Enhanced GATA4 expression in senescent systemic lupus erythematosus monocytes promotes high levels of IFNα production.

Enhanced interferon α (IFNα) production has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). We previously reported IFNα production by monocytes upon activation of the stimulator of IFN genes (STING) pathway was enhanced in patients with SLE. We investigated the mechanism of enhanced IFNα production in SLE monocytes. Monocytes enriched from the peripheral blood of SLE patients and healthy controls (HC) were stimulated with 2'3'-cyclic GAMP (2'3'-cGAMP), a ligand of STING. IFNα positive/negative cells were FACS-sorted for RNA-sequencing analysis. Gene expression in untreated and 2'3'-cGAMP-stimulated SLE and HC monocytes was quantified by real-time PCR. The effect of GATA binding protein 4 (GATA4) on IFNα production was investigated by overexpressing GATA4 in monocytic U937 cells by vector transfection. Chromatin immunoprecipitation was performed to identify GATA4 binding target genes in U937 cells stimulated with 2'3'-cGAMP. Differentially expressed gene analysis of cGAS-STING stimulated SLE and HC monocytes revealed the enrichment of gene sets related to cellular senescence in SLE. CDKN2A, a marker gene of cellular senescence, was upregulated in SLE monocytes at steady state, and its expression was further enhanced upon STING stimulation. GATA4 expression was upregulated in IFNα-positive SLE monocytes. Overexpression of GATA4 enhanced IFNα production in U937 cells. GATA4 bound to the enhancer region of IFIT family genes and promoted the expressions of IFIT1, IFIT2, and IFIT3, which promote type I IFN induction. SLE monocytes with accelerated cellular senescence produced high levels of IFNα related to GATA4 expression upon activation of the cGAS-STING pathway.

Immunoglobulin directly enhances differentiation of oligodendrocyte-precursor cells and remyelination.

Multiple sclerosis (MS) is an inflammatory demyelinating disease characterized by multiple lesions in the central nervous system. Although the role of B cells in MS pathogenesis has attracted much attention, but the detailed mechanisms remain unclear. To investigate the effects of B cells on demyelination, we analyzed a cuprizone-induced demyelination model, and found that demyelination was significantly exacerbated in B cell-deficient mice. We next investigated whether immunoglobulin affected the myelin formation process using organotypic brain slice cultures and revealed that remyelination was improved in immunoglobulin-treated groups compared with the control group. Analysis of oligodendrocyte-precursor cell (OPC) monocultures showed that immunoglobulins directly affected on OPCs and promoted their differentiation and myelination. Furthermore, OPCs expressed FcγRI and FcγRIII, two receptors that were revealed to mediate the effects of IgG. To the best of our knowledge, this is the first study to demonstrate that B cells act in an inhibitory manner against cuprizone-induced demyelination, while immunoglobulins enhance remyelination following demyelination. Analysis of the culture system revealed that immunoglobulins directly act on OPCs to promote their differentiation and myelination. Future studies to elucidate the effects of immunoglobulins on OPCs in vivo and the detailed mechanisms of these effects may lead to new treatments for demyelinating diseases.

First-in-human clinical trial of the NKT cell-stimulatory glycolipid OCH in multiple sclerosis.

Background: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system that causes the damage to the myelin sheath as well as axonal degeneration. Individuals with MS appear to have changes in the numbers and functions of T-cell subsets, leading to an immunological imbalance accompanied by enhanced autoreactivity. In previous preclinical studies, (2 S,3 S,4R)-1-O-(α-D-Galactopyranosyl)-N-tetracosanoyl-2-amino-1,3,4-nonanetriol (OCH), a synthetic analog of α-galactosylceramide stimulatory for invariant NKT (iNKT) cells, has shown therapeutic or disease-preventive immunoregulatory effects in autoimmune disease models such as experimental autoimmune encephalomyelitis (EAE). Objectives: This study is the first-in-human study of oral OCH to evaluate the pharmacokinetics and to examine the effects on immune cells as well as related gene expression profiles. Methods: Fifteen healthy volunteers and 13 MS patients who met the study criteria were enrolled. They were divided into five cohorts and received oral administration of various doses of granulated powder of OCH (0.3-30 mg), once per week for 4 or 13 weeks. Plasma OCH concentrations were measured by high-performance liquid chromatography. Frequencies of lymphocyte subsets in peripheral blood were evaluated by flow cytometry, and microarray analysis was performed to determine OCH-induced changes in gene expression. Results: Oral OCH was well tolerated, and its bioavailability was found to be sufficient. Six hours after a single dose of OCH, increased frequencies of Foxp3+ regulatory T-cells were observed in some cohorts of healthy subjects and MS patients. Furthermore, gene expression analysis demonstrated an upregulation of several immunoregulatory genes and downregulation of pro-inflammatory genes following OCH administration. Conclusion: This study has demonstrated immunomodulatory effects of the iNKT cell-stimulatory drug OCH in human. Safety profiles together with the presumed anti-inflammatory effects of oral OCH encouraged us to conduct a phase II trial.

Different Spatial and Temporal Roles of Monocytes and Monocyte-Derived Cells in the Pathogenesis of an Imiquimod Induced Lupus Model.

Mounting evidence indicates the importance of aberrant Toll-like receptor 7 (TLR7) signaling in the pathogenesis of systemic lupus erythematosus (SLE). However, the mechanism of disease progression remains unclear. An imiquimod (IMQ)-induced lupus model was used to analyze the lupus mechanism related to the aberrant TLR7 signals. C57BL/6 mice and NZB/NZW mice were treated with topical IMQ, and peripheral blood, draining lymph nodes, and kidneys were analyzed focusing on monocytes and monocyte-related cells. Monocytes expressed intermediate to high levels of TLR7, and the long-term application of IMQ increased Ly6Clo monocytes in the peripheral blood and Ly6Clo monocyte-like cells in the lymph nodes and kidneys, whereas Ly6Chi monocyte-like cell numbers were increased in lymph nodes. Ly6Clo monocyte-like cells in the kidneys of IMQ-induced lupus mice were supplied by bone marrow-derived cells as demonstrated using a bone marrow chimera. Ly6Clo monocytes obtained from IMQ-induced lupus mice had upregulated adhesion molecule-related genes, and after adoptive transfer, they showed greater infiltration into the kidneys compared with controls. RNA-seq and post hoc PCR analyses revealed Ly6Clo monocyte-like cells in the kidneys of IMQ-induced lupus mice had upregulated macrophage-related genes compared with peripheral blood Ly6Clo monocytes and downregulated genes compared with kidney macrophages (MF). Ly6Clo monocyte-like cells in the kidneys upregulated Il6 and chemoattracting genes including Ccl5 and Cxcl13. The higher expression of Il6 in Ly6Clo monocyte-like cells compared with MF suggested these cells were more inflammatory than MF. However, MF in IMQ-induced lupus mice were characterized by their high expression of Cxcl13. Genes of proinflammatory cytokines in Ly6Chi and Ly6Clo monocytes were upregulated by stimulation with IMQ but only Ly6Chi monocytes upregulated IFN-α genes upon stimulation with 2'3'-cyclic-GMP-AMP, an agonist of stimulator of interferon genes. Ly6Chi and Ly6Clo monocytes in IMQ-induced lupus mice had different features. Ly6Chi monocytes responded in the lymph nodes of locally stimulated sites and had a higher expression of IFN-α upon stimulation, whereas Ly6Clo monocytes were induced slowly and tended to infiltrate into the kidneys. Infiltrated monocytes in the kidneys likely followed a trajectory through inflammatory monocyte-like cells to MF, which were then involved in the development of nephritis.

Dysregulated B cell differentiation towards antibody-secreting cells in neuromyelitis optica spectrum disorder.

BACKGROUND: Anti-aquaporin 4 (AQP4) antibody (AQP4-Ab) is involved in the pathogenesis of neuromyelitis optica spectrum disorder (NMOSD). However, the mechanism involved in AQP4-Ab production remains unclear. METHODS: We analyzed the immunophenotypes of patients with NMOSD and other neuroinflammatory diseases as well as healthy controls (HC) using flow cytometry. Transcriptome analysis of B cell subsets obtained from NMOSD patients and HCs was performed. The differentiation capacity of B cell subsets into antibody-secreting cells was analyzed. RESULTS: The frequencies of switched memory B (SMB) cells and plasmablasts were increased and that of naïve B cells was decreased in NMOSD patients compared with relapsing-remitting multiple sclerosis patients and HC. SMB cells from NMOSD patients had an enhanced potential to differentiate into antibody-secreting cells when cocultured with T peripheral helper cells. Transcriptome analysis revealed that the profiles of B cell lineage transcription factors in NMOSD were skewed towards antibody-secreting cells and that IL-2 signaling was upregulated, particularly in naïve B cells. Naïve B cells expressing CD25, a receptor of IL-2, were increased in NMOSD patients and had a higher potential to differentiate into antibody-secreting cells, suggesting CD25+ naïve B cells are committed to differentiate into antibody-secreting cells. CONCLUSIONS: To the best of our knowledge, this is the first study to demonstrate that B cells in NMOSD patients are abnormally skewed towards antibody-secreting cells at the transcriptome level during the early differentiation phase, and that IL-2 might participate in this pathogenic process. Our study indicates that CD25+ naïve B cells are a novel candidate precursor of antibody-secreting cells in autoimmune diseases.

Gut dysbiosis and multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) and T cell-mediated autoimmune processes are assumed to be involved in its pathogenesis. Recently, accumulating evidence has indicated that commensal bacteria interact with the host immune system and that the alteration of commensal bacteria composition, termed dysbiosis, is associated with various autoimmune diseases including CNS autoimmune diseases. In this review, we introduce recent findings regarding the association between gut microbiota and MS and related diseases and microbiota function in an animal model of MS.

[Gut environment and multiple sclerosis].

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) and T cell-mediated autoimmune processes are assumed to be involved in its pathogenesis. Recently, accumulating evidence has indicated that commensal bacteria interact with the host immune system and that the alteration of commensal bacteria composition, termed dysbiosis, is associated with various autoimmune diseases including CNS autoimmune diseases. The effect of gut microbiota on disease has been initially shown in experimental autoimmune encephalomyelitis (EAE), animal model of MS. Recent analysis of microbiota revealed dysbiosis in patients with MS including the reduction of short chain fatty acid (SCFA). Administration of SCFA ameliorates disease severity of EAE in association with the induction of regulatory T cells. Moreover metabolites of microbiota such as SCFA and tryptophan have been shown to influence glial functions in CNS. In this review, we introduce recent findings regarding the interaction between gut microbiota and MS both in EAE and MS.

An autopsy case of corticobasal degeneration with inferior olivary hypertrophy.

We report autopsy results of a female patient who was confirmed pathologically as having corticobasal degeneration (CBD). This patient presented with progressive gait disturbance at the age of 66 years, and subsequently showed parkinsonism with a right-sided predominance and dementia. She was clinically diagnosed as having possible corticobasal syndrome without palatal myoclonus throughout the disease course. An autopsy at 72 years of age revealed that neuronal loss with gliosis was severe in the substantia nigra and the portion from hippocampal cornu ammonis (CA1) region to the parahippocampal gyrus, and mild-to-moderate in the basal ganglia, thalamus, red nucleus, dentate nucleus, and cerebral cortices, predominantly in the frontal lobe. Myelin pallor was observed in the pyramidal tract of the brainstem and central tegmental tract. Neurodegenerative or axonal degenerative findings were observed predominantly on the left side, except for the dentate nucleus, which was more affected on the right side. The inferior olivary nucleus exhibited hypertrophic degeneration predominantly on the left side. The topography of neurodegeneration was likely to correspond to the dentate nucleus and inferior olivary nucleus. Phosphorylated tau-immunoreactive pretangles, neurofibrillary tangles, coiled bodies, and threads were diffusely observed in the whole brain. The distribution of tau deposits was prominent in the deeper affected lesions of the dentate nucleus and inferior olivary nucleus. Inferior olivary hypertrophy is unusual in patients with CBD. It is highly possible that the neurodegeneration of the inferior olivary nucleus followed that of the dentate nucleus in our patient. Moreover, these results indicate not only the severity of neurodegenerative changes, but also that of tau deposition that could be related to the topography of the projections of the dentato-olivary pathway. Tau propagation and subsequent neurodegeneration along the fiber connections may have occurred. Our results support the possibility that progression of CBD lesions can be mediated by tau propagation.

Mucosal-Associated Invariant T Cells Are Involved in Acute Ischemic Stroke by Regulating Neuroinflammation.

Background Mucosal-associated invariant T (MAIT) cells have been associated with inflammation in several autoimmune diseases. However, their relation to ischemic stroke remains unclear. This study attempted to elucidate the role of MAIT cells in acute ischemic stroke in mice. Methods and Results We used MR1 knockout C57BL/6 (MR1-/-) mice and wild-type littermates (MR1+/+). After performing a transient middle cerebral artery occlusion (tMCAO), we evaluated the association with inflammation and prognosis in the acute cerebral ischemia. Furthermore, we analyzed the tMCAO C57BL/6 mice administered with the suppressive MR1 ligand and the vehicle control. We also evaluated the infiltration of MAIT cells into the ischemic brain by flow cytometry. Results showed a reduction of infarct volume and an improvement of neurological impairment in MR1-/- mice (n=8). There was a reduction in the number of infiltrating microglia/macrophages (n=3-5) and in their activation (n=5) in the peri-infarct area of MR1-/- mice. The cytokine levels of interleukin-6 and interleukin-17 at 24 hours after tMCAO (n=3-5), and for interleukin-17 at 72 hours after tMCAO (n=5), were lower in the MR1-/- mice. The administration of the suppressive MR1 ligand reduced the infarct volume and improved functional impairment (n=5). Flow cytometric analysis demonstrated there was a reduction of MAIT cells infiltrating into the ischemic brain at 24 hours after tMCAO (n=17). Conclusions Our results showed that MAIT cells play an important role in neuroinflammation after focal cerebral ischemia and the use of MAIT cell regulation has a potential role as a novel neuroprotectant for the treatment of acute ischemic stroke.

Butyrate suppresses demyelination and enhances remyelination.

BACKGROUND: The association of gut microbiota and diseases of the central nervous system (CNS), including multiple sclerosis (MS), has attracted much attention. Although a previous analysis of MS gut microbiota revealed a reduction in species producing short-chain fatty acids (SCFAs), the influence of these metabolites on demyelination and remyelination, the critical factors of MS pathogenesis, remains unclear. METHODS: To investigate the relationship between demyelination and gut microbiota, we administered a mixture of non-absorbing antibiotics or SCFAs to mice with cuprizone-induced demyelination and evaluated demyelination and the accumulation of microglia. To analyze the direct effect of SCFAs on demyelination or remyelination, we induced demyelination in an organotypic cerebellar slice culture using lysolecithin and analyzed the demyelination and maturation of oligodendrocyte precursor cells with or without SCFA treatment. RESULTS: The oral administration of antibiotics significantly enhanced cuprizone-induced demyelination. The oral administration of butyrate significantly ameliorated demyelination, even though the accumulation of microglia into demyelinated lesions was not affected. Furthermore, we showed that butyrate treatment significantly suppressed lysolecithin-induced demyelination and enhanced remyelination in an organotypic slice culture in the presence or absence of microglia, suggesting that butyrate may affect oligodendrocytes directly. Butyrate treatment facilitated the differentiation of immature oligodendrocytes. CONCLUSIONS: We revealed that treatment with butyrate suppressed demyelination and enhanced remyelination in an organotypic slice culture in association with facilitating oligodendrocyte differentiation. Our findings shed light on a novel mechanism of interaction between the metabolites of gut microbiota and the CNS and may provide a strategy to control demyelination and remyelination in MS.

Expanded circulating peripheral helper T cells in systemic lupus erythematosus: association with disease activity and B cell differentiation.

OBJECTIVE: Peripheral helper T (TPH) cells are a recently identified Th cell subset that promotes B cell differentiation and antibody production in inflamed tissues. This study investigated circulating TPH cells to determine their involvement in systemic lupus erythematosus (SLE). METHODS: Peripheral blood mononuclear cells collected from SLE patients and healthy individuals were analysed. TPH cells were identified as CD3+CD4+CD45RA-CXCR5- cells with a high expression of PD-1. The frequency, activation status and subsets of TPH cells were evaluated by flow cytometry. The production of IL-21 was assessed by intracellular staining and the association of TPH cells with disease activity and B cell populations was determined. RESULTS: Circulating TPH cells, identified as CD3+CD4+CD45RA-PD-1highCXCR5- cells were increased in the peripheral blood of SLE patients compared with controls. Circulating TPH cells produced similar amounts of IL-21 compared with follicular Th cells. The expansion and activation of TPH cells were correlated with SLE disease activity. Activated TPH cells, particularly Th1-type TPH cells, were associated with the promotion of B cell differentiation in SLE patients. CONCLUSION: The association of TPH cells with disease activity suggests the involvement of extrafollicular T-B cell interactions in the pathogenesis of SLE. TPH cells promote autoantibody production in aberrant lymphoid organs and therefore might be a novel therapeutic target in autoantibody-producing disorders.

Unique primed status of microglia under the systemic autoimmune condition of lupus-prone mice.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of various autoantibodies. This disease causes disabling neuropsychiatric symptoms even in the absence of apparent inflammation in the central nervous system (CNS), but the mechanisms involved remain unknown. Innate immune-mediated inflammation has attracted attention as a pathogenic mechanism in neuropsychiatric diseases. METHODS: We investigated the CNS of lupus-prone mice focusing on innate immunity. Three strains of lupus-prone mice, FcγRIIB-/-Yaa, an F1 hybrid of NZB and NZW (NZB/NZW) mice, and MRL/Faslpr (MRL/lpr) mice were used to analyze CNS immunopathology. RESULTS: Flow cytometry analysis demonstrated the numbers of brain CD45+ cells were increased compared with controls in lupus-prone mice. Upregulation of MHC class I and PDCA1 was observed in microglia and CD11b+ myeloid cells of lupus-prone mice, indicating they were activated in response to interferons (IFN). Microglial gene expression analysis of FcγRIIB-/-Yaa mice revealed the upregulation of IFN-responsive genes and inflammation-related genes including Axl, Clec7a, and Itgax, which were previously reported in neurodegenerative conditions and primed conditions. Upregulated chemokine gene expressions including Ccl5 and Cxcl10 were concurrent with increased numbers of T cells and monocytes, especially Ly6Clo monocytes in the CNS. Upregulation of Axl, Clec7a, Itgax, Ccl5, and Cxcl10 was also observed in NZB/NZW mice, indicating common lupus pathology. The primed status of microglia in FcγRIIB-/-Yaa mice was also demonstrated by morphological changes such as enlarged cell bodies with hypertrophic processes, and hyperreactivity to lipopolysaccharide. Immunohistochemistry of FcγRIIB-/-Yaa mice indicated reactive responses of astrocytes and vascular endothelium. Behavioral studies of FcγRIIB-/-Yaa mice revealed depressive-like behavior and heat hyperalgesia in the forced swim test and the tail-flick test, respectively. CONCLUSIONS: Our data indicated that microglia in lupus exhibit a unique primed phenotype characterized by the upregulated expressions of neurodegeneration-related genes and IFN-responsive genes. Interaction with peripheral cells and brain resident cells was presumed to orchestrate neuroinflammation. Targeting innate immune cells, such as microglia and monocytes, may be a promising therapeutic approach for neuropsychiatric SLE.

MicroRNA-101a regulates microglial morphology and inflammation.

BACKGROUND: Microglia, as well as other tissue-resident macrophages, arise from yolk sac progenitors. Thus, it is likely that the central nervous system environment is critical for the acquisition of a distinct microglial phenotype. Several microRNAs that are enriched in the brain play crucial roles in brain development and may also play a role in the differentiation of microglia. METHODS: To track the differentiation of hematopoietic cells into microglia, lineage-negative bone marrow cells were co-cultured with astrocytes in the absence or presence of microRNAs or their inhibitors. Microglia-like cells were identified as small, round cells that were immunopositive for CD11b, Iba1, CX3CR1, and triggering receptor expressed on myeloid cells (TREM)-2. RESULTS: Five microRNAs (miR-101a, miR-139-3p, miR-214*, miR-218, and miR-1186) were identified as modifiers of the differentiation of bone marrow-derived microglia-like cells. Among them, miR-101a facilitated the differentiation of bone marrow cells into microglia-like cells most potently. Small, round cells expressing CD11b, Iba1, CX3CR1, and TREM-2 were predominant in cells treated by miR-101a. miR-101a was abundantly expressed in non-microglial brain cells. Transfection of miR-101a into microglia significantly increased the production of IL-6 in response to LPS. Finally, miR-101a downregulated the expression of MAPK phosphatase-1. CONCLUSIONS: miR-101a, which is enriched in the brain, promotes the differentiation of bone marrow cells into microglia-like cells.

The dual role of short fatty acid chains in the pathogenesis of autoimmune disease models.

Autoimmune diseases are influenced by both genetic and environmental factors. The gut environment has attracted much attention as an essential component that modulates immune responses, and therefore immune-mediated disorders, such as autoimmune diseases. Growing evidence suggests that microbiota and their metabolites are critical factors for immune modulation. Recently, we reported that the microbiome in patients with multiple sclerosis, an autoimmune disease targeting the myelin sheath of the central nervous system, is characterized by a reduction of bacteria belonging to Clostridia clusters IV and XIVa, which are potent producers of short-chain fatty acids (SCFAs) by fermentation of indigestible carbohydrates. In the present study, we investigated the role of SCFAs in the regulation of inflammation. We demonstrated that oral administration of SCFAs ameliorated the disease severity of systemic autoimmune inflammatory conditions mediated by lymphocytes such as experimental autoimmune encephalitis and collagen-induced arthritis. Amelioration of disease was associated with a reduction of Th1 cells and an increase in regulatory T cells. In contrast, SCFAs contributed to the exaggeration of K/BxN serum transfer arthritis, representing the effector phase of inflammation in rheumatoid arthritis. An increased understanding of the effect of microbiota metabolites will lead to the effective treatment and prevention of systemic inflammatory disorders.

Granuloma formation in a patient with GNE myopathy: A case report.

We report a patient with GNE myopathy with a homozygous mutation (c.1505-4G>A) in GNE gene. The patient recognized progressive weakness of extremities at age 60. Neurological examination at age 65 revealed severe weakness and atrophy in the tibialis anterior muscles and distal predominant moderate weakness in the extremities. Muscle biopsy performed at age 65 showed myopathic changes with rimmed vacuoles, and the noteworthy finding was non-caseating epithelioid cell granuloma formation surrounded by numerous inflammatory cells. Granuloma formation has never been reported in patients with GNE myopathy. We presume that aggregation of abnormal proteins and autophagy dysregulation in the myocytes of GNE myopathy could induce granuloma formation.

Development of a culture system to induce microglia-like cells from haematopoietic cells.

AIMS: Microglia are the resident immune cells in the central nervous system, originating from haematopoietic-derived myeloid cells. A microglial cell is a double-edged sword, which has both pro-inflammatory and anti-inflammatory functions. Although understanding the role of microglia in pathological conditions has become increasingly important, histopathology has been the only way to investigate microglia in human diseases. METHODS: To enable the study of microglial cells in vitro, we here establish a culture system to induce microglia-like cells from haematopoietic cells by coculture with astrocytes. The characteristics of microglia-like cells were analysed by flow cytometry and functional assay. RESULTS: We show that triggering receptor expressing on myeloid cells-2-expressing microglia-like cells could be induced from lineage negative cells or monocytes by coculture with astrocytes. Microglia-like cells exhibited lower expression of CD45 and MHC class II than macrophages, a characteristic similar to brain microglia. When introduced into brain slice cultures, these microglia-like cells changed their morphology to a ramified shape on the first day of the culture. Moreover, we demonstrated that microglia-like cells could be induced from human monocytes by coculture with astrocytes. Finally, we showed that interleukin 34 was an important factor in the induction of microglia-like cells from haematopoietic cells in addition to cell-cell contact with astrocytes. Purified microglia-like cells were suitable for further culture and functional analyses. CONCLUSION: Development of in vitro induction system for microglia will further promote the study of human microglial cells under pathological conditions as well as aid in the screening of drugs to target microglial cells.

Coagulation and fibrinolysis abnormalities in familial amyloid polyneuropathy.

OBJECTIVE: Familial amyloid polyneuropathy (FAP) is an autosomal dominant form of hereditary amyloidosis. Several studies reported coagulation factor X deficiency and excessive fibrinolysis in immunoglobulin light chain amyloidosis. However, few have investigated coagulation and fibrinolysis in FAP. The objective of this study was to determine abnormalities in plasma biomarkers of coagulation and fibrinolysis in FAP. METHODS: We prospectively recruited eight FAP patients with transthyretin mutations and ten age-matched control patients with other neuropathies in our university. We examined plasma biomarkers of coagulation and fibrinolysis including prothrombin time, activated partial thromboplastin time, fibrinogen, fibrin/fibrinogen degradation products, D-dimer, α2-antiplasmin, antithrombin, plasminogen, thrombin-antithrombin complex, plasmin-α2-antiplasmin complex, prothrombin fragment 1+2, and coagulation factor X. The Mann-Whitney U test was performed for statistical comparisons between FAP and control groups. RESULTS: FAP patients exhibited significantly decreased levels of coagulation factor X, plasminogen and α2-antiplasmin, and significantly increased levels of prothrombin fragment 1+2 compared to control patients. CONCLUSION: Our results indicate abnormalities of coagulation and fibrinolysis in FAP patients.

In vitro differentiation of lineage-negative bone marrow cells into microglia-like cells.

Microglia are believed to be the only resident immune cells in the CNS, originating from hematopoietic-derived myeloid cells and invading the CNS during development. However, the detailed mechanisms of differentiation and transformation of microglial cells are not fully understood. Here, we demonstrate that murine microglial cells show two morphological forms in vitro, namely, small round cells expressing CD11b, Iba1, triggering receptor expressing on myeloid cells-2 (TREM2), and weakly expressing major histocompatibility complex class II and large flat cells expressing only CD11b and Iba1. Moreover, lineage-negative bone marrow (LN) cells cultured with primary mixed glial culture cells could differentiate into only the small round microglia-like cells, despite the absence of CCR2 and Gr-1 expression. Addition of macrophage colony stimulating factor (M-CSF) to LN cell culture allowed the proliferation and expression of TREM2 in LN cells, and the addition of neutralizing anti-M-CSF antibodies suppressed the proliferation of LN cells despite the expression of TREM2. When LN cells were cultured with M-CSF, the number of small round cells in the culture was considerably low, indicating that the small round morphology of the immature cells is not maintained in the presence of only M-CSF. On the other hand, when LN cells were grown in the presence of astrocytes, the small round cells were maintained at a concentration of approximately 30% of the total population. Therefore, cell-cell contact with glial cells, especially astrocytes, may be necessary to maintain the small round shape of the immature cells expressing TREM2.