Basophil activation test: food challenge in a test tube or specialist research tool?

Oral food challenge (OFC) is the gold-standard to diagnose food allergy; however, it is a labour and resource-intensive procedure with the risk of causing an acute allergic reaction, which is potentially severe. Therefore, OFC are reserved for cases where the clinical history and the results of skin prick test and/or specific IgE do not confirm or exclude the diagnosis of food allergy. This is a significant proportion of patients seen in Allergy clinics and results in a high demand for OFC. The basophil activation test (BAT) has emerged as a new diagnostic test for food allergy. With high diagnostic accuracy, it can be particularly helpful in the cases where skin prick test and specific IgE are equivocal and may allow reducing the need for OFC. BAT has high specificity, which confers a high degree of certainty in confirming the diagnosis of food allergy and allows deferring the performance of OFC in patients with a positive BAT. The diagnostic utility of BAT is allergen-specific and needs to be validated for different allergens and in specific patient populations. Standardisation of the laboratory methodology and of the data analyses would help to enable a wider clinical application of BAT

Biocompatibility of a self-assembled crosslinkable hyaluronic acid nanogel

Hyaluronic acid nanogel (HyA-AT) is a redox sensitive crosslinkable nanogel, obtained through the conjugation of a thiolated hydrophobic molecule to the hyaluronic acid chain. Engineered nanogel was studied for its biocompatibility, including immunocompatibility and hemocompatability. The nanogel did not compromise the metabolic activity or cellular membrane integrity of 3T3, microvascular endothelial cells, and RAW 264.7 cell lines, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase release assays. Also, we didn't observe any apoptotic effect on these cell lines through the Annexin V-FITC test. Furthermore, the nanogel cell internalization was analyzed using murine bone marrow derived macrophages, and the in vivo and ex vivo biodistribution of the Cy5.5 labeled nanogel was monitored using a non-invasive near-infrared fluorescence imaging system. The HyA-AT nanogel exhibits fairly a long half-live in the blood stream, thus showing potential for drug delivery applications.The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/ BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project “BioHealth – Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124- FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. The authors would like to acknowledge also the support of FCT for the PhD grant reference SFRH/BD/61516/2009. They would also like to thank Bioimaging department on Molecular Medicine Institute (IMM) in Lisbon, namely Dr José Rino and Dr António Temudo. Also thank the animal facilities in IMM (Lisbon), specially Dr. Dolores Bonaparte and Dr. Joana Marques. Finally, the authors thank Dr Africa Gonzalez and Mercedes Pelletero the performance of the studies on the activation of complement

A novel antibody-like material for breast cancer antigen CA15-3, used to track breast cancer by potentiometric transduction

This work presents the development of a low cost sensor device for the diagnosis of breast cancer in point-of-care, made with new synthetic biomimetic materials inside plasticized poly(vinyl chloride), PVC, membranes, for subsequent potentiometric detection. This concept was applied to target a conventional biomarker in breast cancer: Breast Cancer Antigen (CA15-3). The new biomimetic material was obtained by molecularly-imprinted technology. In this, a plastic antibody was obtained by polymerizing around the biomarker that acted as an obstacle to the growth of the polymeric matrix. The imprinted polymer was specifically synthetized by electropolymerization on an FTO conductive glass, by using cyclic voltammetry, including 40 cycles within -0.2 and 1.0 V. The reaction used for the polymerization included monomer (pyrrol, 5.0×10-3 mol/L) and protein (CA15-3, 100U/mL), all prepared in phosphate buffer saline (PBS), with a pH of 7.2 and 1% of ethylene glycol. The biomarker was removed from the imprinted sites by proteolytic action of proteinase K. The biomimetic material was employed in the construction of potentiometric sensors and tested with regard to its affinity and selectivity for binding CA15-3, by checking the analytical performance of the obtained electrodes. For this purpose, the biomimetic material was dispersed in plasticized PVC membranes, including or not a lipophilic ionic additive, and applied on a solid conductive support of graphite. The analytical behaviour was evaluated in buffer and in synthetic serum, with regard to linear range, limit of detection, repeatability, and reproducibility. This antibody-like material was tested in synthetic serum, and good results were obtained. The best devices were able to detect 5 times less CA15-3 than that required in clinical use. Selectivity assays were also performed, showing that the various serum components did not interfere with this biomarker. Overall, the potentiometric-based methods showed several advantages compared to other methods reported in the literature. The analytical process was simple, providing fast responses for a reduced amount of analyte, with low cost and feasible miniaturization. It also allowed the detection of a wide range of concentrations, diminishing the required efforts in previous sample pre-treating stages

Self-assembled nanoparticles made of fucan

Amphiphilic polymers can self-assemble in water due to hydrophilic and hydrophobic interactions, forming nanoparticles (NPs) with unique physicochemical characteristics and thermodynamic stability. A non toxic sulfated Fucan, extracted from Spatoglossum schroederi was chemically modified by the grafting of Hexadecylamine (C16) to the polymer hydrophilic backbone. The resulting modified material (Fucan-C16) formed nanosized particles which were characterized by 1H NMR to assess the substitution degree of the hydrophobic chains, fluorescence spectroscopy to determine the critical aggregation concentration (cac), cryo-field emission scanning electron microscopy (Cryo- FESEM) to evaluate the shape and size of the NPs, and dynamic light scattering (DLS) to verify the size distribution. The (cac) of Fucan-C16 NPs ranged between 0.05 and 0.03mg/mL. Cryo-FESEM revealed that Fucan-C16 formed spherical macromolecular particles with diameters between 120 and 180 nm, which were confirmed by DLS. In addition, the size of the NPs were not affected by the concentration of the polymer or by the variation of the pH.The size of nanoparticles increases with increasing its concentration in solution.CAPES, FCT and CNP

siRNA inhibition of endocytic pathways to characterize the cellular uptake mechanisms of folate-functionalized glycol chitosan nanogels

Glycol chitosan nanogels have been widely used in gene, drug, and contrast agent delivery in an effort to improve disease diagnosis and treatment. Herein, we evaluate the internalization mechanisms and intracellular fate of previously described glycol chitosan nanogels decorated with folate to target the folate receptor. Uptake of the folate-decorated nanogel was impaired by free folate, suggesting competitive inhibition and shared internalization mechanisms via the folate receptor. Nanogel uptake was shown to occur mainly through flotillin-1 and Cdc42-dependent endocytosis. This was determined by inhibition of uptake reduction observed upon siRNA depletion of these two proteins and the pathways that they regulate. The data also suggest the involvement of the actin cytoskeleton in nanogel uptake via macropinocytosis. After 7 h of incubation with HeLa cells, approximately half of the nanogel population was localized in endolysosomal compartments, whereas the remaining 50% of the material was in undefined regions of the cytoplasm. Glycol chitosan nanogels may thus have potential as drug delivery vectors for targeting different intracellular compartments.BioHealth - Biotechnology and Bioengineering approaches to improve health quality, Ref. NORTE-07-0124-FEDER-000027, cofunded by the Programa Operacional Regional do Norte (ON.2 − O Novo Norte), QREN, FEDER. P.P. was funded through an FCT Ph.D. grant (SFRH/BD/64977/2009). Funding is also acknowledged from a Cancer Research UK studentship (C36040/A11652), EPSRC Grant (EP/J021334/1 to A.T.J. and E.S.

In vivo performance of chitosan/soy-based membranes as wound dressing devices for acute skin wounds

Wound management represents a major clinical challenge on what concerns healing enhancement and pain control. The selection of an appropriate dressing plays an important role in both recovery and esthetic ap- pearance of the regenerated tissue. Despite the wide range of available dressings, the progress in the wound care market relies on the increasing interest in using natural-based biomedical products. Herein, a rat wound- dressing model of partial-thickness skin wounds was used to study newly developed chitosan/soy (cht/soy)- based membranes as wound-dressing materials. Healing and repair of nondressed, cht/soy membrane-dressed, and Epigard -dressed wounds were followed macroscopically and histologically for 1 and 2 weeks. cht/soy membranes performed better than the controls, promoting a faster wound repair. Re-epithelialization, ob- served 1 week after wounding, was followed by cornification of the outermost epidermal layer at the second week of dressing, indicating repair of the wounded tissue. The use of this rodent model, although in impaired healing conditions, may enclose some drawbacks regarding the inevitable wound contraction. Moreover, being the main purpose the evaluation of cht/soy-based membranes’ performance in the absence of growth factors, the choice of a clinically relevant positive control was limited to a polymeric mesh, without any growth factor influencing skin healing/repair, Epigard. These new cht/soy membranes possess the desired features regarding healing/repair stimulation, ease of handling, and final esthetic appearance-thus, valuable prop- erties for wound dressings.The author Tircia C. Santos acknowledges the Portuguese Foundation for Science and Technology (FCT) for her PhD grant (SFRH/BD/40861/2007). This work was developed under the scope of the European Network of Excellence EXPERTISSUES (NMP3-CT-2004-5000283)

THE NURSING WORK ON THE HIGH BLOOD PRESSURE´S TRACKING IN CHILDREN AND ADOLESCENTS OF A PUBLIC SCHOOL OF GOIÂNIA-GOIÁS

This paper was written during the development of an assistance project by students of the FederalUniversity of Goiás Nursing College. The purpose was to investigate the prevalence of the high blood pressure in 160children and preadolescents students from 5th to 7th high classes in Goiânia Goiás state school., from August 2002to February - 2003. 4% of the students evaluated had out of standard blood pressure. All of them were lead to healthappointment to the Youth High-Pressure League of the Federal University of Goiás Clinical Hospital

Consenso Técnico para a determinação do status da proteína HER2 por imunocitoquímica em carcinoma da mama

A determinação do status da proteína HER2 por imunocitoquímica é uma metodologia fundamental para o diagnóstico, prognóstico e indicação terapêutica no carcinoma da mama, nomeadamente para o encaminhamento terapêutico com Herceptin®/trastuzumab. O estabelecimento desta terapêutica nas vertentes adjuvante ou neoadjuvante, e até em doença metastática, tem vindo a acentuar a importância da determinação do referido status de modo a melhor responder às necessidades dos doentes. Sendo a imunocitoquímica o método validado para determinação do status HER2 em carcinoma da mama, é de extrema importância definir linhas de orientação para a sua correta performance como tem sido estabelecido em diversos países em todo o mundo. A área científica de Anatomia Patológica, Citológica e Tanatológica da Escola Superior de Tecnologia da Saúde de Lisboa (APCT-ESTeSL) e a Associação Portuguesa de Técnicos de Anatomia Patológica (APTAP) reuniram um painel de especialistas para a construção e estabelecimento de linhas de orientação técnica para a determinação do status HER2 em carcinoma da mama para a realidade portuguesa. Pretende-se com este consenso criar linhas de orientação técnicas para a construção, validação e manutenção do teste imunocitoquímico para determinação do status HER2 em carcinoma da mama, no que diz respeito à realidade portuguesa. Todas as orientações aqui descritas têm em conta o estado da arte atual no que diz respeito à determinação do status HER2 por imunocitoquímica em carcinoma da mama, bem como a experiência pessoal e académica de cada um dos membros do painel de especialistas que a subscrevem

Lactoferrin perturbs lipid rafts and requires integrity of Pma1p-lipid rafts association to exert its antifungal activity against Saccharomyces cerevisiae

"Available online 07 January 2021"Lactoferrin (Lf) is a bioactive milk-derived protein with remarkable wide-spectrum antifungal activity. To deepen our understanding of the molecular mechanisms underlying Lf cytotoxicity, the role of plasma membrane ergosterol- and sphingolipid-rich lipid rafts and their association with the proton pump Pma1p was explored. Pma1p was previously identified as a Lf-binding protein. Results showed that bovine Lf (bLf) perturbs sterol-rich lipid rafts organization by inducing intracellular accumulation of ergosterol. Using yeast mutant strains lacking lipid rafts-associated proteins or enzymes involved in the synthesis of ergosterol and sphingolipids, we found that perturbations in the composition of these membrane domains increase resistance to bLf-induced yeast cell death. Also, when Pma1p-lipid rafts association is compromised in the Pma110 mutant and in the absence of the Pma1p-binding protein Ast1p, the bLf killing activity is impaired. Altogether, results showed that the perturbation of lipid rafts and the inhibition of both Pma1p and V-ATPase activities mediate the antifungal activity of bLf. Since it is suggested that the combination of conventional antifungals with lipid rafts-disrupting compounds is a powerful antifungal approach, our data will help to pave the way for the use of bLf alone or in combination for the treatment/eradication of clinically and agronomically relevant yeast pathogens/fungi.This work was supported by national funds through the Portuguese Foundation for Science and Technology (FCT I.P.) under the scope of the strategic funding of “Contrato-Programa” UIDB/04050/2020 and UIDB/ 04469/2020 unit; by the BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte; and by the Servicio para el Control de la Esterilización, Laboratorio de Microbiología Oral (CN-16-036). Cátia Santos-Pereira acknowledges the PhD fellowship PD/BD/128032/2016 funded by FCT under the scope of the doctoral programme in Applied and Environmental Microbiology (DP_AEM).info:eu-repo/semantics/publishedVersio

Design of a lipid nanovesicle system encasing bacteriophages for inhalational therapy : a proof-of-concept

Inflammatory diseases that occur in the pharynx and involving both the adenoids and tonsils are important not only for being very frequent, but also because they often require minor surgery for their resolution. These structures have immunological functions leading to production of antibodies, and work in the local immunity of the pharynx and protection of the entire body. The most common etiologic agent of sore throats is Streptococcus pyogenes, an important pathogen of the beta-hemolytic group A which causes streptococcal pharyngitis. The emergence of antibiotic-resistant bacterial strains and the poor penetration of chemical antibiotics in bacterial biofilms raise the need for safe and effective options of antimicrobial treatment. The application of bacteriophages (or cocktails therefrom) has been proposed as an alternative (or complement) to conventional chemical antibiotics, allowing the release of natural predators of bacteria directly on these biofilms. The major advantage of bacteriophage-based antibiotherapy relative to its conventional chemical counterpart is that bacteriophages replicate at the site of infection, being available in abundance where they are needed the most. When compared with chemical antibiotics, bacteriophages have other important advantages: (i) strong tissue permeability, (ii) bacteriophage concentration remains high at the focus of infection, continuously increasing with bacterial (host) presence, (iii) elimination of the focus of infection occurs only after eradication of the host bacterium, (iv) bacteriophages are fully compatible with antibiotics and may act synergistically, (v) they are specific against the target bacteria, (vi) have a superior ability to penetrate bacterial biofilms, inducing production of enzymes that hydrolyze the biofilm polymeric matrix, (vii) although bacteria can develop resistance to bacteriophages, isolation of new lytic bacteriophages is much simpler and cheaper than developing a new chemical antibiotic. In this research effort, development of a biotechnological process for the inhalational administration of a bacteriophage cocktail (endotoxin free) was pursued, using strategies of nanoencapsulation within lipid nanovesicles (as forms of protection for the bacteriophage against the immune system) to treat infectious pathologies such as pharyngo-tonsillitis caused by Streptococcus pyogenes. This method of targeting may have a high potential for the treatment of bacterial infections of the respiratory tract, since inhalation therapy is considered to be favorable to certain respiratory infections because the aerosol is delivered directly at the site of infection, accelerating the action of bacterial predators. Additionally, a smaller amount of bioactive substance is needed, thus preventing or reducing possible side effects. As a proof of concept for the nanoencapsulation strategy, and since there is not yet available a strictly lytic bacteriophage cocktail for Streptococcus pyogenes, a well-defined and characterized bacteriophage was utilized, viz. bacteriophage T4. Water-in-oil-in-water (W/O/W) multiple emulsions are nanosystems in which dispersions of small water droplets within larger oil droplets are themselves dispersed in a continuous aqueous phase. Due to their compartimentalized internal structure, multiple emulsions present important advantages over simple O/W emulsions for encapsulation of biomolecules, such as the ability to carry both polar and non-polar molecules, and a better control over releasing of therapeutic molecules. T4 bacteriophage was entrapped within W/O/W multiple nanoemulsions, aiming at mimicking the multifunctional design of biology, optimized with several lipid matrices, poloxamers and stabilizing layer compositions. Physicochemical characterization of the optimized bacteriophage-encasing nanovesicle formulations encompassed determination of particle size, size distribution and particle charge, via Zeta potential analysis, surface morphology via CRYO-SEM, and thermal analysis via DSC, whereas antimicrobial activity of the nanoemulsions produced were evaluated via the “spot-test” using appropriate bacterial cultures