Both C57BL/KsJ (H2d haplotype) and CB10-H2 (H2b haplotype) mice are highly susceptible to congenital toxoplasmosis.

Congenital toxoplasmosis may cause abortion, neonatal death, or foetal abnormalities. Despite little information from human studies, a genetic influence over congenital disease was demonstrated and, host genome have been implicated to resistance/susceptibility to Toxoplasma gondii infection in both human and mice. It was previously shown that BALB/c mice (H2d) were more resistant to congenital toxoplasmosis than C57BL/6 mice (H2b). However, it is unclear whether these differences are attributable to the MHC haplotype or to other components of the mouse's genetic background. Therefore, in this work, we intend to address this question by investigating the pregnancy outcome in H2d -congenic C57BL/6 mice (C57BL/KsJ-H2d) and H2b-congenic BALB/c mice (CB10-H2-H2b). For this, animals were infected by intragastric route on the first day of pregnancy and examined on days 8 (8dP/8dI) or 18 (18dP/18dI) of gestation and infection. The pregnancy outcome, parasite burden, systemic cytokine profile and antibody response to infection were evaluated. Infected mice showed adverse pregnancy outcomes, in parallel low parasite detection in the uterus/placenta, being that the C57BL/KsJ showed the worst results in relation to CB10-H2 mice. Both mouse lineages showed an increase in IFN-γ and TNF levels systemically on 8dP/8dI and on 18dP/18dI, and C57BL/KsJ showed an increase in IL-6 levels in both gestation/infection periods. Additionally, C57BL/KsJ showed 7- and 7-fold increase in IL-6, 4- and 2.5-fold increase in IFN-γ and, 6- and 4-fold increase in TNF production on 8dP/8dI and 18dP/18dI, respectively in association with 1.5-fold decrease in TGF-ß levels on 8dP/8dI compared to CB10-H2 mice. In conclusion, the high IFN-γ and TNF serum levels observed in C57BL/KsJ (H2d) and CB10-H2 (H2b) mice were involved in the poor pregnancy outcomes in congenital toxoplasmosis. In addition, the higher IFN-γ, IL-6 and TNF levels detected in C57BL/KsJ in relation to CB10-H2 mice on 8dP/8dI seem to be related to the genetic background of C57BL/6J mice that may have contributed to the worse pregnancy outcome in this mouse lineage.

Effects of Notch signaling pathway inhibition by dibenzazepine in acute experimental toxoplasmosis.

Notch signaling pathway plays a crucial role in cellular fate across species, being important for the differentiation and development of several cell types. The aim of this study was to evaluate the effect of Notch inhibition pathway by dibenzazepine (DBZ) in histological and inflammatory alterations and, tissue parasitism in acute Toxoplasma gondii infection. For this, C57BL/6 mice were treated with DBZ before infection with T. gondii, and the small intestine, lungs and liver were analyzed. The genes related to Notch signaling pathway were assayed through qPCR in the organs, and cytokine measurement was performed in serum samples. In the small intestine, T. gondii infection impaired the Hes1 and Math1 mRNA expressions, increased the inflammation and decreased goblet and Paneth cell numbers. The DBZ-treatment was able to partially preserve these cells, however, the parasitism and inflammation were not altered. In parallel, the high IL-2, IL-6, TNF and, IFN-γ levels induced by infection were not changed with the DBZ treatment, with the IFN-γ levels even higher. In contrast, in the liver and lungs, the DBZ-treatment diminished parasitism and inflammation. Our results highlight that Notch pathway inhibition in T.gondii infection results in different parasitological and inflammatory outcomes depending on the organ analyzed.

Toxoplasma gondii Infection Decreases Intestinal 5-Lipoxygenase Expression, while Exogenous LTB4 Controls Parasite Growth.

5-Lipoxygenase (5-LO) is an enzyme required for the production of leukotrienes and lipoxins and interferes with parasitic infections. In vitro, Toxoplasma gondii inhibits leukotriene B4 (LTB4) production, and mice deficient in 5-LO are highly susceptible to infection. The aim of this study was to investigate the effects of the pharmacological inhibition of the 5-LO pathway and exogenous LTB4 supplementation during experimental toxoplasmosis. For this purpose, susceptible C57BL/6 mice were orally infected with T. gondii and treated with LTB4 or MK886 (a selective leukotriene inhibitor through inhibition of 5-LO-activating protein [FLAP]). The parasitism, histology, and immunological parameters were analyzed. The infection decreased 5-LO expression in the small intestine, and treatment with MK886 reinforced this reduction during infection; in addition, MK886-treated infected mice presented higher intestinal parasitism, which was associated with lower local interleukin-6 (IL-6), interferon gamma (IFN-γ), and tumor necrosis factor (TNF) production. In contrast, treatment with LTB4 controlled parasite replication in the small intestine, liver, and lung and decreased pulmonary pathology. Interestingly, treatment with LTB4 also preserved the number of Paneth cells and increased α-defensins expression and IgA levels in the small intestine of infected mice. Altogether, these data demonstrated that T. gondii infection is associated with a decrease in 5-LO expression, and on the other hand, treatment with the 5-LO pathway product LTB4 resulted in better control of parasite growth in the organs, adding to the knowledge about the pathogenesis of T. gondii infection.

Inhibition of Heme Oxygenase-1 by Zinc Protoporphyrin IX Improves Adverse Pregnancy Outcomes in Malaria During Early Gestation.

The enzyme heme oxygenase-1 (HO-1) has cytoprotective effects by catalyzing the degradation of heme to produce carbon monoxide, iron and biliverdin. Furthermore, HO-1 activity has been associated with successful pregnancy. On the other hand, in the context of certain inflammatory conditions, HO-1 can induce iron overload and cell death. To investigate the role of HO-1 in gestational malaria, pregnant BALB/c mice were infected with Plasmodium berghei ANKA in early, mid and late gestation. We found that malaria affected the pregnancy outcome in the three periods evaluated. However, only poor pregnancy outcomes in early pregnancy were related to HO-1 upregulation, iron overload, lipid peroxidation and necrosis of the decidua, which were prevented by HO-1 inhibition. In conclusion, HO-1 expression must be finely tuned in gestational malaria to avoid the deleterious effect of increased enzyme activity.

Anti-parasitic activity of Annona muricata L. leaf ethanolic extract and its fractions against Toxoplasma gondii in vitro and in vivo.

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.

IgE AND IgG antibody responses to Dermatophagoides pteronyssinus in dogs with demodicosis and atopic dermatitis / Respostas de anticorpos IgE E IgG específicos à Dermatophagoides pteronyssinus em soros de cães com demodicose e dermatite atópica

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 evaluatedin 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.

The Availability of Iron Is Involved in the Murine Experimental Toxoplasma gondii Infection Outcome.

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.

Biotechnological and Immunological Platforms Based on PGL-I Carbohydrate-Like Peptide of Mycobacterium leprae for Antibodies Detection Among Leprosy Clinical Forms.

Phenolic glycolipid I (PGL-I) is an abundant antigen on the Mycobacterium leprae cell wall, commonly used for operational classification of leprosy patients. Our aim was to develop PGL-I mimotopes with similar characteristics and functions of the native antigen. We have used a random peptide phage display (PD) library for selections against the monoclonal antibody anti-PGL-I. After three selection cycles, six peptides were identified. All sequences were interspersed by a spacer generating a chimeric peptide (PGLI-M3) that was artificially synthesized. The highly reactive peptide was submitted to a reverse PD selection with a single-chain Fv (scFv) antibody fragment combinatorial library. The most reactive scFv was then validated by enzyme-linked immunosorbent assay (ELISA) against both native PGL-I and two derived synthetic (NDO and ND-O-HSA). We have further proved the scFv specificity by detecting M. leprae bacilli in leprosy lesions through immunohistochemistry. We then described its applicability in ELISA for all clinical forms and household contacts (HC). Afterward, we showed differential binding affinities of PGLI-M3 to sera (anti-PGL-I IgM) from all leprosy clinical forms through surface plasmon resonance (SPR). ELISA IgM detection showed 89.1% sensitivity and 100% specificity, considering all clinical forms. Positivity for anti-PGL-I IgM was twofold higher in both HC and patients with paucibacillary forms in hyperendemic regions than in endemic ones. The SPR immunosensor was able to differentiate clinical forms with 100% accuracy. This is the first time that a PGL-I mimotope has efficiently mimicked the carbohydrate group of the M. leprae antigen with successful immunoassay applications and may become a substitute for the native antigen.

Characterization of antennal sensilla, larvae morphology and olfactory genes of Melipona scutellaris stingless bee.

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.

Toxoplasma gondii 70 kDa heat shock protein: systemic detection is associated with the death of the parasites by the immune response and its increased expression in the brain is associated with parasite replication.

The heat shock protein of Toxoplasma gondii (TgHSP70) is a parasite virulence factor that is expressed during T. gondii stage conversion. To verify the effect of dexamethasone (DXM)-induced infection reactivation in the TgHSP70-specific humoral immune response and the presence of the protein in the mouse brain, we produced recombinant TgHSP70 and anti-TgHSP70 IgY antibodies to detect the protein, the specific antibody and levels of immune complexes (ICs) systemically, as well as the protein in the brain of resistant (BALB/c) and susceptible (C57BL/6) mice. It was observed higher TgHSP70-specific antibody titers in serum samples of BALB/c compared with C57BL/6 mice. However, the susceptible mice presented the highest levels of TgHSP70 systemically and no detection of specific ICs. The DXM treatment induced increased parasitism and lower inflammatory changes in the brain of C57BL/6, but did not interfere with the cerebral parasitism in BALB/c mice. Additionally, DXM treatment decreased the serological TgHSP70 concentration in both mouse lineages. C57BL/6 mice presented high expression of TgHSP70 in the brain with the progression of infection and under DXM treatment. Taken together, these data indicate that the TgHSP70 release into the bloodstream depends on the death of the parasites mediated by the host immune response, whereas the increased TgHSP70 expression in the brain depends on the multiplication rate of the parasite.