Subcutaneous, Oral, and Intranasal Immunization of BALB/c Mice with Leishmania infantum K39 Antigen Induces Non-Protective Humoral Immune Response.

Visceral leishmaniasis is a high-burden disease caused by parasites of the Leishmania genus. The K39 kinesin is a highly antigenic protein of Leishmania infantum, but little is known about the immune response elicited by this antigen. We evaluated the humoral immune response of female BALB/c mice (n = 6) immunized with the rK39-HFBI construct, formed by the fusion of the K39 antigen to a hydrophobin partner. The rK39-HFBI construct was administered through subcutaneous, oral, and intranasal routes using saponin as an adjuvant. We analyzed the kinetics of IgG, IgG1, and IgG2a production. The groups were then challenged by an intravenous infection with L. infantum promastigote cells. The rK39-HFBI antigen-induced high levels of total IgG (p < 0.05) in all groups, but only the subcutaneous route was associated with increased production of IgG1 and IgG2a 42 days after immunization (p < 0.05), suggesting a potential secondary immune response following the booster dose. There was no reduction in the splenic parasite load; thus, the rK39-HFBI failed to protect the mice against infection under the tested conditions. The results presented here demonstrate that the high antigenicity of the K39 antigen does not contribute to a protective immune response against visceral leishmaniasis.

Codium isthmocladum lectin 1 (CiL-1): Interaction with N-glycans explains antinociceptive and anti-inflammatory activities in adult zebrafish (Danio rerio).

Inflammation and oxidative stress are processes associated with different human diseases. They are treated using drugs that have several side effects. Seaweed are sources of potentially relevant natural compounds for use as treatment of these disorders. Lectins are able to reversibly interact with complex carbohydrates and modulate cell membrane glycosylated receptors through this interaction. This study aimed to determine the antinociceptive and anti-inflammatory potential of CiL-1 in adult zebrafish by modulation of TRPA1 through lectin-glycan binding. Possible neuromodulation by TRPA1 channel was also evaluated by camphor pretreatment. CiL-1 was efficacious at all tested doses, revealing anti-nociceptive and anti-inflammatory effects in adult zebrafish. This galactose-binding lectin was also able to reduce the content of ROS in brain and liver. In silico analyses showed CiL-1 interactions with both ligands tested. LacNac2 presents the most favorable binding energy with the protein. The interaction occurs at 4 subsites as an extended conformation at the site. LacNac2-Sia had a less favorable curved-shape interaction energy. Based on the predictions made for the oligosaccharides, a tetra-antenate putative glycan was schematically constructed, illustrating an interaction between TRPA1 N-glycan and CiL-1. This binding seems to be related to CiL-1 anti-inflammatory activity as result of receptor modulation.

A review of plant-based expression systems as a platform for single-domain recombinant antibody production.

Monoclonal antibodies have contributed to improving the treatment of several diseases. However, limitations related to pharmacokinetic parameters and production costs have instigated the search for alternative products. Camelids produce functional immunoglobulins G devoid of light chains and CH1 domains, in which the antigenic recognition site is formed by a single domain called VHH or nanobody. VHHs' small size and similarity to the human VH domain contribute to high tissue penetration and low immunogenicity. In addition, VHHs provide superior antigen recognition compared to human antibodies, better solubility and stability. Due to these characteristics and the possibility of obtaining gene-encoding VHHs, applications of this biological tool, whether as a monomer or in related recombinant constructs, have been reported. To ensure antibody efficacy and cost-effectiveness, strategies for their expression, either using prokaryotic or eukaryotic systems, have been utilized. Plant-based expression systems are useful for VHH related constructs that require post-translational modifications. This system has exhibited versatility, low-cost upstream production, and safety. This article presents the main advances associated to the heterologous expression of VHHs in plant systems. Besides, we show insights related to the use of VHHs as a strategy for plant pathogen control and a tool for genomic manipulation in plant systems.

Hypolipidemic and reduced nitrergic effects of p-hydroxycinnamic diesters extracted from Copernicia prunifera in mice challenged by a high-fat diet.

Dyslipidemia is a chronic non-transmissible condition that has increased due to an unhealthy lifestyle. Statins have been used as the standard treatment to control hyperlipidemia. However, side effects and high costs may be associated with its prolonged treatment, so plants derivatives have been an attractive therapy to overcome these problems. Among the compounds extracted from plants, the p-hydroxycinnamic diesters (HCE), present in carnauba wax (CW), have been found with good pharmacological properties. Therefore, this study aimed to evaluate the potential anti-hypercholesterolemic and possible toxicological effects of HCE in C57BL/6J mice under a high-fat (HF) diet. Male C57BL/6J mice were fed during 60 days under the HF diet and therefore were either treated with HCE (200 and 400 mg/kg) or simvastatin (20 mg/kg) or received saline (controls) by gavage for 30 days under the same diet. HCE treatment was able to reduce serum total cholesterol and LDL levels. Besides, this compound increased liver X receptor (LXR) and but not significantly affected IL-1ß and TNF-α liver mRNA transcription activity. In conclusion, HCE treatment was found safe and may attenuate the deleterious effects of dyslipidemia due to chronic feeding with western diets.

Orally hypoglycemic activity of an insulin mimetic glycoprotein isolated from Cnidoscolus quercifolius Pohl. (Euphorbiaceae) seeds, Cq-IMP.

The genus Cnidoscolus (Euphorbiaceae) is widely distributed in tropical areas. In the Northeast of Brazil, the species C. quercifolius is endemic and has been used in traditional medicine. In this study, a novel protein was isolated from C. quercifolius seeds and characterized by its molecular weight, primary structure, isoelectric point (pI), and carbohydrate content. The hypoglycemic activity of this protein was investigated by in vitro assay with the RIN-5F glucose-responsive cell line and in vivo test using alloxan-induced diabetic mice models. In addition, safe use of the protein was also investigated by cytotoxicity, hemagglutinating, and immunogenicity assays. The protein which was named Cq-IMP (Cnidoscolus quercifolius - Insulin Mimetic Protein) showed a single 11.18 KDa glycopolypeptide chain (16.4% of carbohydrates, m/m), pI of 8.0 and N-terminal sequence (TKDPELKQcKKQQKKqQQYDDDDKK) with similarity around 46-62% to sucrose binding protein-like and vicilin-like protein that was confirmed by mass spectrometry tryptic peptides analysis. Besides that, Cq-IMP presented anti-insulin antibody cross-reactivity as hypoglycemic activity in both in vitro and in vivo models. Additionally, it did not present any toxicity by methods tested. In conclusion, Cq-IMP is an insulin-mimetic protein, with a potent hypoglycemic activity and no toxicity showing great potential for therapeutic applications and drug development.

p-Methoxycinnamic Acid Diesters Lower Dyslipidemia, Liver Oxidative Stress and Toxicity in High-Fat Diet Fed Mice and Human Peripheral Blood Lymphocytes.

The pursuit of cholesterol lowering natural products with less side effects is needed for controlling dyslipidemia and reducing the increasing toll of cardiovascular diseases that are associated with morbidity and mortality worldwide. The present study aimed at the examining effects of p-methoxycinnamic acid diesters (PCO-C) from carnauba (Copernicia prunifera)-derived wax on cytotoxic, genotoxic responses in vitro and on dyslipidemia and liver oxidative stress in vivo, utilizing high-fat diet (HFD) chronically fed Swiss mice. In addition, we evaluated the effect of PCO-C on the expression of key cholesterol metabolism-related genes, as well as the structural interactions between PCO-C and lecithin-cholesterol acyl transferase (LCAT) in silico. Oral treatment with PCO-C was able to reduce total serum cholesterol and low-density lipoprotein (LDL) levels following HFD. In addition, PCO-C reduced excessive weight gain and lipid peroxidation, and increased the gene expression of LCAT following HFD. Furthermore, the high affinity of the studied compound (ΔG: -8.78 Kcal/mol) towards the active sites of mutant LCAT owing to hydrophobic and van der Waals interactions was confirmed using bioinformatics. PCO-C showed no evidence of renal and hepatic toxicity, unlike simvastatin, that elevated aspartate aminotransferase (AST) levels, a marker of liver dysfunction. Finally, PCO-C showed no cytotoxicity or genotoxicity towards human peripheral blood lymphocytes in vitro. Our results suggest that PCO-C exerts hypocholesterolemic effects. The safety of PCO-C in the toxicological tests performed and the reports of its beneficial biological effects render this a promising compound for the development of new cholesterol-lowering therapeutics to control dyslipidemia. More work is needed for further elucidating PCO-C role on lipid metabolism to support future clinical studies.

Ethnobotanic, phytochemical uses and ethnopharmacological profile of genus Cnidoscolus spp. (Euphorbiaceae): A comprehensive overview.

The application of medicinal plants are the most important biotechnological alternative in the treatment of numerous diseases, especially in developing countries, such as Brazil. Among them, we specified some specimens of the genus Cnidoscolus used as phytotherapies, with healing properties, analgesic, anti-inflammatory, antibiotic and diuretic, anticancer, among others. Such effects are possibly associated with the presence of terpenoids, alkaloids, coumarins, flavonoids phenolic compounds, among others. Thus, the objective of this work was to evaluate in the literature the studies on the phytochemical, ethnopharmacological and biotechnological applications of this genus, from 1998 to 2017. Among the sixty-one studies reported in this review, ten species are popularly utilized to pharmacological and/or biotechnological applications. Cnidoscolus aconitifolius and Cnidoscolus chayamansa are the most cited species, which were also supported by either animal or cellular investigations indicating some beneficial pharmacological actions like antioxidant, anti-inflammatory and potential cytotoxic activity. The plant parts of this genus under study are important as sources for the isolation and identification of bioactive molecules with biotechnological applications, among the many diseases treated with this phytotherapy. Given these verdicts, ethnopharmacological approaches are significant systematic tools in the determination of plant species that exhibit medicinal and nutritional purposes. The results presented here should further stimulate the development of validation studies to ensure the safe and effective use of these plant species.