Soluble epoxide hydrolase inhibitors for smoking-associated inflammatory lung diseases and chronic obstructive pulmonary disease: a meta-analytical systematic review of preclinical and clinical studies.

An evaluation of the inflammatory enzymatic interactions related to pulmonary function can help identify biomarkers for interventions or prophylactic measures to improve patient prognosis. This study aimed to determine the effect of epoxide hydrolase inhibition by GSK2256294 in different pulmonary inflammation models. A secondary search was performed using Medline/PubMed, Web of Science, SciELO, Cochrane Library, Embase, Academic Google, and gray literature by two independent reviewers, who analyzed the methodological quality and consistency of the data. Different variables were compared using a meta-analysis. A total of 86 studies were found, 4 of which were selected from the gray literature. Based on the eligibility criteria, two clinical and one preclinical studies were evaluated. GSK2256294 inhibited the soluble epoxide hydrolase enzyme in both clinical and preclinical models, exhibiting greater effectiveness in clinical studies and contributing to the anti-inflammatory activity mediated by the eicosatrienoic pathway by reducing the levels of dihydroxyeicosatrienoic acids and leukotoxin-diol. Overall, GSK2256294 was identified as a promising drug for controlling the deleterious manifestations of lung inflammation. Further clinical and preclinical studies are required to ensure consistency among the evidence and identify other biological activities mediated by GSK2256294.

Comparative genomics of Bordetella pertussis and prediction of new vaccines and drug targets.

Pertussis is a highly contagious respiratory disease caused by Bordetella pertussis, a Gram-negative bacterium described over a century ago. Despite broad vaccine coverage and treatment options, the disease is remerging as a public health problem especially in infants and older children. Recent data indicate re-emergence of the disease is related to bacterial resistance to immune defences and decreased vaccine effectiveness, which obviously suggests the need of new effective vaccines and drugs. In an attempt to contribute with solutions to this great challenge, bioinformatics tools were used to genetically comprehend the species of these bacteria and predict new vaccines and drug targets. In fact, approaches were used to analysis genomic plasticity, gene synteny and species similarities between the 20 genomes of Bordetella pertussis already available. Furthermore, it was conducted reverse vaccinology and docking analysis to identify proteins with potential to become vaccine and drug targets, respectively. The analyses showed the 20 genomes belongs to a homogeneous group that has preserved most of the genes over time. Besides that, were found genomics islands and good proteins to be candidates for vaccine and drugs. Taken together, these results suggests new possibilities that may be useful to develop new vaccines and drugs that will help the prevention and treatment strategies of pertussis disease caused by these Bordetella strains. Communicated by Ramaswamy H. Sarma.

Pan-genomic analyses of 47 complete genomes of the Rickettsia genus and prediction of new vaccine targets and virulence factors of the species.

The genus Rickettsia belongs to the Proteobacteria phylum and these bacteria infect animals and humans causing a range of diseases worldwide. The genus is divided into 4 groups and despite the public health threat and the knowledge accumulated so far, the mandatory intracellular bacteria behaviour and limitation for in vitro culture makes it difficult to create new vaccines and drug targets to these bacteria. In an attempt to overcome these limitations, pan-genomic approaches has used 47 genomes of the genus Rickettsia, in order to describe species similarities and genomics islands. Moreover, we conducted reverse vaccinology and docking analysis aiming the identification of proteins that have great potential to become vaccine and drug targets. We found out that the bacteria of the four Rickettsia groups have a high similarity with each other, with about 90 to 100% of identity. A pathogenicity island and a resistance island were predicted. In addition, 8 proteins were also predicted as strong candidates for vaccine and 9 as candidates for drug targets. The prediction of the proteins leads us to believe in a possibility of prospecting potential drugs or creating a polyvalent vaccine, which could reach most strains of this large group of bacteria.Communicated by Ramaswamy H. Sarma.

Metabolomic Evaluation of Chronic Periodontal Disease in Older Adults.

Periodontal disease is an infectious inflammatory disease related to the destruction of supporting tissues of the teeth, leading to a functional loss of the teeth. Inflammatory molecules present in the exudate are catalyzed and form different metabolites that can be identified and quantified. Thus, we evaluated the inflammatory exudate present in crevicular fluid to identify metabolic biological markers for diagnosing chronic periodontal disease in older adults. Research participants were selected from long-term institutions in Brazil. Participants were individuals aged 65 years or older, healthy, or with chronic periodontal disease. Gas chromatography/mass spectrometry was used to evaluate potential biomarkers in 120 crevicular fluid samples. We identified 969 metabolites in the individuals. Of these, 15 metabolites showed a variable importance with projection score > 1 and were associated with periodontal disease. Further analysis showed that among the 15 metabolites, two (5-aminovaleric acid and serine, 3TMS derivative) were found at higher concentrations in the crevicular fluid, indicating their potential diagnostic power for periodontal disease in older adults. Our findings indicated that some metabolites are present at high concentrations in the crevicular fluid in older adults with periodontal disease and can be used as biomarkers of periodontal disease.

Development of an Electrochemical Immunosensor for Specific Detection of Visceral Leishmaniasis Using Gold-Modified Screen-Printed Carbon Electrodes.

Visceral leishmaniasis is a reemerging neglected tropical disease with limitations for its diagnosis, including low concentration of antibodies in the serum of asymptomatic patients and cross-reactions. In this context, this work proposes an electrochemical immunosensor for the diagnosis of visceral leishmaniasis in a more sensitive way that is capable of avoiding cross-reaction with Chagas disease (CD). Crude Leishmania infantum antigens tested in the enzyme-linked immunosorbent assay (ELISA) were methodologically standardized to best engage to the sensor. The antibodies anti-Trypanosoma cruzi and anti-Leishmania sp. Present in serum from patients with diverse types of CD or leishmaniasis were chosen. A screen-printed carbon electrode modified with gold nanoparticles was the best platform to guarantee effective adsorption of all antigens so that the epitope of specific recognition for leishmaniasis occurred efficiently and without cross-reaction with the evaluated CD. The current peaks reduced linearly after the recognition, and still were able to notice the discrimination between different kinds of diseases (digestive, cardiac, undetermined Chagas/acute and visceral chronic leishmaniasis). Comparative analyses with ELISA were performed with the same groups, and a low specificity (44%) was verified due to cross-reactions (high number of false positives) on ELISA tests, while the proposed immunosensor presented high selectivity and specificity (100%) without any false positives or false negatives for the serum samples from isolated patients with different types of CD and visceral leishmaniasis. Furthermore, the biosensor was stable for 5 days and presented a detection limit of 200 ng mL-1.

Mast Cells and Basophils: From Malevolent Design to Coevolutionary Arms Race.

Mast cells and basophils are central to acquired resistance against blood-feeding arthropods which, in turn, counteract these cells by modulating their biological activities. The phenotypic exuberance displayed in this battlefield points to a reciprocal selective pressure suggesting a coevolutionary arms race that shapes both ectoparasites and vertebrate hosts.

TNF-α blockade impairs in vitro tuberculous granuloma formation and down modulate Th1, Th17 and Treg cytokines.

Tuberculosis (TB) is a granulomatous disease that has affected humanity for thousands of years. The production of cytokines, such as IFN-γ and TNF-α, is fundamental in the formation and maintenance of granulomas and in the control of the disease. Recently, the introduction of TNF-α-blocking monoclonal antibodies, such as Infliximab, has brought improvements in the treatment of patients with chronic inflammatory diseases, but this treatment also increases the risk of reactivation of latent tuberculosis. Our objective was to analyze, in an in vitro model, the influence of Infliximab on the granulomatous reactions and on the production of antigen-specific cytokines (TNF-α, IFN-γ, IL-12p40, IL-10 and IL-17) from beads sensitized with soluble Bacillus Calmette-Guérin (BCG) antigens cultured in the presence of peripheral blood mononuclear cells (PBMC) from TB patients. We evaluated 76 individuals, with tuberculosis active, treated and subjects with positive PPD. Granuloma formation was induced in the presence or absence of Infliximab for up to 10 days. The use of Infliximab in cultures significantly blocked TNF-α production (p <0.05), and led to significant changes in granuloma structure, in vitro, only in the treated TB group. On the other hand, there was a significant reduction in the levels of IFN-γ, IL-12p40, IL-10 and IL-17 after TNF-α blockade in the three experimental groups (p <0.05). Taken together, our results demonstrate that TNF-α blockade by Infliximab directly influenced the structure of granuloma only in the treated TB group, but negatively modulated the production of Th1, Th17 and regulatory T cytokines in the three groups analyzed.

An insight into the salivary gland and fat body transcriptome of Panstrongylus lignarius (Hemiptera: Heteroptera), the main vector of Chagas disease in Peru.

Triatomines are hematophagous arthropod vectors of Trypanosoma cruzi, the causative agent of Chagas Disease. Panstrongylus lignarius, also known as Panstrongylus herreri, is considered one of the most versatile triatomines because it can parasitize different hosts, it is found in different habitats and countries, it has sylvatic, peridomestic and domestic behavior and it is a very important vector of Chagas disease, especially in Peru. Molecules produced and secreted by salivary glands and fat body are considered of important adaptational value for triatomines because, among other functions, they subvert the host haemostatic, inflammatory and immune systems and detoxify or protect them against environmental aggressors. In this context, the elucidation of the molecules produced by these tissues is highly valuable to understanding the ability of this species to adapt and transmit pathogens. Here, we use high-throughput sequencing techniques to assemble and describe the coding sequences resulting from the transcriptome of the fat body and salivary glands of P. lignarius. The final assembly of both transcriptomes together resulted in a total of 11,507 coding sequences (CDS), which were mapped from a total of 164,676,091 reads. The CDS were subdivided according to their 10 folds overexpression on salivary glands (513 CDS) or fat body (2073 CDS). Among the families of proteins found in the salivary glands, lipocalins were the most abundant. Other ubiquitous families of proteins present in other sialomes were also present in P. lignarius, including serine protease inhibitors, apyrase and antigen-5. The unique transcriptome of fat body showed proteins related to the metabolic function of this organ. Remarkably, nearly 20% of all reads mapped to transcripts coded by Triatoma virus. The data presented in this study improve the understanding on triatomines' salivary glands and fat body function and reveal important molecules used in the interplay between vectors and vertebrate hosts.

Phytochemical characterization of the Vochysia rufa (Vochysiaceae) extract and its effects on oxidative stress in the pancreata of streptozotocin-induced diabetic rats.

Aqueous extract of macerated Vochysia rufa stem bark has been commonly used in the treatment of diabetes. Therefore, we evaluated the antihyperglycemic and antioxidant effects of an extract of V. rufa on the pancreata of streptozotocin (STZ)-induced diabetic rats. Animals received one of the following treatments daily by oral gavage: water (diabetic-control), V. rufa extract (diabetic-V. rufa), or glibenclamide (diabetic-GBD). Total antioxidant capacity; levels of thiobarbituric acid reactive substances, reduced glutathione, and sulfhydryls; and superoxide dismutase, catalase, and glutathione peroxidase (GPx) activities were measured in the pancreas. Biochemical analysis of serum total cholesterol and fractions, triglycerides, creatinine, urea, acid uric, ALP, γ-GT, AST, and ALT was performed, and pancreatic ß-cells positive for insulin were evaluated by immunohistochemistry. Rats treated with extract exhibited a decrease in fasting blood glucose compared with levels in diabetic control rats. GPx activity and sulfhydryl levels were significantly lower in diabetic-V. rufa rats compared with those of diabetic-control rats. V. rufa extract acted to normalize the biochemical alterations found in diabetic rats (diabetic-controls), as demonstrated by increases in urea, HDL, ALP, AST, and ALT. Reduction in blood glucose was independent of an increase in insulin. The V. rufa extract was found to be composed of free sugars (inositol, galactose, glucose, mannose, sucrose, arabinose, and ribose) as the main metabolites. Thus, aqueous extract of the stem bark of V. rufa is capable of reducing blood glucose, resulting in an antioxidant effect on the pancreatic tissue of STZ-diabetic rats.

Nucleosides present on phlebotomine saliva induce immunossuppression and promote the infection establishment.

BACKGROUND: Sand fly saliva plays a crucial role in establishing Leishmania infection. We identified adenosine (ADO) and adenosine monophosphate (AMP) as active pharmacologic compounds present in Phlebotomus papatasi saliva that inhibit dendritic cell (DC) functions through a PGE2/IL 10-dependent mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we prepared a mixture of ADO and AMP in equimolar amounts similar to those present in the salivary-gland extract (SGE) form one pair of salivary glands of P. papatasi and co-injected it with Leishmania amazonensis or L. major into mouse ears. ADO+AMP mimicked exacerbative effects of P. papatasi saliva in leishmaniasis, increasing parasite burden and cutaneous lesions. Enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect associated with IL-10 enhancement. Immunosuppressive factors COX2 and IL-10 were upregulated and failed to enhance ear lesion and parasite burden in IL 10-/- infected mice. Furthermore, nucleosides increased regulatory T cell (Treg) marker expression on CD4+CD25- cells, suggesting induction of Tregs on effector T cells (T eff). Treg induction (iTreg) was associated with nucleoside-induced tolerogenic dendritic cells (tDCs) expressing higher levels of COX2 and IL-10. In vitro generation of Tregs was more efficient in DCs treated with nucleosides. Suppressive effects of nucleosides during cutaneous leishmaniasis were mediated through an A2AR-dependent mechanism. Using BALB/c mice deficient in A2A ADO receptor (A2AR-/-), we showed that co-inoculated mice controlled infection, displaying lower parasite numbers at infection sites and reduced iTreg generation. CONCLUSION/SIGNIFICANCE: We have demonstrated that ADO and AMP in P. papatasi saliva mediate exacerbative effects of Leishmania infection by acting preferentially on DCs promoting a tolerogenic profile in DCs and by generating iTregs in inflammatory foci through an A2AR mechanism.

Plasmodium falciparum infection induces expression of a mosquito salivary protein (Agaphelin) that targets neutrophil function and inhibits thrombosis without impairing hemostasis.

BACKGROUND: Invasion of mosquito salivary glands (SGs) by Plasmodium falciparum sporozoites is an essential step in the malaria life cycle. How infection modulates gene expression, and affects hematophagy remains unclear. PRINCIPAL FINDINGS: Using Affimetrix chip microarray, we found that at least 43 genes are differentially expressed in the glands of Plasmodium falciparum-infected Anopheles gambiae mosquitoes. Among the upregulated genes, one codes for Agaphelin, a 58-amino acid protein containing a single Kazal domain with a Leu in the P1 position. Agaphelin displays high homology to orthologs present in Aedes sp and Culex sp salivary glands, indicating an evolutionarily expanded family. Kinetics and surface plasmon resonance experiments determined that chemically synthesized Agaphelin behaves as a slow and tight inhibitor of neutrophil elastase (K(D) ∼ 10 nM), but does not affect other enzymes, nor promotes vasodilation, or exhibit antimicrobial activity. TAXIscan chamber assay revealed that Agaphelin inhibits neutrophil chemotaxis toward fMLP, affecting several parameter associated with cell migration. In addition, Agaphelin reduces paw edema formation and accumulation of tissue myeloperoxidase triggered by injection of carrageenan in mice. Agaphelin also blocks elastase/cathepsin-mediated platelet aggregation, abrogates elastase-mediated cleavage of tissue factor pathway inhibitor, and attenuates neutrophil-induced coagulation. Notably, Agaphelin inhibits neutrophil extracellular traps (NETs) formation and prevents FeCl3-induced arterial thrombosis, without impairing hemostasis. CONCLUSIONS: Blockade of neutrophil elastase emerges as a novel antihemostatic mechanism in hematophagy; it also supports the notion that neutrophils and the innate immune response are targets for antithrombotic therapy. In addition, Agaphelin is the first antihemostatic whose expression is induced by Plasmodium sp infection. These results suggest that an important interplay takes place in parasite-vector-host interactions.

Influence of the CCR-5/MIP-1 α axis in the pathogenesis of Rocio virus encephalitis in a mouse model.

Rocio virus (ROCV) caused an outbreak of human encephalitis during the 1970s in Brazil and its immunopathogenesis remains poorly understood. CC-chemokine receptor 5 (CCR5) is a chemokine receptor that binds to macrophage inflammatory protein (MIP-1 α). Both molecules are associated with inflammatory cells migration during infections. In this study, we demonstrated the importance of the CCR5 and MIP-1 α, in the outcome of viral encephalitis of ROCV-infected mice. CCR5 and MIP-1 α knockout mice survived longer than wild-type (WT) ROCV-infected animals. In addition, knockout mice had reduced inflammation in the brain. Assessment of brain viral load showed mice virus detection five days post-infection in wild-type and CCR5-/- mice, while MIP-1 α-/- mice had lower viral loads seven days post-infection. Knockout mice required a higher lethal dose than wild-type mice as well. The CCR5/MIP-1 α axis may contribute to migration of infected cells to the brain and consequently affect the pathogenesis during ROCV infection.

The protein LJM 111 from Lutzomyia longipalpis salivary gland extract (SGE) accounts for the SGE-inhibitory effects upon inflammatory parameters in experimental arthritis model.

Several studies have pointed out the immunomodulatory properties of the Salivary Gland Extract (SGE) from Lutzomyia longipalpis. We aimed to identify the SGE component (s) responsible for its effect on ovalbumin (OVA)-induced neutrophil migration (NM) and to evaluate the effect of SGE and components in the antigen-induced arthritis (AIA) model. We tested the anti-arthritic activities of SGE and the recombinant LJM111 salivary protein (rLJM111) by measuring the mechanical hypernociception and the NM into synovial cavity. Furthermore, we measured IL-17, TNF-α and IFN-γ released by lymph nodes cells stimulated with mBSA or anti-CD3 using enzyme-linked immunosorbent assay (ELISA). Additionally, we tested the effect of SGE and rLJM111 on co-stimulatory molecules expression (MHC-II and CD-86) by flow cytometry, TNF-α and IL-10 production (ELISA) of bone marrow-derived dendritic cells (BMDCs) stimulated with LPS, chemotaxis and actin polymerization from neutrophils. Besides, the effect of SGE on CXCR2 and GRK-2 expression on neutrophils was investigated. We identified one plasmid expressing the protein LJM111 that prevented NM in OVA-challenged immunized mice. Furthermore, both SGE and rLJM111 inhibited NM and pain sensitivity in AIA and reduced IL-17, TNF-α and IFN-γ. SGE and rLJM111 also reduced MHC-II and CD-86 expression and TNF-α whereas increased IL-10 release by LPS-stimulated BMDCs. SGE, but not LJM 111, inhibited neutrophils chemotaxis and actin polymerization. Additionally, SGE reduced neutrophil CXCR2 expression and increased GRK-2. Thus, rLJM111 is partially responsible for SGE mechanisms by diminishing DC function and maturation but not chemoattraction of neutrophils.

Defibrotide interferes with several steps of the coagulation-inflammation cycle and exhibits therapeutic potential to treat severe malaria.

OBJECTIVE: The coagulation-inflammation cycle has been implicated as a critical component in malaria pathogenesis. Defibrotide (DF), a mixture of DNA aptamers, displays anticoagulant, anti-inflammatory, and endothelial cell (EC)-protective activities and has been successfully used to treat comatose children with veno-occlusive disease. DF was investigated here as a drug to treat cerebral malaria. METHODS AND RESULTS: DF blocks tissue factor expression by ECs incubated with parasitized red blood cells and attenuates prothrombinase activity, platelet aggregation, and complement activation. In contrast, it does not affect nitric oxide bioavailability. We also demonstrated that Plasmodium falciparum glycosylphosphatidylinositol (Pf-GPI) induces tissue factor expression in ECs and cytokine production by dendritic cells. Notably, dendritic cells, known to modulate coagulation and inflammation systemically, were identified as a novel target for DF. Accordingly, DF inhibits Toll-like receptor ligand-dependent dendritic cells activation by a mechanism that is blocked by adenosine receptor antagonist (8-p-sulfophenyltheophylline) but not reproduced by synthetic poly-A, -C, -T, and -G. These results imply that aptameric sequences and adenosine receptor mediate dendritic cells responses to the drug. DF also prevents rosetting formation, red blood cells invasion by P. falciparum and abolishes oocysts development in Anopheles gambiae. In a murine model of cerebral malaria, DF affected parasitemia, decreased IFN-γ levels, and ameliorated clinical score (day 5) with a trend for increased survival. CONCLUSION: Therapeutic use of DF in malaria is proposed.

Nucleosides from Phlebotomus papatasi salivary gland ameliorate murine collagen-induced arthritis by impairing dendritic cell functions.

Among several pharmacological compounds, Phlebotomine saliva contains substances with anti-inflammatory properties. In this article, we demonstrated the therapeutic activity of salivary gland extract (SGE) of Phlebotomus papatasi in an experimental model of arthritis (collagen-induced arthritis [CIA]) and identified the constituents responsible for such activity. Daily administration of SGE, initiated at disease onset, attenuated the severity of CIA, reducing the joint lesion and proinflammatory cytokine release. In vitro incubation of dendritic cells (DCs) with SGE limited specific CD4(+) Th17 cell response. We identified adenosine (ADO) and 5'AMP as the major salivary molecules responsible for anti-inflammatory activities. Pharmacologic inhibition of ADO A2(A) receptor or enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect. Importantly, CD73 (ecto-5'-nucleotidase enzyme) is expressed on DC surface during stage of activation, suggesting that ADO is also generated by 5'AMP metabolism. Moreover, both nucleosides mimicked SGE-induced anti-inflammatory activity upon DC function in vitro and attenuated establishment of CIA in vivo. We reveal that ADO and 5'AMP are present in pharmacological amounts in P. papatasi saliva and act preferentially on DC function, consequently reducing Th17 subset activation and suppressing the autoimmune response. Thus, it is plausible that these constituents might be promising therapeutic molecules to target immune inflammatory diseases.

Regulation of Trypanosoma cruzi-induced myocarditis by programmed death cell receptor 1.

Trypanosoma cruzi infection causes intense myocarditis, leading to cardiomyopathy and severe cardiac dysfunction. Protective adaptive immunity depends on balanced signaling through a T cell receptor and coreceptors expressed on the T cell surface. Such coreceptors can trigger stimulatory or inhibitory signals after binding to their ligands in antigen-presenting cells (APC). T. cruzi modulates the expression of coreceptors in lymphocytes after infection. Deregulated inflammation may be due to unbalanced expression of these molecules. Programmed death cell receptor 1 (PD-1) is a negative T cell coreceptor that has been associated with T cell anergy or exhaustion and persistent intracellular infections. We aimed to study the role of PD-1 during T. cruzi-induced acute myocarditis in mice. Cytometry assays showed that PD-1 and its ligands are strongly upregulated in lymphocytes and APC in response to T. cruzi infection in vivo and in vitro. Lymphocytes infiltrating the myocardium exhibited high levels of expression of these molecules. An increased cardiac inflammatory response was found in mice treated with blocking antibodies against PD-1, PD-L1, and to a lesser extent, PD-L2, compared to that found in mice treated with rat IgG. Similar results in PD-1(-/-) mice were obtained. Moreover, the PD-1 blockade/deficiency led to reduced parasitemia and tissue parasitism but increased mortality. These results suggest the participation of a PD-1 signaling pathway in the control of acute myocarditis induced by T. cruzi and provide additional insight into the regulatory mechanisms in the pathogenesis of Chagas' disease.

A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation.

Platelet aggregation and acute inflammation are key processes in vertebrate defense to a skin injury. Recent studies uncovered the mediation of 2 serine proteases, cathepsin G and chymase, in both mechanisms. Working with a mouse model of acute inflammation, we revealed that an exogenous salivary protein of Ixodes ricinus, the vector of Lyme disease pathogens in Europe, extensively inhibits edema formation and influx of neutrophils in the inflamed tissue. We named this tick salivary gland secreted effector as I ricinus serpin-2 (IRS-2), and we show that it primarily inhibits cathepsin G and chymase, while in higher molar excess, it affects thrombin activity as well. The inhibitory specificity was explained using the crystal structure, determined at a resolution of 1.8 Å. Moreover, we disclosed the ability of IRS-2 to inhibit cathepsin G-induced and thrombin-induced platelet aggregation. For the first time, an ectoparasite protein is shown to exhibit such pharmacological effects and target specificity. The stringent specificity and biological activities of IRS-2 combined with the knowledge of its structure can be the basis for the development of future pharmaceutical applications.

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.

Neospora caninum excreted/secreted antigens trigger CC-chemokine receptor 5-dependent cell migration.

Neospora caninum, the causative agent of neosporosis, is an obligate intracellular parasite considered to be a major cause of abortion in cattle throughout the world. Most studies concerning N. caninum have focused on life cycle, seroepidemiology, pathology and vaccination, while data on host-parasite interaction, such as host cell migration, mechanisms of evasion and dissemination of this parasite during the early phase of infection are still poorly understood. Here we show the ability of excreted/secreted antigens from N. caninum (NcESAs) to attract monocytic cells to the site of primary infection in both in vitro and in vivo assays. Molecules from the family of cyclophilins present on the NcESAs were shown to work as chemokine-like proteins and NcESA-induced chemoattraction involved G(i) protein signaling and participation of CC-chemokine receptor 5 (CCR5). Additionally, we demonstrate the ability of NcESAs to enhance the expression of CCR5 on monocytic cells and this increase occurred in parallel with the chemotactic activity of NcESAs by increasing cell migration. These results suggest that during the first days of infection, N. caninum produces molecules capable of inducing monocytic cell migration to the sites of infection, which will consequently enhance initial parasite invasion and proliferation. Altogether, these results help to clarify some key features involved in the process of cell migration and may reveal virulence factors and therapeutic targets to control neosporosis.