RESUMO
Serum is an important source of proteins that interact with pathogens. Once bound to the cell surface, serum proteins can stimulate the innate immune system. The phagocytosis of Sporothrix schenckii conidia by human macrophages is activated through human serum opsonisation. In this study, we have attempted to characterise human blood serum proteins that bind to the cell wall of S. schenckii conidia. We systematically observed the same four proteins independent of the plasma donor: albumin, serum amyloid protein (SAP), α-1 antitrypsin (AAT), and transferrin were identified with the help of tandem mass spectrometry. Phagocytosis depended on the concentration of the SAP or α-1 antitrypsin that was used to opsonise the conidia; however, transferrin or albumin did not have any effect on conidia internalisation. The presence of mannose did not affect macrophage phagocytosis of the conidia opsonised with SAP or α-1 antitrypsin, which suggests that these proteins are not recognised by the mannose receptor.
Assuntos
Proteínas Sanguíneas/imunologia , Macrófagos/imunologia , Fagocitose , Esporos Fúngicos/imunologia , Sporothrix/imunologia , Esporotricose/imunologia , Proteínas Sanguíneas/química , Humanos , Lectinas Tipo C/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Receptores de Superfície Celular/imunologia , Esporos Fúngicos/genética , Sporothrix/genética , Esporotricose/microbiologiaRESUMO
The development of biotherapeutics requires continuous improvement in analytical methodologies for the assessment of their quality attributes. A subset of biotherapeutics is designed to interact with specific antigens that are exposed on the membranes of target cells or circulating in a soluble form, and effector functions are achieved via recognition of their Fc region by effector cells that induce mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC). Thus, ADCC induction is a critical quality attribute (CQA) that must be evaluated to ensure biotherapeutic efficacy. Induction of ADCC can be evaluated by employing effector cells from different sources, such as peripheral blood mononuclear cells (PBMC) and genetically modified cell lines (e.g., transfected NKs or Jurkat cells), and different approaches can be used for detection and results interpretation depending on the type of effector cells used. In this regard, validation of the assays is relevant to ensure the reliability of the results according to the intended purpose. Herein, we show the standardization and validation of ADCC assays to test the potency of three biotherapeutic proteins using primary NK cells obtained from fresh blood as effector cells and detecting cell death by flow cytometry. The advantage of using primary NKs instead of modified cells is that the response is closer to that occurring in vivo since cytotoxicity is evaluated in a direct manner. Our results indicate that in all cases, the assays exhibited a characteristic sigmoidal dose/response curve complying with accurate, precise and specific parameters. Thereby, the validated ADCC assay is an appropriate alternative to evaluate the biological activities of these type of biotherapeutics.