Multiparametric in vitro and in vivo analysis of the safety profile of self-assembling peptides.

Self-assembling peptides (SAPs) have gained significant attention in biomedicine because of their unique properties and ability to undergo molecular self-assembly driven by non-covalent interactions. By manipulating their composition and structure, SAPs can form well-ordered nanostructures with enhanced selectivity, stability and biocompatibility. SAPs offer advantages such as high chemical and biological diversity and the potential for functionalization. However, studies concerning its potentially toxic effects are very scarce, a limitation that compromises its potential translation to humans. This study investigates the potentially toxic effects of six different SAP formulations composed of natural amino acids designed for nervous tissue engineering and amenable to ready cross-linking boosting their biomechanical properties. All methods were performed in accordance with the relevant guidelines and regulations. A wound-healing assay was performed to evaluate how SAPs modify cell migration. The results in vitro demonstrated that SAPs did not induce genotoxicity neither skin sensitization. In vivo, SAPs were well-tolerated without any signs of acute systemic toxicity. Interestingly, SAPs were found to promote the migration of endothelial, macrophage, fibroblast, and neuronal-like cells in vitro, supporting a high potential for tissue regeneration. These findings contribute to the development and translation of SAP-based biomaterials for biomedical applications.

All About (NK Cell-Mediated) Death in Two Acts and an Unexpected Encore: Initiation, Execution and Activation of Adaptive Immunity.

NK cells are key mediators of immune cell-mediated cytotoxicity toward infected and transformed cells, being one of the main executors of cell death in the immune system. NK cells recognize target cells through an array of inhibitory and activating receptors for endogenous or exogenous pathogen-derived ligands, which together with adhesion molecules form a structure known as immunological synapse that regulates NK cell effector functions. The main and best characterized mechanisms involved in NK cell-mediated cytotoxicity are the granule exocytosis pathway (perforin/granzymes) and the expression of death ligands. These pathways are recognized as activators of different cell death programmes on the target cells leading to their destruction. However, most studies analyzing these pathways have used pure recombinant or native proteins instead of intact NK cells and, thus, extrapolation of the results to NK cell-mediated cell death might be difficult. Specially, since the activation of granule exocytosis and/or death ligands during NK cell-mediated elimination of target cells might be influenced by the stimulus received from target cells and other microenvironment components, which might affect the cell death pathways activated on target cells. Here we will review and discuss the available experimental evidence on how NK cells kill target cells, with a special focus on the different cell death modalities that have been found to be activated during NK cell-mediated cytotoxicity; including apoptosis and more inflammatory pathways like necroptosis and pyroptosis. In light of this new evidence, we will develop the new concept of cell death induced by NK cells as a new regulatory mechanism linking innate immune response with the activation of tumour adaptive T cell responses, which might be the initiating stimulus that trigger the cancer-immunity cycle. The use of the different cell death pathways and the modulation of the tumour cell molecular machinery regulating them might affect not only tumour cell elimination by NK cells but, in addition, the generation of T cell responses against the tumour that would contribute to efficient tumour elimination and generate cancer immune memory preventing potential recurrences.

Normalización de un método inmunoquímico para detectar Papilomavirus humano tipo 16 en lesiones cérvico-uterinas / Standardization of immunochemical method for detection of type 16 human papilomavirus in cervix uterine lesions

Introducción: la infección por Papilomavirus Humano (PVH) es la condición necesaria para la aparición y desarrollo del cáncer cérvico-uterino. Los genotipos de alto riesgo oncogénico son los causantes de este tipo de neoplasia y dentro de ellos el más frecuente es el PVH 16, que se encuentra aproximadamente en el 60 % de los casos. Los métodos de diagnóstico comerciales resultan costosos para países con escasos recursos económicos, lo que sugiere la búsqueda de alternativas empleando protocolos sencillos y baratos. Objetivos: normalizar un método inmunoquímico para la detección del antígeno L1 de PVH tipo 16 en muestras cérvico-uterinas de pacientes con lesiones intraepiteliales escamosas y determinar la coincidencia entre el método normalizado y la Reacción en Cadena de la Polimerasa en Tiempo Real (RCP-TR), como técnica de referencia, para estimar la utilidad de dicho método en el diagnóstico de la infección por este genotipo viral. Métodos: se compararon tres procedimientos de inmunotinción (Indirecto de inmunoperoxidasa en dos pasos, Estreptavidina-Biotina y Amplificación por polímero) respecto a sensibilidad analítica, tinción inespecífica de fondo y tiempo de terminación, para la detección de la proteína L1 de PVH 16 en líneas celulares derivadas de carcinomas cervicales humanos y en muestras cérvico-uterinas utilizadas como controles. El protocolo normalizado se aplicó a muestras cérvico-uterinas de mujeres entre 30 y 59 años, 82 con lesiones intraepiteliales cervicales y 10 sin antecedentes de alteraciones citológicas, a las que además se les determinó PVH 16 mediante RCP-TR. Resultados: el procedimiento de Estreptavidina-Biotina resultó el más sensible y específico. La coincidencia entre el método inmunoquímico y la RCP-TR fue de un 98,6 por ciento, la sensibilidad fue de un 98,57 por ciento y la especificidad de un 91,67 por ciento, con valores predictivos negativo y positivo por encima del 90 por ciento. Conclusiones: se demostró la validez del método inmunoquímico como prueba confirmatoria de la infección por PVH 16. Dicho método probó ser sensible, sencillo y no requiere de una compleja infraestructura para detectar PVH 16 en muestras cervicales. Además, esta técnica permite obtener información rápidamente y evita el uso de métodos invasivos(AU)

Introduction: Human Papillomavirus (HPV) infection is the necessary condition for the occurernce and development of cervical cancer. The high oncogenic risk genotypes are the responsible for this type of neoplasia and the most frequent is HPV 16 that affects roughly 60 percent of cases. Commercial kits for HPV detection are expensive for resource-poor countries, which suggests the search for alternative throguh non-expensive simple protocoles. Objectives: to standardize an immunochemical method for the detection of HPV 16 L1 antigen in cervical samples of patients with squamous intraepithelial lesions and to determine the diagnostic coincidence between the immunochemical method and the real-time polymerase chain reaction to estimate the usefulness of this method for the detection of cervical infection with this viral genotype. Methods: three immunostaining methods (Two-Step Indirect Immunoperoxidase, Labelled Streptavidin-Biotin and Enhanced Polymer) were compared in terms of analytical sensitivity, nonspecific background staining and time of completion, for the detection of protein L1 of HPV-16 in a cell line derived from human cervical carcinoma and clinical samples from uterine cervix. The optimized protocol was applied to 82 cervical samples from women aged 30-59 years with squamous intraepithelial lesions and to 10 samples of sexually active women without previous signals of positive cytology. The presence of type 16 HPV was also detected with the aid of RT-PCR. Results: the Streptavidin-Biotin system was the most sensitive and specific. The diagnostic agreement between the immunochemical method and the real-time polymerase chain reaction reached 98.6 percent, sensitivity was 98.57 percent and specificity was 91.67 %, with positive and negative predictive values above 90 percent. Conclusions: the validity of the immunochemical method as a confirmatory test for infection by HPV-16 has been demonstrated. The normalized immunochemical method proved to be a sensitive, simple, relatively fast method to detect HPV from clinical samples of cervical cells. Furthermore, this method provides information quickly, avoiding the use of invasive methods in patients(AU)