Influence of B7 costimulatory molecules colocalized in extracellular traps (ETs) on CD45RO expression in human autologous cultures
Extracellular traps (ETs) are structures composed of chromatin, histones and granular proteins, and constitute a functional microbicidal mechanism of immune cells. Its participation in the pathophysiology of many pathologies, including COVID-19, autoimmune diseases and cancer, has been studied. Cost...
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Universidad Nacional Córdoba. Facultad de Ciencias Médicas. Secretaria de Ciencia y Tecnología
2022
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| Acceso en línea: | https://revistas.unc.edu.ar/index.php/med/article/view/39025 |
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I10-R327-article-39025 |
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ojs |
| institution |
Universidad Nacional de Córdoba |
| institution_str |
I-10 |
| repository_str |
R-327 |
| container_title_str |
Revista de la Facultad de Ciencias Médicas de Córdoba |
| format |
Artículo revista |
| topic |
extracellular traps human leukocytes, B7 molecules, CD45RO, autologous culture trampas extracelulares leucocitos humanos moléculas B7 CD45RO cultivos autólogos |
| spellingShingle |
extracellular traps human leukocytes, B7 molecules, CD45RO, autologous culture trampas extracelulares leucocitos humanos moléculas B7 CD45RO cultivos autólogos Castellani Austerlitz, MP Rodríguez, FM Carabajal Miotti , CL Ruiz de Frattari , S Vargas , AH González Silva, N Novak , ITC Influence of B7 costimulatory molecules colocalized in extracellular traps (ETs) on CD45RO expression in human autologous cultures |
| topic_facet |
extracellular traps human leukocytes, B7 molecules, CD45RO, autologous culture trampas extracelulares leucocitos humanos moléculas B7 CD45RO cultivos autólogos |
| author |
Castellani Austerlitz, MP Rodríguez, FM Carabajal Miotti , CL Ruiz de Frattari , S Vargas , AH González Silva, N Novak , ITC |
| author_facet |
Castellani Austerlitz, MP Rodríguez, FM Carabajal Miotti , CL Ruiz de Frattari , S Vargas , AH González Silva, N Novak , ITC |
| author_sort |
Castellani Austerlitz, MP |
| title |
Influence of B7 costimulatory molecules colocalized in extracellular traps (ETs) on CD45RO expression in human autologous cultures |
| title_short |
Influence of B7 costimulatory molecules colocalized in extracellular traps (ETs) on CD45RO expression in human autologous cultures |
| title_full |
Influence of B7 costimulatory molecules colocalized in extracellular traps (ETs) on CD45RO expression in human autologous cultures |
| title_fullStr |
Influence of B7 costimulatory molecules colocalized in extracellular traps (ETs) on CD45RO expression in human autologous cultures |
| title_full_unstemmed |
Influence of B7 costimulatory molecules colocalized in extracellular traps (ETs) on CD45RO expression in human autologous cultures |
| title_sort |
influence of b7 costimulatory molecules colocalized in extracellular traps (ets) on cd45ro expression in human autologous cultures |
| description |
Extracellular traps (ETs) are structures composed of chromatin, histones and granular proteins, and constitute a functional microbicidal mechanism of immune cells. Its participation in the pathophysiology of many pathologies, including COVID-19, autoimmune diseases and cancer, has been studied. Costimulatory molecules B7 (B7-1:CD80 and B7-2:CD86) provide the second activation signal for T cells, and their absence is a mechanism of peripheral tolerance. The objectives were to study the influence of B7 molecules colocalized in ETs generated in vitro from leukocytes, on the expression of common leukocyte antigen CD45RO in autologous human blood cell cultures. This molecule is an indicator of T cell activation, and its expression in neutrophils is related to adhesion and activation.
For that reason, we set out to generate and isolate ETs in autologous cultures of leukocytes exposed to lipopolysaccharide (LPS); to mark B7 molecules in isolated ETs and observe the expression of the CD4 coreceptor and CD45RO in interaction assays of ETs and autologous leukocytes in culture. Autologous leukocyte cultures were performed from healthy human blood samples anticoagulated with heparin, (n = 10) (with informed consent, Ethics Committee, HNC, FCM), stimulated with LPS for generation of ETs. We performed: isolation of ETs and ETs interaction assays with autologous cultures. Immunofluorescence (IF) with anti-CD80, anti-CD86, anti-CD4 and anti-CD45RO, DNA staining with DAPI. Controls: paired culture samples. Quantification of labeled cells was performed with FIJI program. Statistical treatment: t-test for paired samples.
At 24 hours of culture, no significant differences (p<0.05) were observed in the percentages of positive cells for CD4 and CD45RO between the paired control samples and those with added ETs. At 72 hours, significant differences (p<0.05) were observed in the expression of CD45RO between the controls and the samples with added ETs. In one donor, significant differences (p<0.05) were observed in the percentages of CD4-positive cells.
The presence of B7 molecules in ETs could give the second signal required for the activation of autoreactive T lymphocytes present in the autologous culture of total leukocytes, implying the possibility of breaking self-tolerance. |
| publisher |
Universidad Nacional Córdoba. Facultad de Ciencias Médicas. Secretaria de Ciencia y Tecnología |
| publishDate |
2022 |
| url |
https://revistas.unc.edu.ar/index.php/med/article/view/39025 |
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I10-R327-article-390252024-04-15T16:14:45Z Influence of B7 costimulatory molecules colocalized in extracellular traps (ETs) on CD45RO expression in human autologous cultures Influencia de moléculas coestimuladoras B7 colocalizadas en trampas extracelulares (ETs) en la expresión de CD45RO en cultivos autólogos humanos Castellani Austerlitz, MP Rodríguez, FM Carabajal Miotti , CL Ruiz de Frattari , S Vargas , AH González Silva, N Novak , ITC extracellular traps human leukocytes, B7 molecules, CD45RO, autologous culture trampas extracelulares leucocitos humanos moléculas B7 CD45RO cultivos autólogos Extracellular traps (ETs) are structures composed of chromatin, histones and granular proteins, and constitute a functional microbicidal mechanism of immune cells. Its participation in the pathophysiology of many pathologies, including COVID-19, autoimmune diseases and cancer, has been studied. Costimulatory molecules B7 (B7-1:CD80 and B7-2:CD86) provide the second activation signal for T cells, and their absence is a mechanism of peripheral tolerance. The objectives were to study the influence of B7 molecules colocalized in ETs generated in vitro from leukocytes, on the expression of common leukocyte antigen CD45RO in autologous human blood cell cultures. This molecule is an indicator of T cell activation, and its expression in neutrophils is related to adhesion and activation. For that reason, we set out to generate and isolate ETs in autologous cultures of leukocytes exposed to lipopolysaccharide (LPS); to mark B7 molecules in isolated ETs and observe the expression of the CD4 coreceptor and CD45RO in interaction assays of ETs and autologous leukocytes in culture. Autologous leukocyte cultures were performed from healthy human blood samples anticoagulated with heparin, (n = 10) (with informed consent, Ethics Committee, HNC, FCM), stimulated with LPS for generation of ETs. We performed: isolation of ETs and ETs interaction assays with autologous cultures. Immunofluorescence (IF) with anti-CD80, anti-CD86, anti-CD4 and anti-CD45RO, DNA staining with DAPI. Controls: paired culture samples. Quantification of labeled cells was performed with FIJI program. Statistical treatment: t-test for paired samples. At 24 hours of culture, no significant differences (p<0.05) were observed in the percentages of positive cells for CD4 and CD45RO between the paired control samples and those with added ETs. At 72 hours, significant differences (p<0.05) were observed in the expression of CD45RO between the controls and the samples with added ETs. In one donor, significant differences (p<0.05) were observed in the percentages of CD4-positive cells. The presence of B7 molecules in ETs could give the second signal required for the activation of autoreactive T lymphocytes present in the autologous culture of total leukocytes, implying the possibility of breaking self-tolerance. Las trampas extracelulares (ETs) son estructuras compuestas de cromatina, histonas y proteínas granulares y constituyen un mecanismo funcional microbicida de células inmunes. Se ha estudiado su participación en la fisiopatogenia de diversas patologías, entre ellas COVID-19, enfermedades autoinmunes y cáncer. Las moléculas coestimuladoras B7 (B7-1:CD80 y B7-2:CD86) brindan la segunda señal de activación para células T y su ausencia es un mecanismo de tolerancia periférica. Los objetivos fueron estudiar la influencia de las moléculas B7 colocalizadas en ETs generadas in vitro a partir de leucocitos sobre la expresión del antígeno común leucocitario CD45RO en cultivos celulares autólogos de sangre humana. Esta molécula es un marcador de activación de células T, y su expresión en neutrófilos, es relacionada a adhesión y activación. Para ello nos propusimos generar y aislar ETs en cultivos autólogos de leucocitos expuestos a lipopolisacarido (LPS); marcar moléculas B7 en ETs aisladas y observar la expresión del correceptor CD4 y CD45RO en ensayos de interacción de ETs y leucocitos autólogos en cultivo. Se realizaron cultivos autólogos de leucocitos de muestras de sangre humana sana anticoagulada con heparina, (n = 10) (con consentimiento informado, Comité de Ética, HNC, FCM), estimulados con LPS para generación de ETs. Se realizó: aislamiento de ETs y ensayos de interacción ETs con cultivos autólogos. Inmunofluorescencia con anti-CD80, anti CD86, anti CD4 y anti CD45RO, tinción de ADN: DAPI. Controles: muestras de cultivos apareados. Cuantificación de células marcadas con programa FIJI. Tratamiento estadístico: test t para muestras apareadas. A 24 horas de cultivo no se observaron diferencias significativas (p<0,05) en los porcentajes de células positivas para CD4 y CD45RO entre las muestras apareadas control y con agregado de ETs. A 72 horas se observaron diferencias significativas (p<0,05) en la expresión de CD45RO entre los controles y las muestras con agregado de ETs. En un donante se observaron diferencias significativas (p<0,05) en los porcentajes de células positivas para CD4. La presencia de moléculas B7 en ETs podría dar la segunda señal requerida para la activación de linfocitos T autorreactivos presentes en el cultivo autólogo de leucocitos totales implicando la posibilidad de ruptura de autotolerancia. Universidad Nacional Córdoba. Facultad de Ciencias Médicas. Secretaria de Ciencia y Tecnología 2022-10-26 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion . https://revistas.unc.edu.ar/index.php/med/article/view/39025 Revista de la Facultad de Ciencias Médicas de Córdoba.; Vol. 79 No. Suplemento JIC XXIII (2022): Suplemento JIC XXIII Revista de la Facultad de Ciencias Médicas de Córdoba; Vol. 79 Núm. Suplemento JIC XXIII (2022): Suplemento JIC XXIII Revista da Faculdade de Ciências Médicas de Córdoba; v. 79 n. Suplemento JIC XXIII (2022): Suplemento JIC XXIII 1853-0605 0014-6722 http://creativecommons.org/licenses/by-nc/4.0 |