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SummaryMotor skill learning stimulates and requires generation of myelinating oligodendrocytes (OLs) from their precursors. We asked whether OL production is also required for non-motor learning and cognition, using T-maze and radial arm maze tasks that tax spatial working memory. Maze training stimulated OL production in the medial prefrontal cortex, anterior corpus callosum (genu), hippocampus and fimbria; myelin sheath formation was also stimulated in the genu. Genetic blockade of OL differentiation and neo-myelination inMyrfconditional knockout mice strongly impaired training-induced improvements in maze performance. Remarkably, working memory performance of individual mice correlated closely with the scale of OL precursor proliferation and OL generation in their genu and anterior cingulate cortex during training, indicating a key role for adaptive OL genesis and myelination in cognitive processing.

Generally, it is difficult to observe IAV particles in postmortem samples from patients with seasonal influenza. In fact, only a few viral antigens are detected in bronchial epithelial cells from autopsied lung sections. Previously, we detected many viral antigens in AEC-IIs from the lung. This was because the majority of A/H1N1/pdm09 in the lung tissue harbored an aspartic acid-to-glycine substitution at position 222 (D222G) of the hemagglutinin protein. A/H1N1/pdm09 harboring the D222G substitution has a receptor-binding preference for α-2,3-linked sialic acids expressed on human AECs and infects them in the same way as H5N1 and H7N9 avian IAVs. Here, we report the first successful observation of virus particles, not only in AEC-IIs, but also in Ms/Mϕs and Neus, using electron microscopy. The finding of a M/Mϕ harboring numerous virus particles within vesicles and at the cell surface suggests that Ms/Mϕs are involved in the pathogenesis of IAV primary pneumonia., A/H1N1 2009 pandemic influenza virus (A/H1N1/pdm09) was first identified as a novel pandemic influenza A virus (IAV) in 2009. Previously, we reported that many viral antigens were detected in type II alveolar epithelial cells (AEC-IIs) within autopsied lung tissue from a patient with A/H1N1/pdm09 pneumonia. It is important to identify the association between the virus and host cells to elucidate the pathogenesis of IAV pneumonia. To investigate the distribution of virus particles and morphological changes in host cells, the autopsied lung specimens from this patient were examined using transmission electron microscopy (TEM) and a novel scanning electron microscopy (SEM) method. We focused on AEC-IIs as viral antigen-positive cells and on monocytes/macrophages (Ms/Mϕs) and neutrophils (Neus) as innate immune cells. We identified virus particles and intranuclear dense tubules, which are associated with matrix 1 (M1) proteins from IAV. Large-scale two-dimensional observation was enabled by digitally “stitching” together contiguous SEM images. A single whole-cell analysis using a serial section array (SSA)-SEM identified virus particles in vesicles within the cytoplasm and/or around the surfaces of AEC-IIs, Ms/Mϕs, and Neus; however, intranuclear dense tubules were found only in AEC-IIs. Computer-assisted processing of SSA-SEM images from each cell type enabled three-dimensional (3D) modeling of the distribution of virus particles within an ACE-II, a M/Mϕ, and a Neu. IMPORTANCE Generally, it is difficult to observe IAV particles in postmortem samples from patients with seasonal influenza. In fact, only a few viral antigens are detected in bronchial epithelial cells from autopsied lung sections. Previously, we detected many viral antigens in AEC-IIs from the lung. This was because the majority of A/H1N1/pdm09 in the lung tissue harbored an aspartic acid-to-glycine substitution at position 222 (D222G) of the hemagglutinin protein. A/H1N1/pdm09 harboring the D222G substitution has a receptor-binding preference for α-2,3-linked sialic acids expressed on human AECs and infects them in the same way as H5N1 and H7N9 avian IAVs. Here, we report the first successful observation of virus particles, not only in AEC-IIs, but also in Ms/Mϕs and Neus, using electron microscopy. The finding of a M/Mϕ harboring numerous virus particles within vesicles and at the cell surface suggests that Ms/Mϕs are involved in the pathogenesis of IAV primary pneumonia.

Background: Applying recent advances in electron microscope technology, cells and cell-cell interactions are observable using virtual slides, three-dimensional (3D) imaging, and serial sections of ultrathin thickness. Inflammation in rheumatoid arthritis (RA) is caused by multiple cell types, including infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and spindle-shaped fibroblasts. Especially, we are focusing on fibroblast-like synoviocytes (FLSs). These cells have been reported to be positive for multiple markers including CD14, CD68, HLA-DR and vimentin, and we have shown that these cells are dendritic cells in our previous study using electron microscopy. Therefore, FLSs are presumed to function as dendritic cells. and these cells involved in the nursing phenomenon. Objectives: The objective of this study is to investigate the chronicity mechanism of immunological inflammation in RA by using electron microscopy. Methods: Synovial tissues collected from RA patients undergoing elbow or knee joint surgery were prepared for this study. Using wide field of view images of the synovial tissue, areas containing the nursing phenomenon were selected by using multiscale electron microscopy. 3D structures of the features were constructed from series of micrographs obtained by using focused ion beam-scanning electron microscopy and those by using transmission electron microscopy with double-axis electron tomography (DA-ET). Results: From multiscale electron microscopy, many synovial dendritic cells and plasma cells were present in various areas of the lymphnodular infiltration. Serial cross-sectional observation with an FIB-SEM and 3D images revealed that synovial dendritic cells patronized the plasma cells, wrapping around the plasma cells, by the long slender axes. A tomogram of an ultra-thin sliced section created by using DA-ET revealed that synovial dendritic cells formed a membranous fusion with the plasma cells. Conclusion: The nursing phenomenon involves characteristic histopathological features, which may be a factor in the chronicity of the inflammation mechanism in RA synovial tissue. References [1] Ochi T, Yoshikawa H, Toyosaki-Maeda T, Lipsky PE. Mesenchymal stromal cells. Nurse-like cells reside in thesynovial tissue and bone marrow in rheumatoid arthritis. Arthritis ResearchaTherapy2007; 9(1): 201. [2] Ochi T, Sawai T, Murakami K, Kamataki A, Uzuki M, Tomita T, et al. Nurse-like cells in rheumatoid arthritis: Formation of survival niches cooperating between the cell types. Mod Rheum2018; 29: 1-5. Disclosure of Interests: None declared

Background:Inflammation in rheumatoid arthritis (RA) is caused by multiple cell types, including infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and spindle-shaped fibroblasts. Especially, we are focusing on fibroblast-like synoviocytes (FLSs). In our previous study, we have reported that FLSs were positive for multiple markers including CD14, CD68 and HLA-DR, and were dendritic-shaped cells constituting nursing phenomenon between lymph or plasma cells. In addition, in our recent study, we found that CD14+FLSs were positive for vimentin (VIM), which is a marker for mesenchymal cells. There are still many issues to be discussed regarding CD14+VIM+ cells.Objectives:To investigate the relationship between CD14+VIM+ cells and the degree of synovitis in rheumatoid arthritis.Methods:Synovial tissues collected from RA patients who underwent joint surgeries were prepared for this study. First, the proportions of CD14+ cells in RA synovial tissue and control were analyzed using flow cytometry and the concentrations of inflammatory cytokines released by CD14+ cells in RA synovial tissue and control were examined by ELISA. Next, the proportions of CD14+VIM+ cells in RA synovial tissue and control were examined immunohistologically and then we analyzed the results using image analysis software. Also, we statistically analyzed the relationship between the proportion of CD14+VIM+ cells, the degree of synovitis, and clinical data.Results:Results of flow cytometry showed that CD14+ cells were frequently observed in RA synovial tissue than control. Cultured CD14+ cells released more inflammatory cytokines than cultured CD14- cells. Also, results of immunohistological staining showed that many CD14+VIM+ cells were observed in RA synovial tissue than in control. The proportion of CD14+VIM+ cells was correlated with Krenn synovitis score. High proportion cases significantly showed high level of CRP and MMP-3.Conclusion:CD14+VIM+ cells might be involved in the mechanism of chronic immunological inflammation in RA and the proportion of these cells might influence the clinical data.References:[1]Ochi T, Yoshikawa H, Toyosaki-Maeda T, Lipsky PE. Mesenchymal stromal cells. Nurse-like cells reside in thesynovial tissue and bone marrow in rheumatoid arthritis. Arthritis Research&Therapy 2007; 9(1): 201.[2]Ochi T, Sawai T, Murakami K, Kamataki A, Uzuki M, Tomita T, et al. Nurse-like cells in rheumatoid arthritis: Formation of survival niches cooperating between the cell types. Mod Rheum 2018; 29: 1-5.[3]Krenn V, Morawietz L, Burmester GR, Kinne RW, Muller-Ladner U, Muller B, Haupl T. Synovitis score: discrimination between chronic low-grade and high-grade synovitis. Histopathology 2006; 49: 358-64.Disclosure of Interests:None declared

Summary The lymphatic system comprises blind-ended tubes that collect interstitial fluid and return it to the circulatory system. In mammals, unidirectional lymphatic flow is driven by muscle contraction working in conjunction with valves. Accordingly, defective lymphatic valve morphogenesis results in backflow leading to edema. In fish species, studies dating to the 18th century failed to identify lymphatic valves, a precedent that currently persists, raising the question of whether the zebrafish could be used to study the development of these structures. Here, we provide functional and morphological evidence of valves in the zebrafish lymphatic system. Electron microscopy revealed valve ultrastructure similar to mammals, while live imaging using transgenic lines identified the developmental origins of lymphatic valve progenitors. Zebrafish embryos bearing mutations in genes required for mammalian valve morphogenesis show defective lymphatic valve formation and edema. Together, our observations provide a foundation from which to further investigate lymphatic valve formation in zebrafish.

The dermis of sea cucumbers is a catch connective tissue or a mutable collagenous tissue that shows rapid, large and reversible stiffness changes in response to stimulation. The main component of the dermis is the extracellular material composed of collagen fibrils embedded in a hydrogel of proteoglycans. The stiffness of the extracellular material determines that of the dermis. The dermis has three mechanical states: soft (Sa), standard (Sb) and stiff (Sc). We studied the ultrastructural changes associated with the stiffness changes. Transverse sections of collagen fibrils in the dermis showed irregular perimeters with electron-dense protrusions or arms that cross-bridged between fibrils. The number of cross-bridges increased in stiffer dermis. The distance between the fibrils was shorter in Sc than that in other states, which was in accord with the previous report that water exuded from the tissue in the transition Sb→Sc. The ultrastructure of collagen fibrils that had been isolated from the dermis was also studied. Fibrils aggregated by tensilin, which causes the transition Sa→Sb possibly through an increase in cohesive forces between fibrils, had larger diameter than those dispersed by softenin, which antagonizes the effect of tensilin. No cross-bridges were found in isolated collagen fibrils. From the present ultrastructural study we propose that three different mechanisms work together to increase the dermal stiffness. 1.Tensilin makes collagen fibrils stronger and stiffer in Sa→Sb through an increase in cohesive forces between subfibrils that constituted fibrils; 2. Cross-bridging by arms caused the fibrils to be a continuous network of bundles both in Sa→Sb and in Sb→Sc; 3. The matrix embedding the fibril network became stiffer in Sb→Sc, which was produced by bonding associated with water exudation.

ALAS2 gene mutations cause X-linked sideroblastic anemia. The presence of ring sideroblasts in a patient's bone marrow is the hallmark of sideroblastic anemia, but the precise mechanisms underlying sideroblast formation are largely unknown. Using a genome-editing system, a mutation was introduced in the erythroid-specific enhancer of the ALAS2 gene in HUDEP2 cells, which were derived from human umbilical stem cells and can produce erythrocytes. The established cell line, termed HA2low, expressed less ALAS2 mRNA than did wild-type cells, even after erythroid differentiation. Although the mRNA expression of α-globin, β-globin, and the mitochondrial iron importer mitoferrin-1 was induced similarly in wild-type and HA2low cells, hemoglobinization of differentiated cells was limited in HA2low cells compared with wild-type cells. Importantly, Prussian blue staining revealed that approximately one-third of differentiated HA2low cells exhibited intracellular iron deposition and these cells looked like ring sideroblasts. Electron microscopy confirmed that the mitochondria in HA2low cells contained high-density deposits that might contain iron. Ring sideroblastic cells appeared among HA2low cells only after differentiation, whereas the induced expression of mitochondrial ferritin was observed in both cell types during differentiation. These results suggest that the induction of mitochondrial ferritin expression might be essential for, but not the primary cause of, ring sideroblast formation. Our results also suggest that the insufficient supply of protoporphyrin IX due to ALAS2 deficiency in combination with increased iron import into mitochondria during erythroid differentiation results in the formation of ring sideroblasts. Furthermore, HA2low cells are a useful tool for characterizing ring sideroblasts in vitro.