Antigen production using heterologous expression of dengue virus-2 non-structural protein 1 (NS1) in Nicotiana tabacum (Havana) for immunodiagnostic purposes.

KEY MESSAGE: Expression of dengue-2 virus NS1 protein in Nicotiana tabacum plants for development of dengue immunodiagnostic kits. Dengue is one of the most important diseases caused by arboviruses in the world. A significant increase in its geographical distribution has been noticed over the last 20 years, with continuous transmission of several serotypes and emergence of the hemorrhagic fever in areas where the disease was previously not prevalent. Although the methodological processes for dengue diagnosis are in deep development and improvement, a limitation for the realization of dengue diagnostic tests is the difficulty of large-scale production of the antigen to be used in diagnostic tests. Due to this demand, the purpose of this study was to obtain the non-structural protein 1 (NS1) from dengue-2 serotype by heterologous expression in Nicotiana tabacum (Havana). After confirmation of the NS1 protein gene integration in the plant genome, the heterologous protein was characterized using SDS-PAGE and immunoblotting. In an immunoenzymatic test, the recombinant NS1 protein presents an antigen potential for development of dengue immunodiagnostic kits.

Dengue-1 envelope protein domain III produced in Pichia pastoris: potential use for serological diagnosis.

Dengue is a major international public health concern. There is no drug to treat dengue virus infections and a vaccine is yet to be licensed. The laboratory diagnosis of dengue virus infection has been greatly improved during the last decade; therefore, the main limiting factor is the production of recombinant viral antigens on a large scale. Domain III of dengue virus envelope protein contains multiplex conformation-dependent neutralizing epitopes, making it an attractive diagnostic candidate. In this work, we have demonstrated the expression of dengue virus type 1 envelope domain III protein (EDIII-D1) in methylotrophic yeast, Pichia pastoris GS115. The recombinant secreted protein (sEDIII-D1) was purified by affinity chromatography and characterized by SDS-PAGE. Purified protein was recognized in immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) with dengue-infected human serum samples. In conclusion, secreted expressions of domain III protein can be obtained in P. pastoris by methanol induction. This product has the potential to be used for the diagnosis of dengue infections.

Purinergic Antagonist Suramin Aggravates Myocarditis and Increases Mortality by Enhancing Parasitism, Inflammation, and Reactive Tissue Damage in Trypanosoma cruzi-Infected Mice.

Suramin (Sur) acts as an ecto-NTPDase inhibitor in Trypanosoma cruzi and a P2-purinoceptor antagonist in mammalian cells. Although the potent antitrypanosomal effect of Sur has been shown in vitro, limited evidence in vivo suggests that this drug can be dangerous to T. cruzi-infected hosts. Therefore, we investigated the dose-dependent effect of Sur-based chemotherapy in a murine model of Chagas disease. Seventy uninfected and T. cruzi-infected male C57BL/6 mice were randomized into five groups: SAL = uninfected; INF = infected; SR5, SR10, and SR20 = infected treated with 5, 10, or 20 mg/kg Sur. In addition to its effect on blood and heart parasitism, the impact of Sur-based chemotherapy on leucocytes myocardial infiltration, cytokine levels, antioxidant defenses, reactive tissue damage, and mortality was analyzed. Our results indicated that animals treated with 10 and 20 mg/kg Sur were disproportionally susceptible to T. cruzi, exhibiting increased parasitemia and cardiac parasitism (amastigote nests and parasite load (T. cruzi DNA)), intense protein, lipid and DNA oxidation, marked myocarditis, and mortality. Animals treated with Sur also exhibited reduced levels of nonprotein antioxidants. However, the upregulation of catalase, superoxide dismutase, and glutathione-S-transferase was insufficient to counteract reactive tissue damage and pathological myocardial remodeling. It is still poorly understood whether Sur exerts a negative impact on the purinergic signaling of T. cruzi-infected host cells. However, our findings clearly demonstrated that through enhanced parasitism, inflammation, and reactive tissue damage, Sur-based chemotherapy contributes to aggravating myocarditis and increasing mortality rates in T. cruzi-infected mice, contradicting the supposed relevance attributed to this drug for the treatment of Chagas disease.

Synergism of Plant Compound With Traditional Antimicrobials Against Streptococcus spp. Isolated From Bovine Mastitis.

Mastitis is an inflammation of the mammary gland that causes major losses in the dairy industry. Streptococcus spp. are among the main agents of this disease. Increased resistance to antibiotics is one of the causes of therapeutic failure. Plants, due to their broad chemodiversity, are an interesting source of new molecules with antibacterial activity. Using these compounds along with traditional antibiotics is a possible method for reversing resistance. The objective of this work was to determine the interactions between the activities of guttiferone-A and 7-epiclusianone, two active substances isolated from the fruits of Garcinia brasiliensis, and traditional antibiotics against Streptococcus spp. isolated from bovine mastitis and known to be resistant to them. First, the MIC for the antibiotics and bioactive compounds was determined, followed by their activities, alone and in combination. Then, their cytotoxicity was measured in bovine mammary epithelial cells. Finally, molecular docking simulations were performed to elucidate molecular details of the interactions between ß-lactamase and the compounds binding to it (clavulanic acid, ampicillin, 7-epiclusianone, and guttiferone-A). The bacterial isolates were resistant to ampicillin and gentamicin. Both antibiotics showed predominantly synergistic antibacterial activities in combination with guttiferone-A or 7-epiclusianone. These two active substances were not cytotoxic at synergistic concentrations and both showed strong binding to ß-lactamase, which may explain the reversal of ampicillin resistance. These substances are promising for the treatment of bovine mastitis.

A new TRAF-like protein from B. oleracea ssp. botrytis with lectin activity and its effect on macrophages.

Lectins are involved in a wide range of biological mechanisms, like immunomodulatory agent able to activate the innate immunity. In this study, we purified and characterized a new lectin from cauliflower (Brassica oleracea ssp. botrytis - BOL) by three sequential chromatographic steps and confirmed the purity by SDS-PAGE. Additionally, we evaluated the role of the lectin in innate immunity by a phagocytosis assay, production of H2O2 and NO. BOL was characterized like a non-glycosylated protein that showed a molecular mass of ∼34kDa in SDS-PAGE. Its N-terminal sequence (ETRAFREERPSSKIVTIAG) did not reveal any similarity to the other lectins; nevertheless, it showed 100% homology to a putative TRAF-like protein from Brassica rapa and Brassica napus. This is a first report of the TRAF-protein with lectinic activity. The BOL retained its complete hemagglutination activity from 4°C up to 60°C, with stability being more apparent between pH 7.0 and 8.0. Moreover, the lectin was able to stimulate phagocytosis and induce the production of H2O2 and NO. Therefore, BOL can be explored as an immunomodulatory agent by being able to activate the innate immunity and favor antigen removal.

Vaccination of Mice with Virulence-Associated Protein G (VapG) Antigen Confers Partial Protection against Rhodococcus equi Infection through Induced Humoral Immunity.

Rhodococcus equi is a facultative intracellular bacterium causing severe pyogranulomatous pneumonia, ulcerative enterocolitis, and mesenteric lymphadenopathy in foals aged less than 6 months. Less frequently, this pathogen affects various other species, such as pigs, cattle, cats, and even humans. Although rhodococcosis is treated with a combination of antimicrobial agents, resistance is developed in some cases, and thus, antimicrobial susceptibility must be monitored and managed. Considering these limitations of the current therapy and unavailability of a vaccine to prevent the disease, research is particularly focused on the development of an effective vaccine against rhodococcosis. Most vaccines undergoing development utilize the virulence-associated protein (Vap) A antigen, which was identified previously as a key virulence factor of R. equi. Nevertheless, other proteins, such as VapG, present in most virulent R. equi strains, are also encoded by vap genes located on the R. equi bacterial virulence plasmid. In the present study, we evaluated the effect of VapG immunization on the survival of R. equi-challenged mice. We used attenuated Salmonella as a carrier for VapG (Salmonella-vapG+), a procedure previously adopted to develop a VapA-based vaccine. We observed that vaccination with Salmonella-vapG+ induced both an increased IFN-γ, IL-12, and TNF-α production, and a decreased bacterial burden in organs of the R. equi-challenged mice. Nevertheless, Salmonella-vapG+ vaccination protected only 50% of the mice challenged with a lethal dose of R. equi. Interestingly, we observed an increased frequency of B cells in the spleen of Salmonella-vapG+-vaccinated mice and showed that Salmonella-vapG+-vaccinated R. equi-challenged B-cell-knockout mice did not reduce the bacterial burden. Given these results, we discussed the potential role of the humoral immune response induced by Salmonella-vapG+ vaccination in conferring protection against R. equi infection, as well as the employment of VapG antigen for obtaining hyperimmune plasma to prevent rhodoccocosis in young foals.

Modulation of oxidative and inflammatory cardiac response by nonselective 1- and 2-cyclooxygenase inhibitor and benznidazole in mice.

OBJECTIVES: This study investigated the combined effects of benznidazole (BZ) and ibuprofen (IB) on the oxidative and inflammatory status of the cardiac tissue in vivo. METHODS: Swiss mice were randomized in groups receiving BZ (100 mg/kg) and IB (400 mg/kg) alone or combined (BZ + IB 200 or 400 mg/kg). Control animals were concurrently treated with 1% carboxymethyl cellulose. All treatments were administered orally for 7 days. KEY FINDINGS: BZ treatment increased cardiac production of nitrogen/oxygen-reactive species, malondialdeyde, carbonyl proteins, prostaglandins as well as the activities of catalase, superoxide dismutase and glutathione peroxidase. These parameters were attenuated by IB, with the best results at higher dose. Individually, BZ and IB significantly reduced the tissue levels of chemokine ligand 2, tumour necrosis factor-α and IL-10, but no reduction was observed when the treatments were combined. CONCLUSIONS: BZ triggers an oxidative and nitrosative route, which is associated with increased prostaglandin synthesis and marked damages to the lipids and proteins of the cardiac tissue. IB treatment attenuated reactive stresses triggered by BZ, which was an independent effects of this drug on the endogenous antioxidant enzymes. Individually, but not together, BZ and IB reduced the cardiac inflammatory status, indicating a beneficial and complex drug interaction.

Identification and characterization of an antimicrobial peptide of Hypsiboas semilineatus (Spix, 1824) (Amphibia, Hylidae).

The multidrug-resistant bacteria have become a serious problem to public health. In this scenery the antimicrobial peptides (AMPs) derived from animals and plants emerge as a novel therapeutic modality, substituting or in addition to the conventional antimicrobial. The anurans are one of the richest natural sources of AMPs. In this work several cycles of cDNA cloning of the skin of the Brazilian treefrog Hypsiboas semilineatus led to isolation of a precursor sequence that encodes a new AMP. The sequence comprises a 27 residue signal peptide, followed by an acidic intervening sequence that ends in the mature peptide at the carboxy terminal. The AMP, named Hs-1, has 20 amino acids residues, mostly arranged in an alpha helix and with a molecular weight of 2144.6 Da. The chemically synthesized Hs-1 showed an antimicrobial activity against all Gram-positive bacteria tested, with a range of 11-46 µM, but it did not show any effect against Gram-negative bacteria, which suggest that Hs-1 may have a selective action for Gram-positive bacteria. The effects of Hs-1 on bacterial cells were also demonstrated by transmission electron microscopy. Hs-1 is the first AMP to be described from H. semilineatus.

Vaccination of mice with salmonella expressing VapA: mucosal and systemic Th1 responses provide protection against Rhodococcus equi infection.

Conventional vaccines to prevent the pneumonia caused by Rhodococcus equi have not been successful. We have recently demonstrated that immunization with Salmonella enterica Typhimurium expressing the VapA antigen protects mice against R. equi infection. We now report that oral vaccination of mice with this recombinant strain results in high and persistent fecal levels of antigen-specific IgA, and specific proliferation of the spleen cells of immunized mice in response to the in vitro stimulation with R. equi antigen. After in vitro stimulation, spleen cells of immunized mice produce high levels of Th1 cytokines and show a prominent mRNA expression of the Th1 transcription factor T-bet, in detriment of the Th2 transcription factor GATA-3. Following R. equi challenge, a high H2O2, NO, IL-12, and IFN-gamma content is detected in the organs of immunized mice. On the other hand, TNF-alpha and IL-4 levels are markedly lower in the organs of vaccinated mice, compared with the non-vaccinated ones. The IL-10 content and the mRNA transcription level of TGF-beta are also higher in the organs of immunized mice. A greater incidence of CD4+ and CD8+ T cells and B lymphocytes is verified in vaccinated mice. However, there is no difference between vaccinated and non-vaccinated mice in terms of the frequency of CD4+CD25+Foxp3+ T cells. Finally, we show that the vaccination confers a long-term protection against R. equi infection. Altogether, these data indicate that the oral vaccination of mice with S. enterica Typhimurium expressing VapA induces specific and long-lasting humoral and cellular responses against the pathogen, which are appropriately regulated and allow tissue integrity after challenge.

Synthesis and "in Vitro" Trypanocidal Activity Evaluation of Some Organo-iron Compounds.

Eight organo-iron ferrocene derivatives and arenocenium salts were prepared and evaluated by "in vitro" assay against one strain of Trypanosoma cruzi (Y). Six of the eight organo-iron compounds assayed, piperazinium diferrocenoate 1, eta(6)-(o-xylene)-eta(5)-(cyclopentadienyl) Iron(II) hexafluorophosphate 3, eta(6)-(mesitylene)-eta(5)-(cyclopentadienyl) iron(II) hexafluorphosphate 5, eta(6)-(durene)-eta(5)-(cyclopentadienyl) iron(II) hexafluorphosphate 6, eta(6)-(rho-chlorotoluene)-eta(5)-(cyclopentadienyl) Iron(II) hexafluorphosphate 7 and eta(6)-(chlorobenzene)-eta(5)-(cyclopentadienyl) iron(II) picrate 8 , were poorly active in the "in vitro" assays. Only two compounds 1,1'-(N-pyperidinocarbonyl) ferrocene 2(IC(50)=2.4 mug/mL) and eta(6)-(o-xylene)-eta(5)(cyclopentadienyl) iron(II) picrate 4 (IC(50)=12.08 mug/mL), were more active. Thus, some of the compounds are promising to be used against Chagas' disease as a prophylactic agents.