Centro de Investigaciones básicas y aplicadas http://repositorio.unnoba.edu.ar/xmlui/handle/23601/90 2026-04-05T23:27:30Z 2026-04-05T23:27:30Z http://repositorio.unnoba.edu.ar/xmlui/handle/23601/927 2025-10-21T21:17:06Z 2025-02-28T00:00:00Z

Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1 La evaluación de la interacción directa entre patógenos y receptores inmunitarios suele implicar técnicas sofisticadas o el uso de cepas transgénicas y células modificadas genéticamente. En este trabajo, se describe un método alternativo para detectar la interacción bioquímica entre el sensor microbiano de macrófagos SLAMF1 y Mycobacterium tuberculosis. Se desarrollaron dos enfoques técnicos que emplean citometría de flujo y microscopía de fluorescencia. Se generaron extractos de proteínas celulares totales de macrófagos humanos, que luego se incubaron con células completas de M. tuberculosis (WCMtb) o antígenos de M. tuberculosis (Ag Mtb) durante la noche a 4 °C y finalmente se entrecruzaron mediante un tratamiento con formaldehído/glicina/etilenglicol bis (succinimidil succinato). La interacción de SLAMF1 con WCMtb mediante citometría de flujo se detectó con un anticuerpo anti-SLAMF1 PE. La existencia de interacción mediante microscopía de fluorescencia se determinó mediante la fijación de Ags de Mtb teñidos con rodamina-PE a cubreobjetos recubiertos con poli-D-lisina, que se incubaron con el extracto proteico total de macrófagos derivados de monocitos. Tras el tratamiento de entrecruzamiento, se visualizó SLAMF1 mediante anticuerpos primarios (anti-SLAMF1) y secundarios (Alexa Fluor 488). Los ensayos proporcionaron una sólida herramienta bioquímica para medir las interacciones patógeno-inmunorreceptor, superando las dificultades asociadas con las líneas celulares transgénicas y los experimentos de modulación de la expresión génica de proteínas.; The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic strains and genetically engineered cells. Here, an alternative method to detect biochemical interaction between the macrophage microbial sensor SLAMF1 and Mycobacterium tuberculosis is described. Two technical approaches employing flow cytometry and fluorescence microscopy were developed. Total cell protein extracts from human macrophages were generated, then incubated with whole cells of M. tuberculosis (WCMtb) or M. tuberculosis antigens (Mtb Ags) overnight at 4 °C and finally cross-linked using formaldehyde/glycine/ethylene glycol bis (succinimidyl succinate) treatment. SLAMF1 interaction with WCMtb by flow cytometry was detected with a PE-specific anti-SLAMF1 antibody. The existence of interaction by fluorescence microscopy was performed by attaching Rhodamine-PE stained Mtb Ags to poly-D-lysine coated slides, which were incubated with the total protein extract from monocyte-derived macrophages. After cross-linking treatment, SLAMF1 was visualized using primary (anti-SLAMF1) and secondary (Alexa Fluor 488) antibodies. The assays provided a strong biochemical tool to measure pathogen-immunoreceptor interactions, overcoming the difficulties associated with transgenic cell lines and protein gene expression modulation experiments.

2025-02-28T00:00:00Z http://repositorio.unnoba.edu.ar/xmlui/handle/23601/835 2025-10-21T21:16:16Z 2024-01-05T00:00:00Z

Platelets promote human macrophages-mediated macropinocytosis of Clostridioides difficile; Las plaquetas promueven la macropinocitosis de Clostridioides difficile mediada por macrófagos humanos Clostridioides difficile es el principal agente causal de diarreas hospitalarias. La infección por C. difficile (CDI) es una enfermedad potencialmente letal, cuya terapia actual es el uso de antibióticos sin ser totalmente eficaz. Los mecanismos moleculares, las condiciones inflamatorias y las respuestas inmunes del hospedador que podrían beneficiar la persistencia o eliminación de C. difficile permanece poco explorados. Los macrófagos realizan diferentes formas de endocitosis como parte de sus funciones de vigilancia inmune y las plaquetas, clásicamente conocidas por su papel coagulatorio, también son importantes moduladores del sistema inmune. El objetivo de este estudio fue evaluar la endocitosis de C. difficile en su forma vegetativa por macrófagos humanos y la participación de las plaquetas en este proceso. Nuestros resultados mostraron que tanto los macrófagos como las plaquetas interactúan con C. difficile viva y muerta por calor. Además, las plaquetas forman complejos con monocitos humanos en sangre fresca de donantes sanos y la presencia de C. difficile induce estas interacciones célula-célula. Mediante citometría de flujo y microscopía confocal demostramos que los macrófagos pueden internalizar a C. difficile y que las plaquetas promueven esta internalización. Mediante el uso de inhibidores de diferentes vías endocíticas, demostramos que la macropinocitosis es la vía de entrada de C. difficile a la célula. En conjunto, nuestros hallazgos son la primera evidencia de la internalización de C. difficile vegetativa no toxigénica e hipervirulenta por macrófagos humanos y destacan el papel de las plaquetas en la inmunidad innata durante la CDI. Descifrar la interacción de C. difficile con el sistema inmunológico podría proporcionar nuevas herramientas para comprender la patogénesis de C. difficile y para el desarrollo de terapias dirigidas al hospedador. Clostridioides difficile is the main causative agent of hospital-acquired diarrhea and the potentially lethal disease, C. difficile infection. The cornerstone of the current therapy is the use of antibiotics, which is not fully effective. The molecular mechanisms, inflammatory conditions and host-immune responses that could benefit the persistence or elimination of C. difficile remain unclear. Macrophages perform different ways of endocytosis as part of their immune surveillance functions and platelets, classically known for their coagulatory role, are also important modulators of the immune system. The aim of this study was to evaluate the endocytosis of vegetative C. difficile by human macrophages and the involvement of platelets in this process. Our results showed that both macrophages and platelets interact with live and heat-killed C. difficile. Furthermore, platelets form complexes with human monocytes in healthy donor’s fresh blood and the presence of C. difficile increased these cell-cell interactions. Using flow cytometry and confocal microscopy, we show that macrophages can internalize C. difficile and that platelets improve this uptake. By using inhibitors of different endocytic pathways, we demonstrate that macropinocytosis is the route of entry of C. difficile into the cell. Taken together, our findings are the first evidence for the internalization of vegetative non-toxigenic and hypervirulent C. difficile by human macrophages and highlight the role of platelets in innate immunity during C. difficile infection. Deciphering the crosstalk of C. difficile with immune cells could provide new tools for understanding the pathogenesis of C. difficile infection and for the development of host-directed therapies.

2024-01-05T00:00:00Z http://repositorio.unnoba.edu.ar/xmlui/handle/23601/666 2025-10-21T21:15:19Z 2023-04-07T00:00:00Z

A Novel MSH6 Gene Variant in a Lynch Syndrome Patient with Lipomas Colorectal cancer is one of the most frequently occurring cancers today, with a large percentage of cases having a hereditary basis. Lynch syndrome is the most common cause of hereditary colorectal cancer. The genetic defect characteristics of this syndrome involve mutations in mismatch repair (MMR) genes, which result in microsatellite instability. Early detection of the mutation can help evaluate the cancer risk and, consequently, a proper course of clinical management for the person harboring the mutation. Herein, we describe the first report of a c.1458dup (p.Glu487*) new mutation in a 53-year-old colorectal cancer patient with diagnosed Lynch syndrome. Additionally, the existence of lipomas in this patient and his family could be related to this syndrome. Further investigation may provide a possible visual clue that can indicate a need for genetic screening.

2023-04-07T00:00:00Z http://repositorio.unnoba.edu.ar/xmlui/handle/23601/637 2025-10-21T21:14:40Z 2021-02-09T00:00:00Z

Initial Identification of UDP-Glucose Dehydrogenase as a Prognostic Marker in Breast Cancer Patients, Which Facilitates Epirubicin Resistance and Regulates Hyaluronan Synthesis in MDA-MB-231 Cells UDP-glucose-dehydrogenase (UGDH) synthesizes UDP-glucuronic acid. It is involved in epirubicin detoxification and hyaluronan synthesis. This work aimed to evaluate the effect of UGDH knockdown on epirubicin response and hyaluronan metabolism in MDA-MB-231 breast cancer cells. Additionally, the aim was to determine UGDH as a possible prognosis marker in breast cancer. We studied UGDH expression in tumors and adjacent tissue from breast cancer patients. The prognostic value of UGDH was studied using a public Kaplan–Meier plotter. MDA-MB-231 cells were knocked-down for UGDH and treated with epirubicin. Epirubicin-accumulation and apoptosis were analyzed by flow cytometry. Hyaluronan-coated matrix and metabolism were determined. Autophagic-LC3-II was studied by Western blot and confocal microscopy. Epirubicin accumulation increased and apoptosis decreased during UGDH knockdown. Hyaluronan-coated matrix increased and a positive modulation of autophagy was detected. Higher levels of UGDH were correlated with worse prognosis in triple-negative breast cancer patients that received chemotherapy. High expression of UGDH was found in tumoral tissue from HER2--patients. However, UGDH knockdown contributes to epirubicin resistance, which might be associated with increases in the expression, deposition and catabolism of hyaluronan. The results obtained allowed us to propose UGDH as a new prognostic marker in breast cancer, positively associated with development of epirubicin resistance and modulation of extracellular matrix.

2021-02-09T00:00:00Z