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Characterization of the distribution and dynamics of the antigen-presenting cells using MHC II-EGFP knock-in mouse model
Pačes, Jan ; Černý, Jan (advisor) ; Tlaskalová - Hogenová, Helena (referee)
Results of recent studies indicate that dendritic cells are capable of transporting commensal intestinal bacteria into the mammary glands, which ultimately leads to their occurrence in breast milk. We have therefore decided to evaluate the phenotype of immunologically relevant antigen presenting cells (APCs) present in the mammary glands and the small intestine, respectively and perform a comparison study. We also studied plasticity of these populations during lactation. In situ immunodetection and flow cytometry methods were used to determine phenotype. We succeeded in optimising the methods for preparation of samples for flow cytometry and microscopy. We thoroughly tested protocols for 3D visualisation of APC populations and quantitative image analysis for correlation with flow cytometry, further optimization is nevertheless needed. We found out that during lactation large numbers of MHC II+ cells cluster around the alveoli and milk ducts. These cells are of a distinctly dendritic shape and their phenotype does not correspond to the APCs in the surrounding tissue. A pronounced increase of APC cells in the mammary glands between the fourth and sixth days of lactation was observed, with the majority of these cells expressing the CD103 antigen typical for cell populations of immune cells of the...
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Characterization of the distribution and dynamics of the antigen-presenting cells using MHC II-EGFP knock-in mouse model
Pačes, Jan ; Černý, Jan (advisor) ; Tlaskalová - Hogenová, Helena (referee)
Results of recent studies indicate that dendritic cells are capable of transporting commensal intestinal bacteria into the mammary glands, which ultimately leads to their occurrence in breast milk. We have therefore decided to evaluate the phenotype of immunologically relevant antigen presenting cells (APCs) present in the mammary glands and the small intestine, respectively and perform a comparison study. We also studied plasticity of these populations during lactation. In situ immunodetection and flow cytometry methods were used to determine phenotype. We succeeded in optimising the methods for preparation of samples for flow cytometry and microscopy. We thoroughly tested protocols for 3D visualisation of APC populations and quantitative image analysis for correlation with flow cytometry, further optimization is nevertheless needed. We found out that during lactation large numbers of MHC II+ cells cluster around the alveoli and milk ducts. These cells are of a distinctly dendritic shape and their phenotype does not correspond to the APCs in the surrounding tissue. A pronounced increase of APC cells in the mammary glands between the fourth and sixth days of lactation was observed, with the majority of these cells expressing the CD103 antigen typical for cell populations of immune cells of the...
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The role of Src-family kinases in the immunological synapse of antigen presenting cells.
Kotlabová, Klára ; Brdička, Tomáš (advisor) ; Brábek, Jan (referee)
Antigen presentation during which antigen fragments in complex with MHC glycoproteins are recognized by T cell antigen-specific receptors is necessary for the initiation of adaptive immune response. During this process, immunological synapse is assembled at the site of contact between the T cell and the antigen-presenting cell (APC). This leads to the activation of receptors on the surface of both cells followed by triggering of multiple signaling pathways. However, our knowledge about the signaling occurring at the APC-side of the IS is limited in comparison to the T cell side. Here, we analyze role of Src family kinases in the APC signaling pathways. For this purpose, constructs targeting Csk kinase to the plasma membrane of APCs were prepared to inhibit SFKs there. We show that expression of these constructs inhibits activation of SFKs, calcium mobilization and cell activation of K46 B cell line. Further, expression of these constructs in hematopoietic progenitors attenuates their differentiation into dendritic cells which then results in their decreased ability to stimulate T cells.
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