28 research outputs found

    Innate IFN-γ-Producing Cells in the Spleen of Mice Early after Listeria monocytogenes Infection: Importance of Microenvironment of the Cells Involved in the Production of Innate IFN-γ

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    Production of innate interferon-γ (IFN-γ) is a crucial step in immunological defense against bacteria. However, there is little information regarding cellular mechanisms underlying IFN-γ production in vivo early after bacterial infection. Here we analyze innate IFN-γ production in the spleen of mice early after Listeria monocytogenes (LM) infection ex vivo by flow-cytometry and in situ by immunohistochemistry, and compare them with the IFN-γ-producing cells reported previously in our in vitro coculture system in which cell-cell interaction between lymphocytes and dying bacterial-infected macrophages is required for the production of IFN-γ. In the spleen at 20 h after LM infection, natural killer (NK) cells, a subset of αβ T cells, and subsets of NKT and γδ T cells produced IFN-γ with features similar to the IFN-γ-producing cells in our in vitro coculture system. Immunohistochemistry revealed that LM bacteria were first phagocytosed mainly by ER-TR9+ marginal zone macrophages (MZMs), then forming infectious foci in close vicinity of the marginal zone (MZ) at 20-h postinfection. At this time point, the IFN-γ-producing cells were accumulating at the same site of infectious foci, around which ER-TR9+ MZMs were clustered but most of bacteria were no longer associated with ER-TR9+ MZMs. These results indicate that innate IFN-γ production by innate lymphocytes takes place at infectious foci formed in close vicinity of the MZ, and they also suggest an important role for the microenvironment of the cells accumulated at infectious foci in inducing the production of innate IFN-γ

    Cellular Fragments as Biomaterial for Rapid In Vitro Bone-Like Tissue Synthesis

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    Current stem cell-based techniques for bone-like tissue synthesis require at least two to three weeks. Therefore, novel techniques to promote rapid 3D bone-like tissue synthesis in vitro are still required. In this study, we explored the concept of using cell nanofragments as a substrate material to promote rapid bone formation in vitro. The methods for cell nanofragment fabrication were ultrasonication (30 s and 3 min), non-ionic detergent (triton 0.1% and 1%), or freeze-dried powder. The results showed that ultrasonication for 3 min allowed the fabrication of homogeneous nanofragments of less than 150 nm in length, which mineralized surprisingly in just one day, faster than the fragments obtained from all other methods. Further optimization of culture conditions indicated that a concentration of 10 mM or 100 mM of beta-glycerophosphate enhanced, whereas fetal bovine serum (FBS) inhibited in a concentration-dependent manner, the mineralization of the cell nanofragments. Finally, a 3D collagen-cell nanofragment-mineral complex mimicking a bone-like structure was generated in just two days by combining the cell nanofragments in collagen gel. In conclusion, sonication for three min could be applied as a novel method to fabricate cell nanofragments of less than 150 nm in length, which can be used as a material for in vitro bone tissue engineering

    Surgical resection for advanced bisphosphonate-related osteonecrosis of the jaw associated with fibrous dysplasia: a case report

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    Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is an adverse drug reaction represented by destruction and/or death of bone. Fibrous dysplasia (FD) is a rare bony disorder characterised by abnormal fibro-osseous tissue that has lowered resistance to infection. Effective treatments for BRONJ that follows FD are unclear. Here, we report that advanced BRONJ associated with FD was successfully treated by surgical resection. A 69-year-old woman, whose left maxillary bone showed a ground glass appearance on computed tomography (CT) images, was taking alendronate. At 1 year after teeth within the abnormal bone were extracted, exposed bone was observed in the extraction sites and CT images revealed separated sequestrums. Under the clinical diagnosis of Stage 2 BRONJ with FD, we performed not only sequestrectomy but also a partial resection of the FD. Thereafter, the healing was uneventful without recurrence. In conclusion, our case suggests that surgical resection is useful for advanced BRONJ associated with FD

    Fabrication of initial trabecular bone inspired three-dimensional structure with cell membrane nanofragments

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    Koichi Kadoya, Emilio Satoshi Hara, Masahiro Okada, Yu Yang Jiao, Takayoshi Nakano, Akira Sasaki, Takuya Matsumoto, Fabrication of initial trabecular bone inspired three-dimensional structure with cell membrane nanofragments, Regenerative Biomaterials, 2022, rbac088, https://doi.org/10.1093/rb/rbac08

    Terrein Exhibits Anti-tumor Activity by Suppressing Angiogenin Expression in Malignant Melanoma Cells

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    Background/Aim: Malignant melanoma is a tumor with a poor prognosis that can metastasize distally at an early stage. Terrein, a metabolite produced by Aspergillus terreus, suppresses the expression of angiogenin, an angiogenic factor. However, the pharmacological effects of natural terrein have not been elucidated, because only a small amount of terrein can be extracted from large fungal cultures. In this study, we investigated the antineoplastic effects of terrein on human malignant melanoma cells and its underlying mechanisms. Materials and methods: Human malignant melanoma cell lines were cultured in the presence of terrein and analyzed. Angiogenin production was evaluated using ELISA. Ribosome biosynthesis was evaluated using silver staining of the nucleolar organizer region. Intracellular signaling pathways were analyzed using western blotting. Malignant melanoma cells were transplanted subcutaneously into the backs of nude mice. The tumors were removed at 5 weeks and analyzed histopathologically. Results: Terrein inhibited angiogenin expression, proliferation, migration, invasion, and ribosome biosynthesis in malignant melanoma cells. Terrein was shown to inhibit tumor growth and angiogenesis in animal models. Conclusion: This study demonstrated that terrein has anti-tumor effects against malignant melanoma. Furthermore, chemically synthesized non-natural terrein can be mass-produced and serve as a novel potential anti-tumor drug candidate

    CCN3 (NOV) Drives Degradative Changes in Aging Articular Cartilage

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    Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of Ccn3 mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with p21 and p53 mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 in vitro, either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by H2O2 caused a dose-dependent increase in Ccn3 gene and CCN3 protein expression, along with enhanced expression of p21 and p53 mRNA and proteins, as well as SA-beta gal activity. Overexpression of CCN3 also enhanced p21 promoter activity via p53. Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of p21 and p53 mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21

    CrkL directs ASAP1 to peripheral focal adhesions

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    Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein. In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions

    Immunological characteristics of bronchoalveolar lavage fluid and blood across connective tissue disease-associated interstitial lung diseases

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    Interstitial lung disease (ILD) is a serious complication of connective tissue diseases (CTDs). The heterogeneity of ILDs reflects differences in pathogenesis among diseases. This study aimed to clarify the characteristics of CTD-ILDs via a detailed analysis of the bronchoalveolar lavage fluid (BALF) and blood immune cells. BALF and blood samples were collected from 39 Japanese patients with newly diagnosed ILD: five patients with Sjögren’s syndrome (SS), eight patients with dermatomyositis (DM), six patients with rheumatoid arthritis (RA), six patients with systemic sclerosis, four patients with anti-neutrophil cytoplasmic antibody-associated vasculitis, and 10 patients with idiopathic interstitial pneumonia. We performed single-cell RNA sequencing to analyze the gene expression profiles in these patients’ immune cells. In patients with SS, B cells in the BALF were increased and genes associated with the innate and acquired immunity were enriched in both the BALF and blood. In contrast, patients with DM showed an upregulation of genes associated with viral infection in both the BALF and blood. In patients with RA, neutrophils in the BALF tended to increase, and their gene expression patterns changed towards inflammation. These disease-specific characteristics may help us understand the pathogenesis for each disease and discover potential biomarkers

    Fabrication of initial trabecular bone-inspired three-dimensional structure with cell membrane nano fragments

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    The extracellular matrix of trabecular bone has a large surface exposed to the bone marrow and plays important roles such as hematopoietic stem cell niche formation and maintenance. In vitro reproduction of trabecular bone microenvironment would be valuable not only for developing a functional scaffold for bone marrow tissue engineering but also for understanding its biological functions. Herein, we analyzed and reproduced the initial stages of trabecular bone formation in mouse femur epiphysis. We identified that the trabecular bone formation progressed through the following steps: (i) partial rupture of hypertrophic chondrocytes; (ii) calcospherite formation on cell membrane nano fragments (CNFs) derived from the ruptured cells; and (iii) calcospherite growth and fusion to form the initial three-dimensional (3D) structure of trabecular bones. For reproducing the initial trabecular bone formation in vitro, we collected CNFs from cultured cells and used as nucleation sites for biomimetic calcospherite formation. Strikingly, almost the same 3D structure of the initial trabecular bone could be obtained in vitro by using additional CNFs as a binder to fuse biomimetic calcospherites
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