The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole (PV) in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both acute and chronic infections. Murine macrophages infected with ∆gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro, which was confirmed with reduced IL-12 and interferon-gamma in vivo. This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the nuclear factor-κB (NF-κB) complex. While GRA15 similarly regulates NF-κB, infection with ∆gra83/∆gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labeling experiments to reveal candidate GRA83 interacting T. gondii-derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit the parasite burden. Importance Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma's ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection is important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii 's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both the acute and chronic infection. Murine macrophages infected with Δ gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro , which was confirmed with reduced IL-12 and interferon gamma (IFN-γ) in vivo . This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the NF-κB complex. While GRA15 similarly regulates NF-κB, infection with Δ gra83/ Δ gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labelling experiments to reveal candidate GRA83 interacting T. gondii derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit parasite burden. Importance: Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma' s ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection are important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

A novel peptide-based sensor platform for detection of anti-Toxoplasma gondii immunoglobulins.

Toxoplasma gondii is an intracellular protozoan parasite responsible for toxoplasmosis, which affects humans and animals. Serologic detection of anti-T. gondii immunoglobulins plays a crucial role in the clinical diagnosis of toxoplasmosis. In this work, a novel electrochemical immunosensor for detecting anti-T. gondii immunoglobulins is reported, based on immobilization of an in silico predicted peptide (PepB3), obtained from membrane protein of T. gondii, on the graphite electrode modified with poly(3-hydroxybenzoic acid). Indirect ELISA confirmed infection and binding specificity of peptide PepB3. Molecular modelling and simulations show this peptide binds to the T. gondii human Fab antibody in the surface antigen 1 (SAG1) binding site, remaining a stable complex during the molecular dynamic simulations, especially by hydrogen bonds and hydrophobic interactions. This electrochemical immunosensor was able to discriminate different periods of infection, using infected mouse serum samples, showing selectivity and discriminating infected and uninfected mouse serum.

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

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

Dectin-1 Activation during Leishmania amazonensis Phagocytosis Prompts Syk-Dependent Reactive Oxygen Species Production To Trigger Inflammasome Assembly and Restriction of Parasite Replication.

Protozoan parasites of the genus Leishmania are the causative agents of Leishmaniasis, a disease that can be lethal and affects 12 million people worldwide. Leishmania replicates intracellularly in macrophages, a process that is essential for disease progression. Although the production of reactive oxygen species (ROS) accounts for restriction of parasite replication, Leishmania is known to induce ROS upon macrophage infection. We have recently demonstrated NLRP3 inflammasome activation in infected macrophages, a process that is important for the outcome of infection. However, the molecular mechanisms responsible for inflammasome activation are unknown. In this article, we demonstrate that ROS induced via NADPH oxidase during the early stages of L. amazonensis infection is critical for inflammasome activation in macrophages. We identified that ROS production during L. amazonensis infection occurs upon engagement of Dectin-1, a C-type lectin receptor that signals via spleen tyrosine kinase (Syk) to induce ROS. Accordingly, inflammasome activation in response to L. amazonensis is impaired by inhibitors of NADPH oxidase, Syk, focal adhesion kinase, and proline-rich tyrosine kinase 2, and in the absence of Dectin-1. Experiments performed with Clec7a-/- mice support the critical role of Dectin-1 for inflammasome activation, restriction of parasite replication in macrophages, and mouse resistance to L. amazonensis infection in vivo. Thus, we reported that activation of the Dectin-1/Syk/ROS/NLRP3 pathway during L. amazonensis phagocytosis is important for macrophage restriction of the parasite replication and effectively accounts for host resistance to Leishmania infection.

Eutirucallin: A Lectin with Antitumor and Antimicrobial Properties.

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.

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

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

Lectins from Synadenium carinatum (ScLL) and Artocarpus heterophyllus (ArtinM) Are Able to Induce Beneficial Immunomodulatory Effects in a Murine Model for Treatment of Toxoplasma gondii Infection.

Infection by Toxoplasma gondii affects around one-third of world population and the treatment for patients presenting toxoplasmosis clinically manifested disease is mainly based by a combination of sulfadiazine, pyrimethamine, and folinic acid. However, this therapeutic protocol is significantly toxic, causing relevant dose-related bone marrow damage. Thus, it is necessary to improve new approaches to investigate the usefulness of more effective and non-toxic agents for treatment of patients with toxoplasmosis. It has been described that lectins from plants can control parasite infections, when used as immunological adjuvants in vaccination procedures. This type of lectins, such as ArtinM and ScLL is able to induce immunostimulatory activities, including efficient immune response against parasites. The present study aimed to evaluate the potential immunostimulatory effect of ScLL and ArtinM for treatment of T. gondii infection during acute phase, considering that there is no study in the literature accomplishing this issue. For this purpose, bone marrow-derived macrophages (BMDMs) were treated with different concentrations from each lectin to determine the maximum concentration without or with lowest cytotoxic effect. After, it was also measured the cytokine levels produced by these cells when stimulated by the selected concentrations of lectins. We found that ScLL showed high capacity to induce of pro-inflammatory cytokine production, while ArtinM was able to induce especially an anti-inflammatory cytokines production. Furthermore, both lectins were able to increase NO levels. Next, we evaluated the treatment effect of ScLL and ArtinM in C57BL/6 mice infected by ME49 strain from T. gondii. The animals were infected and treated with ScLL, ArtinM, ArtinM plus ScLL, or sulfadiazine, and the following parameters analyzed: Cytokines production, brain parasite burden and survival rates. Our results demonstrated that the ScLL or ScLL plus ArtinM treatment induced production of pro-inflammatory and anti-inflammatory cytokines, showing differential but complementary profiles. Moreover, when compared with non-treated mice, the parasite burden was significantly lower and survival rates higher in mice treated with ScLL or ScLL plus ArtinM, similarly with sulfadiazine treatment. In conclusion, the results demonstrated the suitable potential immunotherapeutic effect of ScLL and ArtinM lectins to control acute toxoplasmosis in this experimental murine model.

Nucleotide-binding oligomerization domain-containing protein 2 prompts potent inflammatory stimuli during Neospora caninum infection.

Neospora caninum is an apicomplexan parasite responsible for major economic losses due to abortions in cattle. Innate immune responses are crucial for host resistance against the infection, however the molecules involved in parasite recognition are still poorly understood. Nod2 is a cytosolic receptor that recognizes several pathogens and its role during N. caninum infection has not yet been described. In that sense, we evaluated the role of Nod2 in host response against this parasite. We found that infection of macrophages induced increased expression of Nod2, which colocalized with the parasites' vacuoles. Nod2-deficient macrophages showed an impaired induction of pro-inflammatory cytokines, increased production of modulatory molecules, and failure to restrict parasite replication. In vivo, Nod2-knockout mice showed a reduction of MAPK phosphorylation and proinflammatory cytokines, followed by decreased inflammation in target organs and increment in parasite burden. Surprisingly, these mice were partially resistant to lethal doses of tachyzoites. In addition, these phenomena were not observed in Rip2-/- mice. In conclusion, our study indicates that Nod2-dependent responses account for N. caninum elimination. On the other hand, the inflammatory milieu induced by this innate receptor provoked pathogenesis and death in severe experimental neosporosis.

Toxoplasma gondii-Derived Synthetic Peptides Containing B- and T-Cell Epitopes from GRA2 Protein Are Able to Enhance Mice Survival in a Model of Experimental Toxoplasmosis.

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-α 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.

GITR Activation Positively Regulates Immune Responses against Toxoplasma gondii.

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.

Anti-Toxoplasma Activity of Estragole and Thymol in Murine Models of Congenital and Noncongenital Toxoplasmosis.

Toxoplasmosis is caused by Toxoplasma gondii , an obligatory intracellular protozoan. Normally benign, T. gondii infections can cause devastating disease in immunosuppressed patients and through congenital infection of newborn babies. Few prophylactic and therapeutic drugs are available to treat these infections. The goal of the present study was to assess the anti-Toxoplasma effects in a congenital and noncongenital model of toxoplasmosis (using ME49 strain), besides assessing immunological changes, in vitro cytotoxicity, and in vivo acute toxicity of commercial estragole and thymol. The congenital experimental model was used with intermediate stages of maternal infection. The serum levels of immunoglobulin (Ig)M, IgG, interleukin (IL)-10, IL-12, and interferon-gamma (IFN-γ) were quantified from infected and treated C57Bl/6 mice. Estragole and thymol respectively exhibited low to moderate in vivo toxicity and cytotoxicity. Animals treated with estragole showed high IFN-γ and strong type 1 helper T cell response. Both compounds were active against T. gondii ME49 strain. Furthermore, orally administered estragole in infected pregnant mice improved the weight of offspring compared with untreated controls. Subcutaneous administration of both compounds also increased the weight of mouse offspring born to infected mothers, compared with untreated controls. Estragole and thymol display important anti-Toxoplasma activity. Further studies are needed to elucidate the mechanism of action of these compounds.

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

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

CCp5A Protein from Toxoplasma gondii as a Serological Marker of Oocyst-driven Infections in Humans and Domestic Animals.

Considering that the current immunoassays are not able to distinguish the infective forms that cause Toxoplasma gondii infection, the present study was carried out to evaluate the reactivity of two recombinant proteins (CCp5A and OWP1) from oocyst/sporozoite, in order to differentiate infections occurring by ingestion of oocysts or tissue cysts. The reactivity of the recombinant proteins was assessed against panels of serum samples from animals (chickens, pigs, and mice) that were naturally or experimentally infected by different infective stages of the parasite. Also, we tested sera from humans who have been infected by oocysts during a well-characterized toxoplasmosis outbreak, as well as sera from pregnant women tested IgM(+)/IgG(+) for T. gondii, which source of infection was unknown. Only the sporozoite-specific CCp5A protein was able to differentiate the parasite stage that infected chickens, pigs and mice, with specific reactivity for oocyst-infected animals. Furthermore, the CCp5A showed preferential reactivity for recent infection by oocyst/sporozoite in pigs and mice. In humans, CCp5A showed higher reactivity with serum samples from the outbreak, compared with serum from pregnant women. Altogether, these findings demonstrate the usefulness of the CCp5A protein as a new tool to identify the parasite stage of T. gondii infection, allowing its application for diagnosis and epidemiological investigations in animals and humans. The identification of parasite infective stage can help to design effective strategies to minimize severe complications in immunocompromised people and, particularly, in pregnant women to prevent congenital infection.

Effects of morphine on the expression of cytokines and inflammatory mediators in a rabbit model of endotoxin-induced experimental uveitis.

PURPOSE: To evaluate the effects of 1% morphine instillation on clinical parameters, aqueous humor turbidity, and expression levels of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1beta), prostaglandin E2 (PGE2), and myeloperoxidase (MPO) in rabbits with endotoxin-induced experimental uveitis. METHODS: Twenty four New Zealand white rabbits were divided into four groups (n=6 each): control (CG), morphine (MG), naloxone (NG), and morphine-naloxone (MNG) groups. Under dissociative anesthesia, 0.1 mL of solution containing 0.2 µg of lipopolysaccharide (LPS) endotoxin from the Salmonella typhimurium cell wall was injected in the vitreous chamber. Clinical evaluations (conjunctical hyperemia, chemosis blepharospasm, and ocular discharge) and laser flaremetry were performed before (baseline), and 10 and 20 hours after induction of uveitis. Rabbits were subsequently euthanized and eyes were enucleated to quantify expression levels of TNF-α, IL-1 beta, PGE2, and MPO. RESULTS: No significant differences in clinical parameters and flare values were observed between the study groups. TNF-α and IL-1 beta levels increased significantly in the CG, MG, NG, and MNG groups compared to baseline (P<0.05). Significant differences in PGE2 levels were observed between the MG and NMG groups (P<0.05). A trend toward increased MPO activity was observed in response to uveitis induction; however, this trend did not reach statistical significance (P>0.05). CONCLUSIONS: Morphine has no effect on clinical parameters, flare, or expression levels of inflammatory mediators in a rabbit model of uveitis induced by intravitreal injection of LPS.

Effects of morphine on the expression of cytokines and inflammatory mediators in a rabbit model of endotoxin-induced experimental uveitis / Efeitos da morfina na expressão de citocinas e de mediadores inflamatórios em coelhos com uveítes experimental induzida por endotoxina

ABSTRACT Purpose: To evaluate the effects of 1% morphine instillation on clinical parameters, aqueous humor turbidity, and expression levels of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1beta), prostaglandin E2 (PGE2), and myeloperoxidase (MPO) in rabbits with endotoxin-induced experimental uveitis. Methods: Twenty four New Zealand white rabbits were divided into four groups (n=6 each): control (CG), morphine (MG), naloxone (NG), and morphine-naloxone (MNG) groups. Under dissociative anesthesia, 0.1 mL of solution containing 0.2 µg of lipopolysaccharide (LPS) endotoxin from the Salmonella typhimurium cell wall was injected in the vitreous chamber. Clinical evaluations (conjunctical hyperemia, chemosis blepharospasm, and ocular discharge) and laser flaremetry were performed before (baseline), and 10 and 20 hours after induction of uveitis. Rabbits were subsequently euthanized and eyes were enucleated to quantify expression levels of TNF-α, IL-1 beta, PGE2, and MPO. Results: No significant differences in clinical parameters and flare values were observed between the study groups. TNF-α and IL-1 beta levels increased significantly in the CG, MG, NG, and MNG groups compared to baseline (P<0.05). Significant differences in PGE2 levels were observed between the MG and NMG groups (P<0.05). A trend toward increased MPO activity was observed in response to uveitis induction; however, this trend did not reach statistical significance (P>0.05). Conclusions: Morphine has no effect on clinical parameters, flare, or expression levels of inflammatory mediators in a rabbit model of uveitis induced by intravitreal injection of LPS.

RESUMO Objetivo: Estudaram-se os efeitos da instilação de morfina 1% sobre parâmetros clínicos, turbidez do humor aquoso e expressão de fator de necrose tumoral alfa (TNF-alfa), de interleucina-1 beta (IL-1beta), de prostaglandina E2 (PGE2) e de mieloperoxidase (MPO), em olhos de coelhos com uveíte induzida por endotoxina. Material e Métodos: Vinte e quatro coelhos da raça Nova Zelândia Branco foram distribuídos em quatro grupos (n=6, em cada): grupo controle (GC), morfina (GM), naloxona (GN) e morfina-naloxona (GMN). Sob anestesia dissociativa, injetou-se 0,1 mL de solução contendo 0,2 µg de lipossacarídeo (LPS) endotóxico da parede celular de Salmonella typhimurium na câmara vítrea. Realizou-se avaliação clínica (hiperemia conjuntival, quemose, blefaroespasmo e secreção ocular) e a flaremetria a “laser” antes (basal) e após 10 e 20 horas da indução da uveíte. No final, os coelhos foram submetidos à eutanásia e os olhos com uveíte foram enucleados para a quantificação dos níveis de TNF-alfa, IL-1 beta, PGE2 e MPO. Diferenças foram consideradas significativas quando p<0,05. Resultados: Os grupos da pesquisa não diferiram quanto aos parâmetros clínicos e os valores de “flare”. Observou-se elevação significativa nos níveis de TNF-alfa e de IL-1 beta, comparativamente ao basal, nos grupos GC, GM, GN e GMN (p<0,05). Valores de PGE2 variaram entre os grupos GM e GNM (p<0,05). A atividade de MPO aumentou após a indução da uveíte, porém, sem significância estatística (p>0,05). Conclusões: A morfina não atuou sobre parâmetros clínicos, “flare” e expressão dos mediadores inflamatórios estudados, quando instilada em olhos de coelhos com uveíte induzida por injeção intravítrea de LPS.

Epitope-based vaccines with the Anaplasma marginale MSP1a functional motif induce a balanced humoral and cellular immune response in mice.

Bovine anaplasmosis is a hemoparasitic disease that causes considerable economic loss to the dairy and beef industries. Cattle immunized with the Anaplasma marginale MSP1 outer membrane protein complex presents a protective humoral immune response; however, its efficacy is variable. Immunodominant epitopes seem to be a key-limiting factor for the adaptive immunity. We have successfully demonstrated that critical motifs of the MSP1a functional epitope are essential for antibody recognition of infected animal sera, but its protective immunity is yet to be tested. We have evaluated two synthetic vaccine formulations against A. marginale, using epitope-based approach in mice. Mice infection with bovine anaplasmosis was demonstrated by qPCR analysis of erythrocytes after 15-day exposure. A proof-of-concept was obtained in this murine model, in which peptides conjugated to bovine serum albumin were used for immunization in three 15-day intervals by intraperitoneal injections before challenging with live bacteria. Blood samples were analyzed for the presence of specific IgG2a and IgG1 antibodies, as well as for the rickettsemia analysis. A panel containing the cytokines' transcriptional profile for innate and adaptive immune responses was carried out through qPCR. Immunized BALB/c mice challenged with A. marginale presented stable body weight, reduced number of infected erythrocytes, and no mortality; and among control groups mortality rates ranged from 15% to 29%. Additionally, vaccines have significantly induced higher IgG2a than IgG1 response, followed by increased expression of pro-inflammatory cytokines. This is a successful demonstration of epitope-based vaccines, and protection against anaplasmosis may be associated with elicitation of effector functions of humoral and cellular immune responses in murine model.

Survey for natural Neospora caninum infection in wild and captive birds.

Neospora caninum is a protozoan parasite that presents worldwide distribution and is mainly implicated as responsible for bovine abortion. Although the presence of birds in cattle-raising properties is positively correlated to higher infection rates, very little has been described about the role of these animals in the parasite's life cycle. In that sense, this work aimed to investigate the serological and histological positivity of different avian species sampled in its natural habitat or in captivity. No serological positivity was observed in the 294 tested serum samples. On the other hand, Apicomplexa-like cysts found in muscular tissues of two Psittaciformes were immunostained with N. caninum antisera. These findings indicate that N. caninum may infect a wider range of hosts than described to date, and that further studies should be performed in order to determine the presence of the infection in different avian species.

Differential susceptibility of human trophoblastic (BeWo) and uterine cervical (HeLa) cells to Neospora caninum infection.

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-ß), 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.

Recognition by Toll-like receptor 2 induces antigen-presenting cell activation and Th1 programming during infection by Neospora caninum.

Neospora caninum is an apicomplexan parasite responsible for major economic losses due to abortions in cattle. Toll-like receptors (TLRs) sense specific microbial products and direct downstream signaling pathways in immune cells, linking innate, and adaptive immunity. Here, we analyze the role of TLR2 on innate and adaptive immune responses during N. caninum infection. Inflammatory peritoneal macrophages and bone marrow-derived dendritic cells exposed to N. caninum-soluble antigens presented an upregulated expression of TLR2. Increased receptor expression was correlated to TLR2/MyD88-dependent antigen-presenting cell maturation and pro-inflammatory cytokine production after stimulation by antigens. Impaired innate responses observed after infection of mice genetically deficient for TLR2((-/-)) was followed by downregulation of adaptive T helper 1 (Th1) immunity, represented by diminished parasite-specific CD4(+) and CD8(+) T-cell proliferation, IFN-γ:interleukin (IL)-10 ratio, and IgG subclass synthesis. In parallel, TLR2(-/-) mice presented higher parasite burden than wild-type (WT) mice at acute and chronic stages of infection. These results show that initial recognition of N. caninum by TLR2 participates in the generation of effector immune responses against N. caninum and imply that the receptor may be a target for future prophylactic strategies against neosporosis.