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Neospora caninum is a parasite relevant to the veterinary field. Innate and adaptive responses against N. caninum induce effector mechanisms that limit parasite replication, but little is known about their role in humoral response. Our work aimed to verify whether key molecules in the TLR2/MyD88-mediated response would impact the production of specific IgM and IgG antibodies in mice during immunization with soluble antigens of N. caninum. We observed that lack of IFN-gamma did not negatively affect the production of specific antibodies. However, mice genetically deficient in Toll-like receptor 2, Myeloid differentiation factor 88, Interleukin 12 and inducible nitric oxide synthase presented significant decrease in antibody levels against N. caninum antigens, which also reflected in the diversity of the antigen recognized by their serum. In that sense, we show here that molecules within this innate recognition pathway may present a direct impact in the induction of an antibody response against N. caninum.

Toxoplasma gondii is a protozoan with worldwide prevalence, known to affect a large variety of warm-blooded hosts. However, its ability to induce long-lasting infections in cold-blooded animals remains unclear. The most likely source of infection is through consumption of meat containing tissue cysts or by ingestion of food or water contaminated with oocysts. The current global climate change trend and the progressive degradation of natural habitats are prone to alter the distribution of ectotherm populations over a short period of time, which may favor contact between these animals and the protozoan. In association, alligator meat is considered a delicacy in many regions and its consumption has been previously related to a diversity of foodborne diseases. In that sense, we proposed in this study to search for specific antibodies against T. gondii in serum samples of two common species of alligators from the Brazilian fauna (Melanosuchus niger and Caimam crocodilus). We obtained the serum samples from 84 alligators from the Araguaia region, which were tested by agglutination assays that do not require species-specific secondary antibodies (Modified Agglutination Test – MAT; Indirect Hemagglutination Assay – IHA). From the 84 samples tested, eight (9.5%) were positive by MAT. From those, seven (87.5% of MAT+, 8.3% of the total) were also positive by IHA, reassuring a probable exposure of these animals to the parasite. Direct parasite detection in muscle fragments of one serologically reactive alligator did not yield positive results. Our results provide serological evidence that Brazilian alligators may be exposed to T. gondii and further studies should be performed to elucidate whether alligators are natural hosts of this ubiquitous protozoan parasite., Graphical abstract Image 1, Highlights • Exposure to T. gondii was verified in sera of 84 Brazilian alligators. • The serum samples were analyzed to 2 serological assays: MAT and HAI. • 7 samples were found to be positive in bothserological tests.

Neospora caninum is a protozoan associated with abortions in ruminants and neuromuscular disease in dogs. Classically, the immune response against apicomplexan parasites is characterized by the production of proinflammatory cytokines, such as IL-12, IFN-γ and TNF. TNF is mainly produced during the acute phases of the infections and binds to TNF receptor 1 (CD120a, p55, TNFR1) activating a variety of cells, hence playing an important role in the induction of the inflammatory process against diverse pathogens. Thus, in this study, we aimed to evaluate the role of TNF in cellular and humoral immune responses during N. caninum infection. For this purpose, we used a mouse model of infection based on wildtype (WT) and genetically deficient C57BL/6 mice in TNFR1 (Tnfr1 -/-). We observed that Tnfr1 -/- mice presented higher mortality associated with inflammatory lesions and increased parasite burden in the brain after the infection with N. caninum tachyzoites. Moreover, Tnfr1 -/- mice showed a reduction in nitric oxide (NO) levels in vivo. We also observed that Tnfr1 -/- mice showed enhanced serum concentration of antigen-specific IgG2 subclass, while IgG1 production was significantly reduced compared to WT mice, suggesting that TNFR1 is required for regular IgG subclass production and antigen recognition. Based on our results, we conclude that the TNF-TNFR1 complex is crucial for mediating host resistance during the infection by N. caninum.

Made available in DSpace on 2019-10-04T11:56:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-02-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Ministerio de Ciencias e Tecnologia (MCT) of Brazil Phospholipases A(2) represent a family of enzymes with important application in medicine. However, direct tracking is difficult due to the absence of a stable, effective and specific marker for these enzymes. Magic-sized quantum dots (MSQDs) are inorganic semiconducting nanocrystals with unique physical properties. They have the ability to conjugate to proteins, making them excellent markers for biological systems. In this work, we labelled phospholipase A(2) from Bothrops alternatus snake venom with Cadmium selenide (CdSe)/cadmium sulphate (CdS) MSQDs-a biocompatible and luminescent probe. Bioconjugation was confirmed using infrared spectra and fluorescence microscopy, which demonstrated that the CdSe/CdS MSQDs interact with phospholipase A(2) without interfering with its activity. This probe may be an important tool for the elucidation of many biological mechanisms, because it allows the pathway of phospholipase A(2) to be tracked from its entry through the plasma membrane until its incorporation into the nucleus of myoblasts. (C) 2018 Elsevier B.V. All rights reserved. Univ Fed Uberlandia, Inst Genet & Bioquim, Uberlandia, MG, Brazil Univ Fed Uberlandia, Inst Ciencias Biomed, Uberlandia, MG, Brazil Univ Fed Uberlandia, Inst Fis, Uberlandia, MG, Brazil Univ Estadual Paulista, Inst Quim, Araraquara, SP, Brazil Inst Nacl Ciencia & Tecnol Nanobiofarmaceut N Bio, Belo Horizonte, MG, Brazil Univ Estadual Paulista, Inst Quim, Araraquara, SP, Brazil

Neospora caninum poses as a considerable threat to animal health and generates significant economic impact in livestock production worldwide. Here, we have investigated the mechanism that underlies the participation of the inflammasome complex and Reactive Oxygen Species (ROS) in the regulation of immune responses during N. caninum infection. For that purpose, we used in vitro (bone marrow derived macrophages) and in vivo mouse models of infection. Our results show that NLRP3 and NLRC4 receptors, alongside with ASC and Caspase-1, are required for proper activation of the inflammasome during N. caninum infection. As expected, the engagement of these pathways is crucial for IL-1α, IL-1β, and IL-18 production, as well as the induction of pyroptosis. Our results also show that N. caninum induces ROS production dependent of the inflammasome assembly, which in its turn also depends on MyD88/NF-κB-induced ROS to maintain its activation and, ultimately, lead to restriction of parasite replication.

Activities of phospholipases (PLAs) have been linked to pathogenesis in various microorganisms, and implicated in cell invasion and so the interest in these enzymes as potential targets that could contribute to the control of parasite survival and proliferation. Chicken eggs immunized with BnSP-7, a Lys49 phospholipase A2 (PLA2) homologue from Bothrops pauloensis snake venom, represent an excellent source of polyclonal antibodies with potential inhibitory activity on parasite PLAs. Herein, we report the production, characterization and anti-parasitic effect of IgY antibodies from egg yolks of hens immunized with BnSP-7. Produced antibodies presented increasing avidity and affinity for antigenic toxin epitopes throughout immunization, attaining a plateau after 4weeks. Pooled egg yolks-purified anti-BnSP-7 IgY antibodies were able to specifically recognize different PLA2s from Bothrops pauloensis and Bothrops jararacussu venom. Antibodies also neutralized BnSP-7 cytotoxic activity in C2C12 cells. Also, the antibodies recognized targets in Leishmania (Leishmania) amazonensis and Toxoplasma gondii extracts by ELISA and immunofluorescence assays. Anti-BnSP-7 IgY antibodies were cytotoxic to T. gondii tachyzoite and L. (L.) amazonensis promastigotes, and were able to decrease proliferation of both parasites treated before infection. These data suggest that the anti-BnSP-7 IgY is an important tool for discovering new parasite targets and blocking parasitic effects.

Objective The purpose of this study was to evaluate the effects of oropharyngeal colostrum administration in the incidence of late-onset clinical and proven sepsis and in concentrations of immunoglobulin A (IgA) in very-low-birth-weight (VLBW) infants. Methods We conducted a double-blinded, randomized, placebo-controlled trial and assigned 113 VLBW infants to receive 0.2 mL of maternal colostrum or sterile water (placebo) via oropharyngeal route every 2 hours for 48 hours, beginning in the first 48 to 72 hours of life. Neonates of both groups were fed breast milk from the first 3 days of life until a volume of at least 100 mL · kg · day. IgA was measured in serum and urine before and after treatment. Clinical data during hospitalization were collected. Results We found no statistically significant differences between colostrum and placebo groups in the incidence of late-onset clinical sepsis (odds ratio 0.7602; CI 95% 0.3-1.6) and proven sepsis (odds ratio 0.7028; CI 95% 0.3-1.6). The measurement of IgA was similar in serum before (P value 0.87) and after treatment (P value 0.26 day 4 and 0.77 day 18). No differences were also observed in IgA in urine before (P value 0.8) and after treatment (P value 0.73 day 4 and 0.52). Conclusions This study could not confirm the hypothesis that oropharyngeal administration of maternal colostrum to VLBW could reduce the incidence of late-onset sepsis and increase the levels of IgA. We believe that this finding can be justified by the practice of feeding VLBW infants exclusively with breast milk in the first days of life and reinforces the prior knowledge of the importance of early nutrition, especially, with human milk. It also suggests that oropharyngeal administration of colostrum should be reserved for neonates who cannot be fed in first few days of life.

Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii and has been studied for causing neuromuscular disease in dogs and abortions in cattle. It is recognized as one of the main transmissible causes of reproductive failure in cattle and consequent economic losses to the sector. In that sense, this study aimed to evaluate the role of Toll-like receptor 3 (TLR3)-TRIF-dependent resistance against N. caninum infection in mice. We observed that TLR3-/- and TRIF-/- mice presented higher parasite burdens, increased inflammatory lesions, and reduced production of interleukin 12p40 (IL-12p40), tumor necrosis factor (TNF), gamma interferon (IFN-γ), and nitric oxide (NO). Unlike those of T. gondii, N. caninum tachyzoites and RNA recruited TLR3 to the parasitophorous vacuole (PV) and translocated interferon response factor 3 (IRF3) to the nucleus. We also observed that N. caninum upregulated the expression of TRIF in murine macrophages, which in turn upregulated IFN-α and IFN-β in the presence of the parasite. Furthermore, TRIF-/- infected macrophages produced lower levels of IL-12p40, while exogenous IFN-α replacement was able to completely restore the production of this key cytokine. Our results show that the TLR3-TRIF signaling pathway enhances resistance against N. caninum infection in mice, since it improves Th1 immune responses that result in controlled parasitism and reduced tissue inflammation, which are hallmarks of the disease.

Toxoplasma gondii infection can cause abortions or congenital infection for a vast number of domestic animals and humans, leading to economic loss in veterinary sciences, as well as severe consequences for immunocompromised patients. Bidens pilosa Linné has been used in ethnopharmacology for treatment of diseases, as malaria, diabetes and hepatitis, in addition to its use as antioxidant, antiallergic, anti-inflammatory, and antiviral. The components of this plant have never been studied before for treatment of toxoplasmosis, and the conventional drugs currently used to treat this disease have high degree of toxicity. Thus, the aim of this study was to evaluate the effect of B. pilosa against T. gondii, by analyzing a total extract of this plant in parallel with a fraction obtained by precipitation in acetone. Also, it was assessed if the acetonic fraction could present lectinic activity, followed by its identification by mass spectrometry. It was observed with the experimental models designed that both total extract and acetonic fraction of B. pilosa were able to control T. gondii infection by in vitro and in vivo experiments, in addition to their low toxicity to host cells. Both total extract and acetonic fraction of this plant display capacity to impair replication of T. gondii tachyzoites. Interesting, the B. pilosa acetonic fraction treatment for 10 days after infection decreases significantly the number of T. gondii brain cyst in comparison with controls. The protein isolated from B. pilosa acetonic fraction was characterized as a novel lectin identified as maturase K. Taken together, these findings open new perspectives to treat patients infected by T. gondii. Future studies will be necessary to investigate the precise mechanism underlying the control of T. gondii infection to impair the replication of this parasite in the host cells after treatment with B. pilosa maturase K.

Background In last decades, snake venoms have aroused great interest of the medicine due to the pathophysiological effects caused by their toxins. These include the phospholipases A2, low molecular weight proteins capable of causing haemorrhagic, myotoxic, inflammatory and neurotoxic effects after an ophidian accident. The present work describes the isolation and biochemical characterization of a new PLA2 isolated from the B. alternatus snake venom, which was named BaltPLA2. Method The rapid and efficient purification of this toxin was performed using only two chromatography steps (anion exchange and hydrophobic chromatography). Results BaltPLA2 is an acidic protein (pI 4.4) with an apparent molecular mass of 17000 (SDSPAGE) and 14074.74 Da (MALDI TOF/TOF). Analysis of fragments ion by MS / MS showed the following internal amino acid sequence SGVIICGEGTPCEK, which did not exhibit homology with other PLA2 from the same venom. BaltPLA2 is a catalytically active, which displayed an anticoagulant action, inhibition of platelet aggregation induced by epinephrine (~ 80%) and ADP (24%). BaltPLA2 also was able to induce myonecrosis and the release of cytokines (IL-10, IL-12 and TNF- α) in macrophages culture. Conclusion The results presented in this work greatly contribute to a better understanding of the mechanism of enzymatic and pharmacological actions of PLA2s from snake venoms and they may contribute to its application in medical research.

Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii and has been studied for causing neuromuscular disease in dogs and abortions in cattle. It is recognized as the major cause of economic losses in bovine products. In that sense, this study aimed to evaluate the role of TLR3-TRIF dependent resistance against N. caninum infection. We observed that TLR3−/− and TRIF−/− mice presented higher parasite burden, increased inflammatory lesions and reduced production of IL-12p40, TNF, IFN-γ, and NO. Differently from T. gondii, N. caninum tachyzoites and its RNA recruited TLR3 and IRF3 to the parasitophorous vacuole (PV). We observed that N. caninum upregulated the expression of TRIF in macrophages, which by its turn upregulated IFN-α and IFN-β in the presence of the parasite. Furthermore, TRIF−/− infected macrophages produced lower levels of IL-12p40 and IFN-α replacement was able to completely restore the production of this key cytokine. Our results have shown that TLR3-TRIF signaling pathway enhances resistance against N. caninum infection, since it improves Th1 immune responses that control parasitism and tissue inflammation, which are hallmarks of the disease.

Activities of phospholipases (PLAs) have been linked to pathogenesis in various microorganisms, and implicated in cell invasion and so the interest in these enzymes as potential targets that could contribute to the control of parasite survival and proliferation. Chicken eggs immunized with BnSP-7, a Lys49 phospholipase A

Hymenoptera venoms constitute an interesting source of natural toxins that may lead to the development of novel therapeutic agents. The present study investigated the enzymatic and biological characteristics of the crude venom of the ant Odontomachus bauri. Its crude venom presents several protein bands, with higher staining for six proteins with gelatinolytic activity (17, 20, 26, 29, 43 and 48 kDa). The crude venom showed high proteolytic activity on azocasein at optimal pH 8.0 and 37 °C. In the presence of protease inhibitors as aprotinin, leupeptin and EDTA, the azocaseinolytic activity was reduced by 45%, 29% and 9%, respectively, suggesting that the enzymes present in the crude venom belong to the three classes of proteases, with the serine proteases in greater intensity. The crude venom degraded the fibrinogen α-chain faster than the β-chain, while the fibrinogen γ-chain remained unchanged. In biological assays, O. bauri venom showed hemolytic and coagulant activity in vitro, and defibrinating activity in vivo. In addition, the venom showed antimicrobial activity against Staphylococcus aureus and Escherichia coli as well as antiparasitic activity on Toxoplasma gondii infection in vitro. In that sense, this study sheds perspectives for pharmacological applications of O. bauri venom enzymes.

Eutirucallin is a lectin isolated from the latex of Euphorbia tirucalli, a plant known for its medical properties. The present study explores various characteristics of Eutirucallin including stability, cytotoxicity against tumor cells, antimicrobial and antiparasitic activities. Eutirucallin was stable from 2 to 40 days at 4°C, maintained hemagglutinating activity within a restricted range, and showed optimal activity at pH 7.0–8.0. Eutirucallin presented antiproliferative activity for HeLa, PC3, MDA-MB-231, and MCF-7 tumor cells but was not cytotoxic for non-tumorigenic cells such as macrophages and fibroblasts. Eutirucallin inhibited the Ehrlich ascites carcinoma in vivo and it was also observed that Eutirucallin inhibited 62.5% of Escherichia coli growth. Also, Eutirucallin showed to be effective when tested directly against Toxoplasma gondii infection in vitro. Therefore, this study sheds perspectives for pharmacological applications of Eutirucallin.

Neospora caninum is an intracellular protozoan parasite that has drawn increasing interest due to its association with worldwide repetitive bovine abortions, which cause billionaire losses to the meat and dairy industries annually. Innate immunity plays an important role in infection control, and N. caninum activates the production of inflammatory mediators through Toll-like Receptors (TLR), Nod-Like Receptors (NLR) and Mitogen Activated Protein Kinase (MAPK) signaling pathways. Advances in the knowledge of initial host-parasite interactions are desirable for the design of control measures against the infection, obliterating its pathogenesis. In that sense, we here aimed to describe the role of the innate C-type lectin receptor (CLR) Dectin-1 during the infection by N. caninum. With that intent, we observed that the absence of Dectin-1, observed in genetically depleted (Dectin-1-/-) mice or competitively inhibited by an inert agonist (Laminarin, LAM), rescued 50% of the mice infected with lethal doses of N. caninum. Dectin-1-/- and LAM-treated mice also presented a reduction in the parasite load during acute and chronic phases, associated to decreased inflammatory scores in the central nervous system. Amongst all the cell phenotypes that migrated to the initial site of infection, dendritic cells and macrophages gained subpopulations with high Dectin-1 surface expression. The impairment of the receptor in these cells led to a decreased parasite burden, as well as augmented production of IL-12p40. We also found that Dectin-1+ cells produced less reactive oxygen species (ROS) at the initial site of the infection, while mice deficient in NAPDH oxidase isoform 2 (NOX2-/-) were not able to control parasite replication and produce IL-12p40, even upon LAM treatment. Interestingly, the absence of functional Dectin-1 did not alter the susceptibility of mice against closely related T. gondii. In conclusion, the gathered data suggests that Dectin-1 is involved in the parasite-induced downmodulation of ROS and other key molecules triggered for the control of N. caninum infection, and is a promising target for future development of protocols intended for intervention against neosporosis.

Neospora caninum is an intracellular protozoan parasite that has drawn increasing interest due to its association with worldwide repetitive bovine abortions, which cause billionaire losses to the meat and dairy industries annually. Innate immunity plays an important role in infection control, and N. caninum activates the production of inflammatory mediators through toll-like receptors, NOD-like receptors, and mitogen-activated protein kinase signaling pathways. Advances in the knowledge of initial host–parasite interactions are desirable for the design of control measures against the infection, obliterating its pathogenesis. In that sense, we here aimed to describe the role of the innate C-type lectin receptor Dectin-1 during the infection by N. caninum. With that intent, we observed that the absence of Dectin-1, observed in genetically depleted (Dectin-1−/−) mice or competitively inhibited by an inert agonist [laminarin (LAM)], rescued 50% of the mice infected with lethal doses of N. caninum. Dectin-1−/− and LAM-treated mice also presented a reduction in the parasite load during acute and chronic phases, associated with decreased inflammatory scores in the central nervous system. Among all the cell phenotypes that migrated to the initial site of infection, dendritic cells and macrophages gained subpopulations with high Dectin-1 surface expression. The impairment of the receptor in these cells led to a decreased parasite burden, as well as augmented production of IL-12p40. We also found that Dectin-1+ cells produced less reactive oxygen species (ROS) at the initial site of the infection, while mice deficient in NADPH oxidase isoform 2 (NOX2−/−) were not able to control parasite replication and produce IL-12p40, even upon LAM treatment. Interestingly, the absence of functional Dectin-1 did not alter the susceptibility of mice against closely related Toxoplasma gondii. In conclusion, the gathered data suggest that Dectin-1 is involved in the parasite-induced downmodulation of ROS, and other key molecules triggered for the control of N. caninum infection and are a promising target for future development of protocols intended for intervention against neosporosis.

Artemisia annua is used as a source of artemisinin, a potent therapeutic agent used for the treatment of infectious diseases, chiefly malaria. However, the low concentration (from 0.01 to 1.4% of dried leaf matter) of artemisinin in the plant obtained with the traditional cropping system makes it a relatively expensive drug, especially in developing countries. Considering that artemisinin and silicon (Si) are both stored in A. annua glandular trichomes, and that Si accumulation has never been investigated, this study aimed to look into Si effects on A. annua trichome artemisinin concentration, and whether leaf infusion from Si-treated A. annua plants is able to control Toxoplasma gondii growth. T. gondii is the etiologic agent of toxoplasmosis, a zoonotic parasitic disease whose traditional treatment shows significant side effects. The experimental design consisted of A. annua seedlings randomly planted in soil treated with different doses of calcium/magnesium silicate (0, 200, 400, 800, and 1600 kg ha-1). Analysis of foliar macronutrients showed significant increases of nitrogen content only at the highest dose of silicate. Foliar micronutrients, Si concentrations, and plant height were not affected by any of the silicate doses. However, the dose of 400 kg ha-1 of silicate increased the trichome size, which in turn raised artemisinin concentration in leaves and the infusion. In contrast, the 800 and 1600 kg ha-1 doses dramatically decreased artemisinin concentration. HeLa cell treatment with the infusion of A. annua grown in soil treated with 400 kg ha-1 of silicate decreased parasite proliferation in a dose-dependent manner when the treatment was carried out after or along with T. gondii infection. However, this effect was similar to A. annua grown in soil without silicate treatment. Thus, it can be concluded that, even though Si applied to the soil at 400 kg ha-1 has a positive effect on the A. annua glandular trichome size and the artemisinin concentration, this outcome cannot be directly associated with the efficiency of A. annua infusion on T. gondii growth, suggesting that other components from A. annua leaves could be acting in synergy with artemisinin.

Tumor necrosis factor (TNF) is a major cytokine in inflammatory processes and its deregulation plays a pivotal role in several diseases. Here, we report that a zinc metalloprotease extracted from Bothrops moojeni venom (BmooMP-alpha-I) inhibits TNF directly by promoting its degradation. This inhibition was demonstrated by both in vitro and in vivo assays, using known TLR ligands. These findings are supported by molecular docking results, which reveal interaction between BmooMP-alpha-I and TNF. The major cluster of interaction between BmooMP-alpha-I and TNF was confirmed by the structural alignment presenting Ligand Root Mean Square Deviation LRMS = 1.05 A and Interactive Root Mean Square Deviation IRMS = 1.01 A, this result being compatible with an accurate complex. Additionally, we demonstrated that the effect of this metalloprotease on TNF is independent of cell cytotoxicity and it does not affect other TLR-triggered cytokines, such as IL-12. Together, these results indicate that this zinc metalloprotease is a potential tool to be further investigated for the treatment of inflammatory disorders involving TNF deregulation.

Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2) is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN), as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b), mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-alpha and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii.

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.

OBJECTIVE To evaluate the detergent and aqueous phases of crude saline extract (crude) of Taenia solium metacestodes extracted with Triton X-114, for the detection of immunoglobulin G (IgG) antibodies by enzyme-linked immunosorbent assay (ELISA) and immunoblot for the laboratory diagnosis of human neurocysticercosis. METHOD Serum samples were collected from 138 individuals and tested; 40 were diagnosed with neurocysticercosis (NC; group 1), 58 presented other parasitic diseases (group 2) and 40 were apparently healthy (group 3). RESULTS The detergent phase showed only one dominant component (70-50 kDa), while the aqueous phase showed four (116, 110, 97 and 77 kDa) by silver staining. ELISA sensitivity and specificity were 92.5% and 84.5% for crude phase, 92.5% and 93.3% for detergent phase and 72.5% and 72.6% for aqueous phase. CONCLUSIONS The immunoblot of the antigens confirmed the results obtained by ELISA. Detergent phase purified from T. solium metacestodes was an important source of specific peptides and was efficient in detecting T. solium metacestodes antibodies in serum samples. This phase can be used for detecting IgG antibodies to T. solium metacestodes in seroepidemiological investigations and in the diagnostic screening of NC patients.

Background Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, an intracellular protozoan parasite able to infect a wide range of hosts, including humans. Congenital infection can cause severe damage to the fetus. Thus, it is important to detect antibodies against the parasite to confirm clinical manifestations. Considering that all immunoglobulin isotypes may be present in biological samples from newborns and their mothers, this study aimed to evaluate the ability to diagnose recent toxoplasmosis by using colostrum, as an alternative noninvasive way to obtain biological samples, as well as to determine correlation rates between antibodies from serum samples to detect IgG, IgM and IgA isotypes against T. gondii. Methods A total of 289 puerperal women from Clinical Hospital of Federal University of Uberlândia (mean age: 24.8 years, range: 14 – 43 years) took part in this study. Serum and colostrum samples from these patients were analyzed using ELISA and immunoblotting assays for soluble antigens from T. gondii. Results ELISA immunoassays with serum samples showed reactivity in 47.0, 6.9 and 2.8 % of samples to anti-T. gondii IgG, IgM and IgA, respectively, in comparison with colostrum samples, which showed reactivity in 46.0, 7.9 and 2.8 % of samples to the same isotypes. Also, significant correlation rates of anti-T. gondii antibody levels between serum and colostrum samples were observed. Interestingly, reactivity to IgM and/or IgA in colostrum and/or serum confirmed clinical manifestations of congenital toxoplasmosis in three newborns. Immunoblotting assays showed that it is possible to detect IgG, IgM and IgA antibodies against various antigens of T. gondii in serum and colostrum samples. IgG antibodies in serum and colostrum samples recognized more antigenic fractions than IgM and IgA antibodies. Serum IgG detected more antigenic fractions than IgG antibodies present in the colostrum of the same patient. In contrast, specific IgA present in colostrum recognized a higher number of antigens than IgA present in serum samples of the same patient. Conclusions Overall, the results show that it is important to investigate the occurrence of congenital toxoplasmosis, even at puerperal period. Furthermore, this study demonstrates that T. gondii-specific IgG, IgM and IgA antibodies in serum and colostrum samples from puerperal women may be detected with a significant correlation, suggesting that colostrum may also be used as an alternative biological sample to efficiently diagnose recent human toxoplasmosis.

According to the World Health Organization, cancer is the second leading cause of worldwide death and breast cancer is among the most frequent types. Conventional therapies show many side effects, high cost and sometimes are inefficient in the total eradication of tumors. Looking for new alternatives to cure this disease a lot of studies have been developed in the field of medicinal plants, due to the vast distribution and use of folk medicine by patients as a single treatment or as a supplement to conventional treatment, obtaining positive effects in several cases. Currently, research has been directed for the characterization and purification of molecules and substances in order to identify the responsible components for therapeutic activity and minimizing side effects. Euphorbia tirucalli popularly known as Labyrinth, is a frequently cited plant in traditional medicine for its use as an antitumor agent. The present study aims to purify the lectin fraction obtained from the latex of E. tirucalli, and verify the relation and contribution of this bioactive substance, as well as the nonbonding fractions and crude extract, the activity against tumor Erhlich.

Toxoplasma gondii is a widespread parasite responsible for causing clinical diseases especially in pregnant and immunosuppressed individuals. Glucocorticoid-induced TNF receptor (GITR), which is also known as TNFRS18 and belongs to the TNF receptor superfamily, is found to be expressed in various cell types of the immune system and provides an important costimulatory signal for T cells and myeloid cells. However, the precise role of this receptor in the context of T. gondii infection remains elusive. Therefore, the current study investigated the role of GITR activation in the immunoregulation mechanisms induced during the experimental infection of mice with T. gondii. Our data show that T. gondii infection slightly upregulates GITR expression in Treg cells and B cells, but the most robust increment in expression was observed in macrophages and dendritic cells. Interestingly, mice infected and treated with an agonistic antibody anti-GITR (DTA-1) presented a robust increase in pro-inflammatory cytokine production at preferential sites of parasite replication, which was associated with the decrease in latent brain parasitism of mice under treatment with DTA-1. Several in vivo and in vitro analysis were performed to identify the cellular mechanisms involved in GITR activation upon infection, however no clear alterations were detected in the phenotype/function of macrophages, Tregs and B cells under treatment with DTA-1. Therefore, GITR appears as a potential target for intervention during infection by the parasite Toxoplasma gondii, even though further studies are still necessary to better characterize the immune response triggered by GITR activation during T. gondii infection.

To evaluate the detergent and aqueous phases of crude saline extract (crude) of Taenia solium metacestodes extracted with Triton X-114, for the detection of immunoglobulin G (IgG) antibodies by enzyme-linked immunosorbent assay (ELISA) and immunoblot for the laboratory diagnosis of human neurocysticercosis.Serum samples were collected from 138 individuals and tested; 40 were diagnosed with neurocysticercosis (NC; group 1), 58 presented other parasitic diseases (group 2) and 40 were apparently healthy (group 3).The detergent phase showed only one dominant component (70-50 kDa), while the aqueous phase showed four (116, 110, 97 and 77 kDa) by silver staining. ELISA sensitivity and specificity were 92.5% and 84.5% for crude phase, 92.5% and 93.3% for detergent phase and 72.5% and 72.6% for aqueous phase.The immunoblot of the antigens confirmed the results obtained by ELISA. Detergent phase purified from T. solium metacestodes was an important source of specific peptides and was efficient in detecting T. solium metacestodes antibodies in serum samples. This phase can be used for detecting IgG antibodies to T. solium metacestodes in seroepidemiological investigations and in the diagnostic screening of NC patients.

BackgroundToxoplasma gondii may cause abortions, ocular and neurological disorders in warm-blood hosts. Immunized mammals are a wide source of hyperimmune sera used in different approaches, including diagnosis and the study of host-parasite interactions. Unfortunately, mammalian antibodies present limitations for its production, such as the necessity for animal bleeding, low yield, interference with rheumatoid factor, complement activation and affinity to Fc mammalian receptors. IgY antibodies avoid those limitations; therefore they could be an alternative to be applied in T. gondii model.Methodology/principal findingsIn this study we immunized hens with soluble tachyzoite antigens of T. gondii (STAg) and purified egg yolk antibodies (IgY) by an inexpensive and simple method, with high yield and purity degree. IgY anti-STAg antibodies presented high avidity and were able to recognize a broad range of parasite antigens, although some marked differences were observed in reactivity profile between antibodies produced in immunized hens and mice. Interestingly, IgY antibodies against Neospora caninum and Eimeria spp. did not react to STAg. We also show that IgY antibodies were suitable to detect T. gondii forms in paraffin-embedded sections and culture cell monolayers.Conclusions/significanceDue to its cost-effectiveness, high production yield and varied range of possible applications, polyclonal IgY antibodies are useful tools for studies involving T. gondii.

Background Toxoplasma gondii is an intracellular parasite that causes relevant clinical disease in humans and animals. Several studies have been performed in order to understand the interactions between proteins of the parasite and host cells. SAG2A is a 22 kDa protein that is mainly found in the surface of tachyzoites. In the present work, our aim was to correlate the predicted three-dimensional structure of this protein with the immune system of infected hosts. Methods To accomplish our goals, we performed in silico analysis of the amino acid sequence of SAG2A, correlating the predictions with in vitro stimulation of antigen presenting cells and serological assays. Results Structure modeling predicts that SAG2A protein possesses an unfolded C-terminal end, which varies its conformation within distinct strain types of T. gondii. This structure within the protein shelters a known B-cell immunodominant epitope, which presents low identity with its closest phyllogenetically related protein, an orthologue predicted in Neospora caninum. In agreement with the in silico observations, sera of known T. gondii infected mice and goats recognized recombinant SAG2A, whereas no serological cross-reactivity was observed with samples from N. caninum animals. Additionally, the C-terminal end of the protein was able to down-modulate pro-inflammatory responses of activated macrophages and dendritic cells. Conclusions Altogether, we demonstrate herein that recombinant SAG2A protein from T. gondii is immunologically relevant in the host-parasite interface and may be targeted in therapeutic and diagnostic procedures designed against the infection.