of Th1 and Th2 cytokines by mononuclear cells after culture with liveT. gondiiand identified Th17 (CD4+) and Tc17 (CD8+) cells in toxoplasma-seronegative and toxoplasma-seropositive parturient and nonpregnant women. Cytometric bead arrays were used to measure cytokine levels (IL-2, TNF-α, IFN-γ, IL-4, IL-5, and IL-10); immunophenotyping was used to characterize Th17 and Tc17 cells, and the cells were stained with antibodies against CD4+and CD8+T cells expressing IL-17. The addition of tachyzoites to cell cultures induced the synthesis of IL-5, IL-10, and TNF-αby cells from seronegative parturient women and of IL-5 and IL-10 by cells from seropositive, nonpregnant women. We observed a lower level of IL-17-expressing CD4+and CD8+T lymphocytes in cultures of cells from seronegative and seropositive parturient and nonpregnant women that were stimulated with tachyzoites, whereas analysis of the CD4+and CD8+T cell populations showed a higher level of CD4+T cells compared with CD8+T cells. These results suggest that the cytokine pattern and IL-17-expressing CD4+and CD8+T lymphocytes may have important roles in the inflammatory response toT. gondii, thus contributing to the maintenance of pregnancy and control of parasite invasion and replication.

In human and murine models strongyloidiasis induce a Th2 type response. In the current study we investigated the role of different loads of Strongyloides venezuelensis in the immune response raised against the parasite and the participation of the major histocompatibility complex (MHC) class II molecule in the disease outcome in face of the different parasite burden. The C57BL/6 wild type (WT) and MHC II−/− mice were individually inoculated by subcutaneous injection with 500 or 3000 S. venezuelensis L3. The MHC II−/− mice infected with 3000L3 were more susceptible to S. venezuelensis infection when compared with WT groups, in which the parasite was completely eliminated. The production of Th2 cytokines and specific IgG1 or IgE antibodies against parasite were significantly lowered in MHC II−/− infected mice with different larvae inoculums. The infection of MHC II−/− mice with S. venezuelensis induced slight inflammatory alterations in the small intestine, and these lesions were lower when compared with WT mice, irrespective of the parasite load utilized to infect animals. Finally, we concluded that MHC class II molecules are essential in the immune response against S. venezuelensis mainly when infection occurs with high parasite inoculum.

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.

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.

After oral infection, Toxoplasma gondii invades intestinal cells, induces breakdown of intestinal physiology and barrier functions, and causes intestinal pathology in some animal species. Although parasites’ invasion into host cells is a known phenomenon, the effects of T. gondii infection in the intestinal barrier are still not well established. To evaluate morphological and physiological modifications on the colorectal adenocarcinoma-derived Caco-2 cell line during T. gondii infection, microvilli, tight junction integrity, and transepithelial electrical resistance (TEER) were investigated under infection. It was observed that the dextran uptake (endocytosis) and distribution were smaller in infected than in noninfected Caco-2 cells. The infection leads to the partial loss of microvilli at the cell surface. Claudin-1, zonula occludens-1 (ZO-1), and occludin expressions were colocalized by immunofluorescence and presented discontinuous net patterns in infected cells. Immunoblotting analysis at 24 hr postinfection revealed decreasing expression of occludin and ZO-1 proteins, whereas claudin-1 presented similar expression level compared with noninfected cells. T. gondii decreased TEER in Caco-2 cells 24 hr after infection. Our results suggest that T. gondii infection may lead to the loss of integrity of intestinal mucosa, resulting in impaired barrier function.

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.

Toxoplasma gondii is an obligate intracellular parasite that can cause variable clinical symptoms or can even be asymptomatic in immunocompetent individuals. More severe symptoms are observed in immunocompromised patients and congenital transmission of the parasite has been reported. The objective of this study was to evaluate the response of peripheral blood mononuclear cells (PBMC) in parturient and non-pregnant women exposed to live tachyzoites of T. gondii strain RH or ME49. PBMC were isolated from parturient and non-pregnant women with negative or positive serology for toxoplasmosis and cultured with live tachyzoites of the two T. gondii strains for 24 h. Next, the cell culture supernatants were collected and levels of CCL2, CCL5, IL-6, IL-10, IL-12, and TNF-α produced by PBMC after tachyzoite exposure were measured. Live tachyzoite forms of T. gondii significantly inhibited the synthesis of CCL2 in seropositive parturient women, whereas a stimulatory effect on CCL5 was observed in seronegative parturient women. Cells from T. gondii-seronegative non-pregnant women produced significantly higher levels of TNF-α and IL-12, demonstrating the proinflammatory profile induced by the presence of the parasite in culture. The results suggest that the immunomodulation seen during pregnancy contributes to the development of an environment that facilitates escape of the parasite from the immune response.

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.

Toxoplasma gondii is an important pathogen which may cause fetal infection if primary infection. Our previous studies have used human choriocarcinoma trophoblastic cells (BeWo cell line) as experimental model of T. gondii infection involving placental microenvironment. This study aimed to examine the effects of azithromycin and spiramycin against T. gondii infection in BeWo cells. Cells were treated with different concentrations of the macrolide antibiotics and analyzed first for cell viability using thiazolyl blue tetrazole (MTT) assay. As cell viability was significantly decreased with drug concentrations higher than 400 μg/mL, the concentration range used in further experiments was from 50 to 400 μg/mL. The number of infected cells and intracellular replication of T. gondii decreased after treatment with each drug. The infection induced up-regulation of the macrophage migration inhibitory factor (MIF), which was also enhanced in infected cells after treatment with azithromycin, but not with spiramycin. Analysis of the cytokine profile showed increase TNF-α, IL-10 and IL-4 production, but decreased IFN-γ levels, were detected in infected cells and treated with each drug. In conclusion, treatment of human trophoblastic BeWo cells with with azithromycin or spiramycin is able to control the infection and replication of T. gondii. In addition, treatment with these macrolides, especially with azityromycin induces an anti-inflammatory response and high MIF production, which can be important for the establishment and maintenance of a viable pregnancy during T. gondii infection.

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.

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.

Neospora caninum is an apicomplexan parasite, closely related to Toxoplasma gondii, and causes abortion and congenital neosporosis in cattle worldwide. Trophoblast cells act in mechanisms of innate immune defense at the fetal-maternal interface and no data are available about the interaction of Neospora with human trophoblasts. Thus, this study aimed to verify the susceptibility of human trophoblastic (BeWo) compared with uterine cervical (HeLa) cell lines to N. caninum. BeWo and HeLa cells were infected with different parasite:cell ratios of N. caninum tachyzoites and analyzed at different times after infection for cell viability using thiazolyl blue tetrazole and lactate dehydrogenase assays. Both cell lines were also evaluated for cytokine production and parasite infection/replication assays when pre-treated or not with Neospora lysate antigen (NLA) or human recombinant IFN-γ. Cell viability was increased up to 48 h of infection in both types of cells, suggesting that infection could inhibit early cell death and/or induce cell proliferation. Neospora infection induced up-regulation of the macrophage migration inhibitory factor (MIF), mainly in HeLa cells, which was enhanced by cell pre-treatment by NLA or IFN-γ. Conversely, parasite infection induced down-regulation of the transforming growth factor (TGF-β), mostly in BeWo cells, which was decreased with NLA or IFN-γ pre-treatment. HeLa cells were more susceptible to Neospora infection than BeWo cells and IFN-γ pre-treatment resulted in reduced infection indices in both cell lines. Control of parasite growth was mediated by IFN-γ through an indoleamine-2,3-dioxygenase-dependent mechanism in HeLa cells alone. Based on these results, we concluded that BeWo and HeLa cells are readily infected by N. caninum, although presenting differences in susceptibility to infection, cytokine production and cell viability. Thus, these host cells can be considered in comparative approaches to understand strategies used by N. caninum to survive at the maternal-fetal interface.

In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-gamma and iNOS expression in the CNS compared to BALB/c mice. The CNS of C57BL/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite.

Toxoplasma gondii surface is coated by closely related antigens that belong to SRS (SAG-1 related sequences) superfamily. Two tachyzoite-specific SRS antigens, SAG1 and SAG2, are immunodominant proteins that apparently modulate the virulence of infection by inducing the host immune response against tachyzoites during the acute phase. In this study, we described a conformationally insensitive monoclonal antibody (A4D12mAb) that recognizes a linear epitope shared by two isoforms of p22 that is expressed in the surface of T. gondii tachyzoites. By using phage display approach and production of recombinant proteins, we clearly demonstrated that the A4D12mAb recognizes an epitope within C-terminal region of SAG2A. This mAb reacts with both T. gondii genotypes (I and II) but not with a closely related parasite, Neospora caninum. Also, the pretreatment of tachyzoites with A4D12 mAb did not inhibit T. gondii infection, suggesting that the epitope herein mapped is not crucial for tachyzoite invasion. However, a panel of human T. gondii positive sera showed significant degree of inhibition of A4D12 mAb reactivity against T. gondii native antigens, indicating that both A4D12 mAb and human sera recognize an overlapping immunodominant epitope within C-terminal region of SAG2A. To our knowledge, this is the first evidence using bioselection by phage display that identifies a T. gondii linear epitope recognized by a mAb specific to SAG2A. In conclusion, the results here presented add a new piece of information concerning T. gondii SAG2A molecule, emphasizing two dissimilar biological roles of this molecule, particularly for A4D12 epitope, suggesting that these characteristics may be important for parasite survival, since it is part of parasite components able to induce a strong immune response enough to allow host survival and establish long-term chronic infection.

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.

CpG oligodeoxynucleotides (ODN) have shown to be potent immunoadjuvants for several pathogens, but there is limited information concerning their use in immunization protocols against neosporosis. This study aimed to evaluate the potential of CpG-ODN combined with Neospora lysate antigen (NLA) or excreted-secreted antigen (NcESA) to induce protective immune response against Neospora caninum infection in mice. C57BL/6 mice were vaccinated subcutaneously three times at 2-week intervals with NLA, NLA + CpG, NcESA, NcESA + CpG, CpG (adjuvant control) or PBS (infection control). Serological assays showed an increased specific IgG2a response in animals immunized with either antigen plus adjuvant and elevated levels of the IgG1 isotype in those vaccinated with antigens alone. Splenocyte proliferative responses upon antigen stimulation were higher in groups immunized with NLA or NcESA combined with CpG, showing increased IL-12 levels. Also, mice vaccinated with NcESA or NcESA + CpG demonstrated higher IFN- levels and IFN-/IL-10 ratio. After lethal challenge, mice immunized with NLA + CpG or NLA had lower morbidity score and body weight changes in comparison to other groups, and animals did not succumb during acute infection. In contrast, NcESA + CpG or NcESA groups exhibited the highest morbidity scores, body weight impairment and mortality rates, associated with greatest brain parasite burden and inflammation. In conclusion, CpG-ODN was able to induce a Th1-type humoral immune response with predominant IgG2a levels for either NLA or NcESA, but resulting in an effective Th1-driven cellular immune response and total protection only when combined with NLA. Vaccination with NcESA alone or combined with CpG resulted in a strong cellular immune response associated with high levels of IFN- and inflammation, rendering mice more susceptible to parasite challenge. © 2009 Published by Elsevier Ltd.

The present study, investigated the mechanisms involved in the immune responses of Major Histocompatibility Complex class I or class II knockout mice, following Strongyloides venezuelensis infection. Wild-type C57BL/6 (WT), MHC II −/− and MHC I −/− mice were individually inoculated with 3000 larvae (L3) of S. venezuelensis and sacrificed on days 1, 3, 5, 8, 13 and 21 post-infection (p.i.). Samples of blood, lungs and small intestines were collected. The tissue samples were stained with hematoxylin–eosin for the pathological analysis. The presence of the parasite was demonstrated by immunoperoxidase analysis. MHC II −/− mice presented a significantly higher number of adult worms recovered from the small intestine on day 5 p.i. and presented elevated numbers of eggs in the feces. The infection by S. venezuelensis was completely eliminated 13 days after infection in WT as well as in MHC I −/− mice. In MHC II −/− mice, eggs and adult worms were still found on day 21 p.i., however, there was a significant reduction in their numbers. In the lung, the parasite was observed in MHC I −/− on day 1 p.i. and in MHC II −/− mice on days 1 and 5 p.i. In the small intestine of WT mice, a larger number of parasites were observed on day 8 p.i. and their absence was observed after day 13 p.i. Through immunohistochemistry analysis, the parasite was detected in the duodenum of WT on days 5 and 8 p.i., and in knockout mice on days 5, 8 and 13 p.i.; as well as in posterior portions of the small intestine in MHC I −/− and MHC II −/− on day 13 p.i., a finding which was not observed in WT mice. We concluded that immunohistochemistry analysis contributed to a more adequate understanding of the parasite localization in immunodeficient hosts and that the findings aid in the interpretation of immunopathogenesis in Strongyloides infection.

Summary The immune response induced by Toxoplasma gondii is characterized by Th1 immune mechanisms. We previously demonstrated that C57BL/6 mice infested with Myocoptes musculinus and infected with T. gondii by intraperitoneal route undergo accelerated mortality according to Th2 immune mechanisms induced by the acarian. To evaluate whether infection with M. musculinus influences T. gondii-induced Th1 response in a resistant mouse lineage, BALB/c, which develops latent chronic toxoplasmosis in a way similar to that observed in immunocompetent humans, this study was done. The animals were infected with T. gondii ME-49 strain 1 month after M. musculinus infestation, being the survival and the immune response monitored. The double-infected displayed higher mortality rate if compared with the mono-infected mice. In addition, infection with M. musculinus changed the T. gondii-specific immune response, converting BALB/c host to a susceptible phenotype. Spleen cells had increased the levels of IL-4 in double-infected mice. This alteration was associated with severe pneumonia, encephalitis and wasting condition. In addition, a higher tissue parasitism was observed in double-infected animals. It can be concluded that infection with these two contrasting parasites, M. musculinus and T. gondii, may convert an immunocompetent host into a susceptible one, and such a host will develop severe toxoplasmosis.

SUMMARYDefinitive 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.

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.

In attempts to investigate the role of galectin-3 in innate immunity, we studied galectin-3-deficient (gal3−/−) mice with regard to their response to Toxoplasma gondii infection, which is characterized by inflammation in affected organs, Th-1-polarized immune response, and accumulation of cysts in the central nervous system. In wild-type (gal3+/+) mice, infected orally, galectin-3 was highly expressed in the leukocytes infiltrating the intestines, liver, lungs, and brain. Compared with gal3+/+, infected gal3−/− mice developed reduced inflammatory response in all of these organs but the lungs. Brain of gal3−/− mice displayed a significantly reduced number of infiltrating monocytes/macrophages and CD8+ cells and a higher parasite burden. Furthermore, gal3−/− mice mounted a higher Th1-polarized response and had comparable survival rates on peroral T. gondii infection, even though they were more susceptible to intraperitoneal infection. Interestingly, splenic cells and purified CD11c+ dendritic cells from gal3−/− mice produced higher amounts of interleukin-12 than cells from gal3+/+ mice, possibly explaining the higher Th1 response verified in the gal3−/− mice. We conclude that galectin-3 exerts an important role in innate immunity, including not only a pro-inflammatory effect but also a regulatory role on dendritic cells, capable of interfering in the adaptive immune response.

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.

Toxoplasma gondii is an intracellular protozoan that elicits a potent inflammatory response during the acute phase of infection. Herein, we evaluate whether T. gondii infection alters the natural course of aortic lesions. ApoE knockout mice were infected with T. gondii , and at 5 weeks of infection, serum, feces, and liver cholesterol; aortic lesion size, cellularity, and inflammatory cytokines; and levels of serum nitrite and gamma interferon (IFN-γ) were analyzed. Our results showed that serum cholesterol and atherogenic lipoproteins were reduced after T. gondii infection. The reduction of serum levels of total cholesterol and atherogenic lipoproteins was associated with increases in the aortic lesion area, numbers of inflammatory cells, and expression of monocyte chemoattractant protein 1 and inducible nitric oxide synthase mRNA in the site of lesions as well as elevated concentrations of IFN-γ and nitrite in sera of T. gondii -infected animals. These results suggest that infection with T. gondii accelerates atherosclerotic development by stimulating the proinflammatory response and oxidative stress, thereby increasing the area of aortic lesion.

Herein we characterized various genetic markers and the biological behavior of a natural recombinant strain ofToxoplasma gondii(P-Br). From nine genetic markers analyzed, three (B1,ROP1, andSAG1) and three (cS10-A6,GRA6, andSAG3) markers belong to parasites from the type I and type III lineages, respectively. TheSAG2andL363loci were shown to be type I-III chimera alleles. The cB2l-4 microsatellite marker showed a unique haplotype. The P-Br strain presented low virulence in the acute phase of infection and was cystogenic during the chronic infection. The interleukin 12/gamma interferon axis and inducible nitric oxide synthase were main determinants of resistance during the acute infection with the P-Br strain. As opposed to infection with the type II strain ofT. gondii(ME-49), peroral infection with the P-Br strain led only to a light inflammatory infiltrate and no major lesions in the intestine of the C57BL/6 mice. In addition, the BALB/c (resistant to ME-49) and C57BL/6 (susceptible to ME-49) mice were shown, respectively, to be more susceptible and more resistant to cyst formation and toxoplasmic encephalitis when infected with the P-Br strain. Further, the C57BL/KsJ and DBA2/J congenic strains containing major histocompatibility complex (MHC) haplotype “d” were more resistant than the parental strains (C57BL/6 and DBA1/J), when infected with the ME-49 but not with the P-Br strain. Together, our results indicate that resistance to cyst formation and toxoplasmic encephalitis induced during infection with P-Br is not primarily controlled by the MHC haplotype d, as previously reported for type II strains ofT. gondii.

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.

The induction of indoleamine 2,3-dioxygenase (INDO) expression and the tryptophan (Trp)-kynurenine (Kyn) metabolic pathway during in vivo infection withToxoplasma gondiiwas investigated. Decreased levels of Trp and increased formation of Kyn were observed in the lungs, brain, and serum from mice infected withT. gondii. Maximal INDO mRNA expression and enzyme activity were detected in the lungs at 10 to 20 days postinfection. Further, the induction of INDO mRNA expression, Trp degradation and Kyn formation were completely absent in tissues from mice deficient in IFN-γ (IFN-γ−/−) or IFN regulatory factor -1 (IRF-1−/−). These findings indicate the important role of endogenous IFN-γ and IRF-1 in the in vivo induction of the Trp-Kyn metabolic pathway during acute infection withT. gondii. In contrast, expression of INDO mRNA and its activity was preserved in the tissues of TNF-receptor p55- or inducible nitric oxide synthase-deficient mice infected withT. gondii. Together with the results showing the extreme susceptibility of the IFN-γ−/−and the IRF-1−/−mice to infection withT. gondii, our results indicate a possible involvement of INDO and Trp degradation in host resistance to early infection with this parasite.

Enteropathogenic Escherichia coli (EPEC) is a major cause of childhood diarrhea in developing countries and is a leading cause of severe diarrheal illness among Brazilian infants. As one approach to constructing a vaccine candidate against diarrhea caused by EPEC, we evaluated whether the pilin subunit (BfpA) of the bundle-forming pilus (BFP) could be expressed by a live Salmonella vaccine strain. Several copies of the coding region of BfpA (bfpA) were amplified by PCR from a preparation of the EAF plasmid of EPEC strain B171 and cloned into plasmid vectors. An intact copy of bfpA was subcloned into the heat inducible prokaryotic expression vector pCYTEXP1, and the resulting pBfpA was used to transform the aroA S. typhimurium strain SL3261, generating SL3261(pBfpA). The recombinant vaccine strain was able to express, but not to process, rBfpA as evidenced by a prominent 21 kDa protein that crossreacted with anti-BFP antiserum found only in extracts of heat-treated SL3261(pBfpA), but not in strains of untreated SL3261(pBfpA) or SL3261 not carrying the plasmid. Furthermore, rBfpA accumulation was not toxic to the Salmonella host, as evidenced by similar plating efficiencies between induced and uninduced strains of SL3261(pBfpA). Finally, SL3261(pBfpA) orally administered to BALB/c mice was capable of eliciting a sustained and vigorous humoral immune response to BfpA, achievable even with a single oral dose of approximately 10(9) organisms. Therefore, this pilin product may serve as a potential immunogen as part of a live combined vaccine strategy to prevent two of the major public health problems in Brazil--salmonellosis and EPEC childhood diahrrea.

Stage conversion between bradyzoites and tachyzoites was investigated in C57BL/6 mice chronically infected with the ME-49 strain of Toxoplasma gondii . In order to promote bradyzoite-tachyzoite conversion, mice were treated in vivo with neutralizing doses of anti-gamma interferon (IFN-γ) or anti-tumor necrosis factor alpha (TNF-α) antibodies. Expression of parasite-specific antigens SAG-1, SAG-2, and heat shock protein 70 (Hsp-70) was visualized in the central nervous system by immunocytochemistry and measured by photometric assay. The immunosuppressive effect of anti-IFN-γ or anti-TNF-α treatment was immediate, leading to parasite stage conversion as indicated by the increased expression of tachyzoite-specific antigens (SAG-1 and SAG-2) and by rapid parasite replication. We also observed expression of high levels of Hsp-70 during a short period of conversion of bradyzoites to tachyzoites. Our data suggest that Hsp-70 may have an important role in the process of bradyzoite-tachyzoite conversion during the reactivation of chronic toxoplasmosis.