Evaluation of an Ionic Calcium Fiber Supplement and Its Impact on Bone Health Preservation in a Dietary Calcium Deficiency Mice Model.

Ionic calcium can help in the prevention of the process of osseous decalcification. This study aimed to evaluate the physicochemical properties and toxic effects of ionic calcium-fiber supplement (ICa+) and its impact on bone health preservation in mice C57/BL6 fed a calcium-deficient diet. Physicochemical properties include FTIR, apparent calcium solubility estimated by the calcium ratio obtained by ionization chromatography and atomic absorption. In vitro genotoxicity and cytotoxicity of the ICa+ were assessed. Twenty-five 7-week-old C57/BL6 mice were fed calcium-free diet (CFD) or CFD plus CaCO3 (1.33 mg Ca) or CFD plus ICa+ (1.33-6.66 mg Ca) for six weeks. After that, bone mass and microstructure parameters were assessed. Histological staining was performed to determine calcium deposits. ICa+ (100%) exhibited an apparent calcium solubility higher than CaCO3 (12.3%). ICa+ showed no cytotoxic and genotoxic in vitro activities. Histomorphometry analysis showed that the ICa+ treated group displayed a higher trabecular number than the trabecular space. Also, the ratio BV/TV was increased compared with all treatments. Ionic calcium-fiber supplementation prevents bone deterioration compared to mice fed a calcium-deficient diet.

Green Metallic Nanoparticles for Cancer Therapy: Evaluation Models and Cancer Applications.

Metal-based nanoparticles are widely used to deliver bioactive molecules and drugs to improve cancer therapy. Several research works have highlighted the synthesis of gold and silver nanoparticles by green chemistry, using biological entities to minimize the use of solvents and control their physicochemical and biological properties. Recent advances in evaluating the anticancer effect of green biogenic Au and Ag nanoparticles are mainly focused on the use of conventional 2D cell culture and in vivo murine models that allow determination of the half-maximal inhibitory concentration, a critical parameter to move forward clinical trials. However, the interaction between nanoparticles and the tumor microenvironment is not yet fully understood. Therefore, it is necessary to develop more human-like evaluation models or to improve the existing ones for a better understanding of the molecular bases of cancer. This review provides recent advances in biosynthesized Au and Ag nanoparticles for seven of the most common and relevant cancers and their biological assessment. In addition, it provides a general idea of the in silico, in vitro, ex vivo, and in vivo models used for the anticancer evaluation of green biogenic metal-based nanoparticles.

Immunoprotection evaluation of the recombinant N-terminal domain of Cys-loop receptors against Rhipicephalus (Boophilus) microplus tick infestation.

Rhipicephalus (Boophilus) microplus ticks are obligatory hematophagous ectoparasites of cattle and act as vectors for disease-causing microorganisms. Conventional tick control is based on the application of chemical acaricides; however, their uncontrolled use has increased resistant tick populations, as well as food and environmental contamination. Alternative immunological tick control has shown to be partially effective. Therefore, there is a need to characterize novel antigens in order to improve immunological protection. The aim of this work was to evaluate Cys-loop receptors as vaccine candidates. N-terminal domains of a glutamate receptor and of a glycine-like receptor were recombinantly produced in Escherichia coli. Groups of BALB/c mice were independently immunized with four doses of each recombinant protein emulsified with Freund's adjuvant. Both vaccine candidates were immunogenic in mice as demonstrated by western blot analysis. Next, recombinant proteins were independently formulated with the adjuvant Montanide ISA 50 V2 and evaluated in cattle infested with Rhipicephalus microplus tick larvae. Groups of three European crossbred calves were immunized with three doses of each adjuvanted protein. ELISA test was used to evaluate the IgG immune response elicited against the recombinant proteins. Results showed that vaccine candidates generated a moderate humoral response on vaccinated cattle. Vaccination significantly affected the number of engorged adult female ticks, having no significant effects on tick weight, egg weight and egg fertility values. Vaccine efficacies of 33% and 25% were calculated for the glutamate receptor and the glycine-like receptor, respectively.

TITLE: Évaluation de l'immunoprotection du domaine N-terminal recombinant des récepteurs Cys-loop contre l'infestation par les tiques Rhipicephalus (Boophilus) microplus. ABSTRACT: Les tiques Rhipicephalus (Boophilus) microplus sont des ectoparasites hématophages obligatoires des bovins et agissent comme vecteurs de micro-organismes pathogènes. Le contrôle conventionnel des tiques est basé sur l'application d'acaricides chimiques, mais leur utilisation incontrôlée a augmenté les populations de tiques résistantes ainsi que la contamination des aliments et de l'environnement. Le contrôle immunologique alternatif des tiques s'est avéré partiellement efficace. Par conséquent, il est nécessaire de caractériser de nouveaux antigènes afin d'améliorer la protection immunologique. Le but de ce travail était d'évaluer les récepteurs Cys-loop comme candidats vaccins. Les domaines N-terminaux d'un récepteur du glutamate et d'un récepteur de type glycine ont été produits par recombinaison chez Escherichia coli. Des groupes de souris BALB/c ont été immunisés indépendamment avec quatre doses de chaque protéine recombinante émulsionnée avec l'adjuvant de Freund. Les deux vaccins candidats étaient immunogènes chez la souris, comme l'a démontré l'analyse par transfert Western. Ensuite, des protéines recombinantes ont été formulées indépendamment avec l'adjuvant Montanide ISA 50 V2 et évaluées chez des bovins infestés de larves de tiques Rhipicephalus microplus. Des groupes de trois veaux croisés européens ont été immunisés avec trois doses de chaque protéine avec adjuvant. Le test ELISA a été utilisé pour évaluer la réponse immunitaire IgG induite contre les protéines recombinantes. Les résultats ont montré que les candidats vaccins généraient une réponse humorale modérée sur les bovins vaccinés. La vaccination a affecté de manière significative le nombre de tiques femelles adultes engorgées mais n'a eu aucun effet significatif sur le poids des tiques, le poids des œufs et les valeurs de fertilité des œufs. Des efficacités vaccinales de 33 % et 25 % ont été calculées pour le récepteur du glutamate et le récepteur de type glycine, respectivement.

Self-assembly of hexahistidine-tagged tobacco etch virus capsid protein into microfilaments that induce IgG2-specific response against a soluble porcine reproductive and respiratory syndrome virus chimeric protein.

BACKGROUND: Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. FINDINGS: Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. CONCLUSIONS: His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-µm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.

Identification of immunogenic proteins from ovarian tissue and recognized in larval extracts of Rhipicephalus (Boophilus) microplus, through an immunoproteomic approach.

Rhipicephalus (Boophilus) microplus ticks are obligatory hematophagous ectoparasites of cattle and act as vectors for disease-causing microorganisms. Conventional tick control is based on the use of chemical acaricides; however, their uncontrolled use has increased tSresistant tick populations, as well as food and environmental contamination. Alternative immunological tick control has shown to be partially effective. The only anti-tick vaccine commercially available at present in the world is based on intestinal Bm86 protein, and shows a variable effectiveness depending on tick strains or geographic isolates. Therefore, there is a need to characterize new antigens in order to improve immunological protection. The aim of this work was to identify immunogenic proteins from ovarian tissue extracts of R. microplus, after cattle immunization. Results showed that ovarian proteins complexed with the adjuvant Montanide ISA 50 V generated a strong humoral response on vaccinated cattle. IgG levels peaked at fourth post-immunization week and remained high until the end of the experiment. 1D and 2D SDS-PAGE-Western blot assays with sera from immunized cattle recognized several ovarian proteins. Reactive bands were cut and analyzed by LC-MS/MS. They were identified as Vitellogenin, Vitellogenin-2 precursor and Yolk Cathepsin. Our findings along with bioinformatic analysis indicate that R. microplus has several Vitellogenin members, which are proteolytically processed to generate multiple polypeptide fragments. This apparent complexity of vitellogenic tick molecular targets gives the opportunity to explore their potential usefulness as vaccine candidates but, at the same time, imposes a challenge on the selection of the appropriate set of antigens.

Virus-like particles from Escherichia Coli-derived untagged papaya ringspot virus capsid protein purified by immobilized metal affinity chromatography enhance the antibody response against a soluble antigen.

There is a growing interest in using virus-like particles (VLPs) as scaffolds for the presentation of antigens of choice to the immune system. In this work, VLPs from papaya ringspot virus capsid protein expressed in Escherichia coli were evaluated as enhancers of antibody response against a soluble antigen. Interestingly, although the capsid protein lacks a histidine tag, its purification by immobilized metal affinity chromatography was achieved. The formation of VLPs was demonstrated by electron microscopy for the first time for this capsid protein. VLPs were enriched by polyethylene glycol precipitation. Additionally, these VLPs were chemically coupled to green fluorescent protein in order to evaluate them as antigen carriers; however, bioconjugate instability was observed. Nonetheless, the adjuvant effect of these VLPs on BALB/c mice was evaluated, using GFP as antigen, resulting in a significant increase in anti-GFP IgG response, particularly, IgG1 class, demonstrating that the VLPs enhance the immune response against the antigen chosen in this study.

Cloning and partial characterization of Entamoeba histolytica PTPases.

Reversible protein tyrosine phosphorylation is an essential signal transduction mechanism that regulates cell growth, differentiation, mobility, metabolism, and survival. Two genes coding for protein tyrosine phophatases, designed EhPTPA and EhPTPB, were cloned from Entamoeba histolytica. EhPTPA and EhPTPB proteins showed amino acid sequence identity of 37%, both EhPTPases showed similarity with Dictyostelium discoideum and vertebrate trasmembranal PTPases. mRNA levels of EhPTPA gene are up-regulated in trophozoites recovered after 96h of liver abscess development in the hamster model. EhPTPA protein expressed as a glutathione S-transferase fusion protein (GST::EhPTPA) showed enzymatic activity with p-nitrophenylphosphate as a substrate and was inhibited by PTPase inhibitors vanadate and molybdate. GST::EhPTPA protein selectively dephosphorylates a 130kDa phosphotyrosine-containing protein in trophozoite cell lysates. EhPTPA gene codifies for a 43kDa native protein. Up-regulation of EhPTPA expression suggests that EhPTPA may play an important role in the adaptive response of trophozoites during amoebic liver abscess development.

Dengue virus inoculation to human skin explants: an effective approach to assess in situ the early infection and the effects on cutaneous dendritic cells.

Although dengue virus (DV) enters through skin while mosquitoes feed, early contacts remain unexplored regarding the cutaneous viral fate and in situ immune responses. We addressed this by exposing healthy, non-cadaveric, freshly obtained human skin explants to a human DV2 isolate. We demonstrated negative-strand DV-RNA and non-structural protein-1, both suggestive of viral replication in skin. Although control, mock-infected and DV-infected explants showed less (MHC-CII(+)/CD1a(+)/Langerin+) Langerhans cells, deranged morphology and decreased frequency were more apparent in DV-infected explants. Whereas DV+ cells were infrequent in epidermis and completely absent in dermis, some areas of basal epidermis were clearly DV+, presumably keratinocytes, cells where TUNEL positivity revealed apoptosis. Unlike fresh, control and mock-infected skin, DV-infected explants expressed CD80 and CD83, indicative of dendritic cell (DC) activation and maturation, respectively. However, sequential sections indicated that these cells were not DV+, suggesting that activated/mature DCs capable of priming T cells, probably, were not infected. Alternatively, the occasionally infected epidermal DC might not have reached maturation. Interestingly, skin DV infection apparently uncouples the DC activation/maturation process from another crucial DC function, the subsequent migration into dermis. This was suggested, because upon cutaneous DV infection, the few emerging CD83+ (mature) DCs remained within the outer epidermis, while no dermal CD83+ DCs were observed. These paradoxical effects might represent unknown DV subversion strategies. This approach is relatively easy, quick (results in 48 h), economical for developing countries where dengue is re-emerging and advantageous to evaluate in situ viral biology, immunity and immunopathology and potential antiviral strategies.