Immunodetection of Cell Wall Components in Studies on Cell Wall Rebuilding in Fagopyrum esculentum and Fagopyrum tataricum.

Immunocytochemical studies of the cell wall are used to visualize specific epitopes of pectins, arabinogalactan proteins, hemicelluloses, extensins, and other wall components using specific primary antibodies. This reaction, combined with calcofluor staining, allows to comprehend how the cell wall is rebuilt during the protoplast culture. In this protocol, the method of immunostaining using antibodies against cell wall components based on Fagopyrum esculentum and Fagopyrum tataricum protoplasts is described.

Immunohistochemical Detection of the Wall Components on the Example of Shoot Apical Meristem of Fagopyrum esculentum and Fagopyrum tataricum.

Immunohistochemistry is a method that allows the detection of individual components of cell walls in an extremely precise way at the level of a single cell and wall domains. The cell wall antibodies detect specific epitopes of pectins, arabinogalactan proteins (AGP), hemicelluloses, and extensins. The presented method visualization of the selected pectic and AGP epitopes using antibodies directed to wall components is described. The method of the analysis of the chemical composition of the wall is present on the example of the shoot apical meristems of Fagopurum esculentum and Fagopyrum tataricum. Recommended protocols for immunostaining and examination on fluorescence microscopy level are presented.

CCL19-Positive Lymph Node Stromal Cells Govern the Onset of Inflammatory Arthritis via Tropomyosin Receptor Kinase.

OBJECTIVE: The study objective was to assess the role of CCL19+ lymph node stromal cells of the joint-draining popliteal lymph node (pLN) for the development of arthritis. METHODS: CCL19+ lymph node stromal cells were spatiotemporally depleted for five days in the pLN before the onset of collagen-induced arthritis (CIA) using Ccl19-Cre × iDTR mice. In addition, therapeutic treatment with recombinant CCL19-immunoglobulin G (IgG), locally injected in the footpad, was used to confirm the results. RNA sequencing of lymph node stromal cells combined with T cell coculture assays using tropomyosin receptor kinase (Trk) family inhibitors together with in vivo local pLN small interfering RNA (siRNA) treatments were used to elucidate the pathway by which CCL19+ lymph node stromal cells initiate the onset of arthritis. RESULTS: Spatiotemporal depletion of CCL19+ lymph node stromal cells prevented disease onset in CIA mice. These inhibitory effects could be mimicked by local CCL19-IgG treatment. The messenger RNA sequencing analyses showed that CCL19+ lymph node stromal cells down-regulated the expression of the tropomyosin receptor kinase A (TrkA) just before disease onset. Blocking TrkA in lymph node stromal cells led to increased T cell proliferation in in vitro coculture assays. Similar effects were observed with the pan-Trk inhibitor larotrectinib in cocultures of lymph node stromal cells of patients with rheumatoid arthritis and T cells. Finally, local pLN treatment with TrkA inhibitor and TrkA siRNA led to exacerbated arthritis scores. CONCLUSION: CCL19+ lymph node stromal cells are crucially involved in the development of inflammatory arthritis. Therefore, targeting of CCL19+ lymph node stromal cells via TRK could provide a tool to prevent arthritis.