Publications by authors named "Takaharu Okada"

49 Publications

A unique mode of keratinocyte death requires intracellular acidification.

Proc Natl Acad Sci U S A 2021 Apr;118(17)

Laboratory for Skin Homeostasis, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan;

The stratum corneum (SC), the outermost epidermal layer, consists of nonviable anuclear keratinocytes, called corneocytes, which function as a protective barrier. The exact modes of cell death executed by keratinocytes of the upper stratum granulosum (SG1 cells) remain largely unknown. Here, using intravital imaging combined with intracellular Ca- and pH-responsive fluorescent probes, we aimed to dissect the SG1 death process in vivo. We found that SG1 cell death was preceded by prolonged (∼60 min) Ca elevation and rapid induction of intracellular acidification. Once such intracellular ionic changes were initiated, they became sustained, irreversibly committing the SG1 cells to corneocyte conversion. Time-lapse imaging of isolated murine SG1 cells revealed that intracellular acidification was essential for the degradation of keratohyalin granules and nuclear DNA, phenomena specific to SC corneocyte formation. Furthermore, intravital imaging showed that the number of SG1 cells exhibiting Ca elevation and the timing of intracellular acidification were both tightly regulated by the transient receptor potential cation channel V3. The functional activity of this protein was confirmed in isolated SG1 cells using whole-cell patch-clamp analysis. These findings provide a theoretical framework for improved understanding of the unique molecular mechanisms underlying keratinocyte-specific death mode, namely corneoptosis.
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http://dx.doi.org/10.1073/pnas.2020722118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092583PMC
April 2021

Development of Sentinel LN Imaging with a Combination of HAase Based on a Comprehensive Analysis of the Intra-lymphatic Kinetics of LPs.

Mol Ther 2021 01 6;29(1):225-235. Epub 2020 Sep 6.

Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 264-0028, Japan. Electronic address:

The sentinel lymph node (LN) is the first LN to which lymph fluid flows from tumor tissue. We identified the key parameters of liposomes (LPs) that affect their accumulation in regional (primary) LNs with minimum leakage to its connecting (secondary) LNs by a comprehensive analysis of the LN-to-LN trafficking of LPs with various surface charges and various sizes. We used a lymphatic flow-modified (LFM) mouse that allows for the chronological analysis of inguinal (primary) LN-to-axillary (secondary) LN at the body surface. As a result, the anionic medium-sized LPs (130 nm on average) exhibited the highest accumulation in the primary LNs. A mechanism-based analysis revealed that CD169-positive macrophages in LNs were the dominant cell population that captures anionic LPs. Sentinel LN imaging was also performed by the intratumoral injection of fluorescent medium-sized anionic LPs using a breast cancer orthotopic model. In comparison with the typically used contrast agent indocyanine green, the anionic LPs were detected in sentinel LNs with a high sensitivity. Additionally, the co-injection of hyaluronidase significantly improved the sensitivity of detection of the fluorescent LPs in sentinel LNs. In conclusion, medium-sized anionic LPs combined with hyaluronidase represents a potent strategy for investigating sentinel LNs.
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http://dx.doi.org/10.1016/j.ymthe.2020.09.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7791005PMC
January 2021

Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells.

EBioMedicine 2020 Aug 22;58:102913. Epub 2020 Jul 22.

Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Minato-ku, Tokyo105-8512, Japan; International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo (IMSUT), Minato-ku, Tokyo108-8639, Japan. Electronic address:

Background: Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disorder with a high prevalence, especially in industrialized countries. Dysbiosis of the intestinal microbiota has been observed in RA patients. For instance, new-onset untreated RA (NORA) is associated with the underrepresentation of the Clostridium cluster XIVa, including Lachnospiraceae, which are major butyrate producers, although the pathological relevance has remained obscure. Follicular regulatory T (T) cells play critical regulatory roles in the pathogenesis of autoimmune diseases, including RA. Reduced number of circulating T cells has been associated with the elevation of autoantibodies and disease severity in RA. However, the contribution of commensal microbe-derived butyrate in controlling T cell differentiation remains unknown.

Methods: We examined the contribution of microbe-derived butyrate in controlling autoimmune arthritis using collagen-induced arthritis (CIA) and SKG arthritis models. We phenotyped autoimmune responses in the gut-associated lymphoid tissues (GALT) in the colon and joint-draining lymph nodes in the CIA model. We developed an in vitro CXCR5Bcl-6Foxp3 T (iT) cell culture system and examined whether butyrate promotes the differentiation of iT cells.

Findings: Microbe-derived butyrate suppressed the development of autoimmune arthritis. The immunization of type II collagen (CII) caused hypertrophy of the GALT in the colon by amplifying the GC reaction prior to the onset of the CIA. Butyrate mitigated these pathological events by promoting T cell differentiation. Butyrate directly induced the differentiation of functional T cells in vitro by enhancing histone acetylation in T cell marker genes. This effect was attributed to histone deacetylase (HDAC) inhibition by butyrate, leading to histone hyperacetylation in the promoter region of the T-cell marker genes. The adoptive transfer of the butyrate-treated iT cells reduced CII-specific autoantibody production and thus ameliorated the symptoms of arthritis.

Interpretation: Accordingly, microbiota-derived butyrate serves as an environmental cue to enhance T cells, which suppress autoantibody production in the systemic lymphoid tissue, eventually ameliorating RA. Our findings provide mechanistic insights into the link between the gut environment and RA risk.

Funding: This work was supported by AMED-Crest (16gm1010004h0101, 17gm1010004h0102, 18gm1010004h0103, and 19gm1010004s0104 to KH), the Japan Society for the Promotion of Science (JP17KT0055, JP16H01369, and JP18H04680 to KH; JP17K15734 to DT), Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects (KH), Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research (DT), the SECOM Science and Technology Foundation (KH), the Cell Science Research Foundation (KH), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (DT), the Suzuken Memorial Foundation (KH and DT), the Takeda Science Foundation (KH and DT), The Science Research Promotion Fund, and The Promotion and Mutual Aid Corporation for Private Schools of Japan (KH).
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http://dx.doi.org/10.1016/j.ebiom.2020.102913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7387783PMC
August 2020

The liver-brain-gut neural arc maintains the T cell niche in the gut.

Nature 2020 09 11;585(7826):591-596. Epub 2020 Jun 11.

Division of Gastroenterology and Hepatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan.

Recent clinical and experimental evidence has evoked the concept of the gut-brain axis to explain mutual interactions between the central nervous system and gut microbiota that are closely associated with the bidirectional effects of inflammatory bowel disease and central nervous system disorders. Despite recent advances in our understanding of neuroimmune interactions, it remains unclear how the gut and brain communicate to maintain gut immune homeostasis, including in the induction and maintenance of peripheral regulatory T cells (pT cells), and what environmental cues prompt the host to protect itself from development of inflammatory bowel diseases. Here we report a liver-brain-gut neural arc that ensures the proper differentiation and maintenance of pT cells in the gut. The hepatic vagal sensory afferent nerves are responsible for indirectly sensing the gut microenvironment and relaying the sensory inputs to the nucleus tractus solitarius of the brainstem, and ultimately to the vagal parasympathetic nerves and enteric neurons. Surgical and chemical perturbation of the vagal sensory afferents at the hepatic afferent level reduced the abundance of colonic pT cells; this was attributed to decreased aldehyde dehydrogenase (ALDH) expression and retinoic acid synthesis by intestinal antigen-presenting cells. Activation of muscarinic acetylcholine receptors directly induced ALDH gene expression in both human and mouse colonic antigen-presenting cells, whereas genetic ablation of these receptors abolished the stimulation of antigen-presenting cells in vitro. Disruption of left vagal sensory afferents from the liver to the brainstem in mouse models of colitis reduced the colonic pT cell pool, resulting in increased susceptibility to colitis. These results demonstrate that the novel vago-vagal liver-brain-gut reflex arc controls the number of pT cells and maintains gut homeostasis. Intervention in this autonomic feedback feedforward system could help in the development of therapeutic strategies to treat or prevent immunological disorders of the gut.
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http://dx.doi.org/10.1038/s41586-020-2425-3DOI Listing
September 2020

Restricted Clonality and Limited Germinal Center Reentry Characterize Memory B Cell Reactivation by Boosting.

Cell 2020 01 19;180(1):92-106.e11. Epub 2019 Dec 19.

Laboratory of Lymphocyte Dynamics, The Rockefeller University, New York, NY, USA. Electronic address:

Repeated exposure to pathogens or their antigens triggers anamnestic antibody responses that are higher in magnitude and affinity than the primary response. These involve reengagement of memory B cell (MBC) clones, the diversity and specificity of which determine the breadth and effectiveness of the ensuing antibody response. Using prime-boost models in mice, we find that secondary responses are characterized by a clonality bottleneck that restricts the engagement of the large diversity of MBC clones generated by priming. Rediversification of mutated MBCs is infrequent within secondary germinal centers (GCs), which instead consist predominantly of B cells without prior GC experience or detectable clonal expansion. Few MBC clones, generally derived from higher-affinity germline precursors, account for the majority of secondary antibody responses, while most primary-derived clonal diversity is not reengaged detectably by boosting. Understanding how to counter this bottleneck may improve our ability to elicit antibodies to non-immunodominant epitopes by vaccination.
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http://dx.doi.org/10.1016/j.cell.2019.11.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6958527PMC
January 2020

The HVEM-BTLA Axis Restrains T Cell Help to Germinal Center B Cells and Functions as a Cell-Extrinsic Suppressor in Lymphomagenesis.

Immunity 2019 08 13;51(2):310-323.e7. Epub 2019 Jun 13.

Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA. Electronic address:

The tumor necrosis factor receptor superfamily member HVEM is one of the most frequently mutated surface proteins in germinal center (GC)-derived B cell lymphomas. We found that HVEM deficiency increased B cell competitiveness during pre-GC and GC responses. The immunoglobulin (Ig) superfamily protein BTLA regulated HVEM-expressing B cell responses independently of B-cell-intrinsic signaling via HVEM or BTLA. BTLA signaling into T cells through the phosphatase SHP1 reduced T cell receptor (TCR) signaling and preformed CD40 ligand mobilization to the immunological synapse, thus diminishing the help delivered to B cells. Moreover, T cell deficiency in BTLA cooperated with B cell Bcl-2 overexpression, leading to GC B cell outgrowth. These results establish that HVEM restrains the T helper signals delivered to B cells to influence GC selection outcomes, and they suggest that BTLA functions as a cell-extrinsic suppressor of GC B cell lymphomagenesis.
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http://dx.doi.org/10.1016/j.immuni.2019.05.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6703922PMC
August 2019

Homeostatic pruning and activity of epidermal nerves are dysregulated in barrier-impaired skin during chronic itch development.

Sci Rep 2019 06 13;9(1):8625. Epub 2019 Jun 13.

Laboratory for Tissue Dynamics, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, 230-0045, Japan.

The epidermal barrier is thought to protect sensory nerves from overexposure to environmental stimuli, and barrier impairment leads to pathological conditions associated with itch, such as atopic dermatitis (AD). However, it is not known how the epidermal barrier continuously protects nerves for the sensory homeostasis during turnover of the epidermis. Here we show that epidermal nerves are contained underneath keratinocyte tight junctions (TJs) in normal human and mouse skin, but not in human AD samples or mouse models of chronic itch caused by epidermal barrier impairment. By intravital imaging of the mouse skin, we found that epidermal nerve endings were frequently extended and retracted, and occasionally underwent local pruning. Importantly, the epidermal nerve pruning took place rapidly at intersections with newly forming TJs in the normal skin, whereas this process was disturbed during chronic itch development. Furthermore, aberrant Ca increases in epidermal nerves were induced in association with the disturbed pruning. Finally, TRPA1 inhibition suppressed aberrant Ca increases in epidermal nerves and itch. These results suggest that epidermal nerve endings are pruned through interactions with keratinocytes to stay below the TJ barrier, and that disruption of this mechanism may lead to aberrant activation of epidermal nerves and pathological itch.
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http://dx.doi.org/10.1038/s41598-019-44866-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565750PMC
June 2019

Regional dissociation between the cerebral blood flow and gray matter density alterations in idiopathic normal pressure hydrocephalous: results from SINPHONI-2 study.

Neuroradiology 2019 Jan 30;61(1):37-42. Epub 2018 Sep 30.

Department of Neurosurgery, Tama-Hokubu Medical Center, Health and Medical Treatment Corporation, Higashimurayamashi, Tokyo, Japan.

Purpose: The purpose of this study was to elucidate the specific regional cerebral blood flow (rCBF) alterations for idiopathic normal pressure hydrocephalus (iNPH) by comparing the proportional rCBF and gray matter change from those of a normal database at the same point of SPECT and MRI examinations.

Methods: Thirty subjects with iNPH underwent both CBF SPECT and MRI. After normalization, voxel-wise two-sample t tests between patients and 11 normal controls were conducted to compare the regional alteration in the gray matter density and rCBF.

Results: The rCBF reduction and the gray matter decrease were seen in almost similar regions surrounding Sylvian fissure, the left parietotemporal region and frontal lobes, whereas we did not find rCBF increase at the top of the high convexity, where the increase of the gray matter density was the highest (p < 0.05).

Conclusion: Our study showed regional associations and dissociations between the relative gray matter density and rCBF in patients with iNPH.
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http://dx.doi.org/10.1007/s00234-018-2106-1DOI Listing
January 2019

T Follicular Helper Cell-Germinal Center B Cell Interaction Strength Regulates Entry into Plasma Cell or Recycling Germinal Center Cell Fate.

Immunity 2018 04;48(4):702-715.e4

Laboratory of Lymphocyte Differentiation, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan; Laboratory of Lymphocyte Differentiation, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Kanagawa 230-0045, Japan. Electronic address:

Higher- or lower-affinity germinal center (GC) B cells are directed either to plasma cell or GC recycling, respectively; however, how commitment to the plasma cell fate takes place is unclear. We found that a population of light zone (LZ) GC cells, Bcl6CD69 expressing a transcription factor IRF4 and higher-affinity B cell receptors (BCRs) or Bcl6CD69 with lower-affinity BCRs, favored the plasma cell or recycling GC cell fate, respectively. Mechanistically, CD40 acted as a dose-dependent regulator for Bcl6CD69 cell formation. Furthermore, we found that expression of intercellular adhesion molecule 1 (ICAM-1) and signaling lymphocytic activation molecule (SLAM) in Bcl6CD69 cells was higher than in Bcl6CD69 cells, thereby affording more stable T follicular helper (Tfh)-GC B cell contacts. These data support a model whereby commitment to the plasma cell begins in the GC and suggest that stability of Tfh-GC B cell contacts is key for plasma cell-prone GC cell formation.
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http://dx.doi.org/10.1016/j.immuni.2018.03.027DOI Listing
April 2018

KLRG1 Effector CD8 T Cells Lose KLRG1, Differentiate into All Memory T Cell Lineages, and Convey Enhanced Protective Immunity.

Immunity 2018 04 3;48(4):716-729.e8. Epub 2018 Apr 3.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA; Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520, USA. Electronic address:

Protective immunity against pathogens depends on the efficient generation of functionally diverse effector and memory T lymphocytes. However, whether plasticity during effector-to-memory CD8 T cell differentiation affects memory lineage specification and functional versatility remains unclear. Using genetic fate mapping analysis of highly cytotoxic KLRG1 effector CD8 T cells, we demonstrated that KLRG1 cells receiving intermediate amounts of activating and inflammatory signals downregulated KLRG1 during the contraction phase in a Bach2-dependent manner and differentiated into all memory T cell linages, including CXCR1 peripheral memory cells and tissue-resident memory cells. "ExKLRG1" memory cells retained high cytotoxic and proliferative capacity distinct from other populations, which contributed to effective anti-influenza and anti-tumor immunity. Our work demonstrates that developmental plasticity of KLRG1 effector CD8 T cells is important in promoting functionally versatile memory cells and long-term protective immunity.
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http://dx.doi.org/10.1016/j.immuni.2018.03.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465538PMC
April 2018

The Rac Activator DOCK2 Mediates Plasma Cell Differentiation and IgG Antibody Production.

Front Immunol 2018 16;9:243. Epub 2018 Feb 16.

Division of Immunogenetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR), which triggers activation of B cells and differentiation into plasma cells (PCs). Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated with anti-IgM F(ab') antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2-Rac axis in PC differentiation and IgG antibody responses.
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http://dx.doi.org/10.3389/fimmu.2018.00243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820292PMC
March 2019

The Eph-related tyrosine kinase ligand Ephrin-B1 marks germinal center and memory precursor B cells.

J Exp Med 2017 03 31;214(3):639-649. Epub 2017 Jan 31.

Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143

Identification of germinal center (GC) B cells is typically reliant on the use of surface activation markers that exhibit a wide range of expression. Here, we identify Ephrin-B1, a ligand for Eph-related receptor tyrosine kinases, as a specific marker of mature GC B cells. The number of Ephrin-B1 GC B cells increases during the course of an immune response with Ephrin-B1 GC B cells displaying elevated levels of Bcl6, , and relative to their Ephrin-B1 counterparts. We further identified a small proportion of recently dividing, somatically mutated Ephrin-B1 GC B cells that have begun to down-regulate Bcl6 and and express markers associated with memory B cells, such as CD38 and EBI2. Transcriptional analysis indicates that these cells are developmentally related to memory B cells, and likely represent a population of GC memory precursor (PreMem) B cells. GC PreMem cells display enhanced survival relative to bulk GC B cells, localize near the edge of the GC, and are predominantly found within the light zone. These findings offer insight into the significant heterogeneity that exists within the GC B cell population and provide tools to further dissect signals regulating the differentiation of GC B cells.
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http://dx.doi.org/10.1084/jem.20161461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339677PMC
March 2017

In vivo multiphoton imaging of immune cell dynamics.

Pflugers Arch 2016 11 22;468(11-12):1793-1801. Epub 2016 Sep 22.

Laboratory for Tissue Dynamics, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, 230-0045, Japan.

Multiphoton imaging has been utilized to analyze in vivo immune cell dynamics over the last 15 years. Particularly, it has deepened the understanding of how immune responses are organized by immune cell migration and interactions. In this review, we first describe the following technical advances in recent imaging studies that contributed to the new findings on the regulation of immune responses and inflammation. Improved multicolor imaging of immune cell behavior has revealed that their interactions are spatiotemporally coordinated to achieve efficient and long-term immunity. The use of photoactivatable and photoconvertible fluorescent proteins has increased duration and volume of cell tracking, even enabling the analysis of inter-organ migration of immune cells. In addition, visualization of immune cell activation using biosensors for intracellular calcium concentration and signaling molecule activities has started to give further mechanistic insights. Then, we also introduce recent imaging analyses of interactions between immune cells and non-immune cells including endothelial, fibroblastic, epithelial, and nerve cells. It is argued that future imaging studies that apply updated technical advances to analyze interactions between immune cells and non-immune cells will be important for thorough physiological understanding of the immune system.
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http://dx.doi.org/10.1007/s00424-016-1882-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138265PMC
November 2016

Stiffness-dependent cellular internalization of matrix-bound BMP-2 and its relation to Smad and non-Smad signaling.

Acta Biomater 2016 12 12;46:55-67. Epub 2016 Sep 12.

CNRS UMR 5628 (LMGP), MINATEC, 3 parvis Louis Néel, 38016 Grenoble, France; Université Grenoble Alpes, Grenoble Institute of Technology, 3 parvis Louis Néel, 38016 Grenoble, France. Electronic address:

Surface coatings delivering BMP are a promising approach to render biomaterials osteoinductive. In contrast to soluble BMPs which can interact with their receptors at the dorsal side of the cell, BMPs presented as an insoluble cue physically bound to a biomimetic matrix, called here matrix-bound (bBMP-2), are presented to cells by their ventral side. To date, BMP-2 internalization and signaling studies in cell biology have always been performed by adding soluble (sBMP-2) to cells adhered on cell culture plates or glass slides, which will be considered here as a "reference" condition. However, whether and how matrix-bound BMP-2 can be internalized by cells and its relation to canonical (SMAD) and non-canonical signaling (ALP) remain open questions. In this study, we investigated the uptake and processing of BMP-2 by C2C12 myoblasts. This BMP-2 was presented either embedded in polyelectrolyte multilayer films (matrix-bound presentation) or as soluble form. Using fluorescently labeled BMP-2, we showed that the amount of matrix-bound BMP-2 internalized is dependent on the level of crosslinking of the polyelectrolyte films. Cav-1-mediated internalization is related to both SMAD and ALP signaling, while clathrin-mediated is only related to ALP signaling. BMP-2 internalization was independent of the presentation mode (sBMP-2 versus bBMP-2) for low crosslinked films (soft, EDC10) in striking contrast with high crosslinked (stiff, EDC70) films where internalization was much lower and slower for bBMP-2. As anticipated, internalization of sBMP-2 barely depended on the underlying matrix. Taken together, these results indicate that BMP-2 internalization can be tuned by the underlying matrix and activates downstream BMP-2 signaling, which is key for the effective formation of bone tissue.

Statement Of Significance: The presentation of growth factors from material surfaces currently presents significant challenges in academic research, clinics and industry. Being able to deliver efficiently these growth factors by a biomaterial will open new perspectives for regenerative medicine. However, to date, very little is known about how matrix-bound growth factors are delivered to cells, especially whether they are internalized and how they are signaling to drive key differentiation events. These initial steps are crucial as they will guide the subsequent processes leading to tissue regeneration. In this work, we investigate the uptake and processing by cells of BMP-2 ligands embedded in polyelectrolyte multilayer films in comparison to soluble BMP-2. We show that BMP-2 responsive cells can internalize matrix-bound BMP-2 and that internalization is dependent on the cross-linking level of the polyelectrolyte films. In addition, we show that internalization is mediated by both clathrin- and caveolin-dependent pathways. While inhibiting clathrin-dependent endocytosis affects only non-canonical signaling, blocking caveolin-1-dependent endocytosis reduces both canonical and non-canonical BMP signaling. The signaling pathways found for matrix-bound BMP-2 are similar to those found for soluble BMP-2. These results highlight that BMP-2 presented by a biomaterial at the ventral side of the cell can trigger major endocytic and associated signaling pathways leading to bone regeneration.
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http://dx.doi.org/10.1016/j.actbio.2016.09.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113753PMC
December 2016

Regulated selection of germinal-center cells into the memory B cell compartment.

Nat Immunol 2016 07 9;17(7):861-9. Epub 2016 May 9.

Laboratory of Lymphocyte Differentiation, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan.

Despite the importance of memory B cells in protection from reinfection, how such memory cells are selected and generated during germinal-center (GC) reactions remains unclear. We found here that light-zone (LZ) GC B cells with B cell antigen receptors (BCRs) of lower affinity were prone to enter the memory B cell pool. Mechanistically, cells in this memory-prone fraction had higher expression of the transcriptional repressor Bach2 than that of their counterparts with BCRs of higher affinity. Haploinsufficiency of Bach2 resulted in reduced generation of memory B cells, independently of suppression of the gene encoding the transcription factor Blimp-1. Bach2 expression in GC cells was inversely correlated with the strength of help provided by T cells. Thus, we propose an instructive model in which weak help from T cells maintains relatively high expression of Bach2, which predisposes GC cells to enter the memory pool.
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http://dx.doi.org/10.1038/ni.3460DOI Listing
July 2016

Imaging of the cross-presenting dendritic cell subsets in the skin-draining lymph node.

Proc Natl Acad Sci U S A 2016 Jan 11;113(4):1044-9. Epub 2016 Jan 11.

Laboratory for Tissue Dynamics, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa 230-0045, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Saitama 332-0012, Japan; Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan

Dendritic cells (DCs) are antigen-presenting cells specialized for activating T cells to elicit effector T-cell functions. Cross-presenting DCs are a DC subset capable of presenting antigens to CD8(+) T cells and play critical roles in cytotoxic T-cell-mediated immune responses to microorganisms and cancer. Although their importance is known, the spatiotemporal dynamics of cross-presenting DCs in vivo are incompletely understood. Here, we study the T-cell zone in skin-draining lymph nodes (SDLNs) and find it is compartmentalized into regions for CD8(+) T-cell activation by cross-presenting DCs that express the chemokine (C motif) receptor 1 gene, Xcr1 and for CD4(+) T-cell activation by CD11b(+) DCs. Xcr1-expressing DCs in the SDLNs are composed of two different populations: migratory (CD103(hi)) DCs, which immigrate from the skin, and resident (CD8α(hi)) DCs, which develop in the nodes. To characterize the dynamic interactions of these distinct DC populations with CD8(+) T cells during their activation in vivo, we developed a photoconvertible reporter mouse strain, which permits us to distinctively visualize the migratory and resident subsets of Xcr1-expressing DCs. After leaving the skin, migratory DCs infiltrated to the deep T-cell zone of the SDLNs over 3 d, which corresponded to their half-life in the SDLNs. Intravital two-photon imaging showed that after soluble antigen immunization, the newly arriving migratory DCs more efficiently form sustained conjugates with antigen-specific CD8(+) T cells than other Xcr1-expressing DCs in the SDLNs. These results offer in vivo evidence for differential contributions of migratory and resident cross-presenting DCs to CD8(+) T-cell activation.
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http://dx.doi.org/10.1073/pnas.1513607113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743831PMC
January 2016

Inactivated Sendai Virus (HVJ-E) Immobilized Electrospun Nanofiber for Cancer Therapy.

Materials (Basel) 2015 Dec 26;9(1). Epub 2015 Dec 26.

Biomaterials Unit, Nano-Life Field, International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan.

Inactivated Hemagglutinating Virus of Japan Envelope (HVJ-E) was immobilized on electrospun nanofibers of poly(-caprolactone) by layer-by-layer (LbL) assembly technique. The precursor LbL film was first constructed with poly-L-lysine and alginic acid via electrostatic interaction. Then the HVJ-E particles were immobilized on the cationic PLL outermost surface. The HVJ-E adsorption was confirmed by surface wettability test, scanning laser microscopy, scanning electron microscopy, and confocal laser microscopy. The immobilized HVJ-E particles were released from the nanofibers under physiological condition. cytotoxic assay demonstrated that the released HVJ-E from nanofibers induced cancer cell deaths. This surface immobilization technique is possible to perform on anti-cancer drug incorporated nanofibers that enables the fibers to show chemotherapy and immunotherapy simultaneously for an effective eradication of tumor cells .
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http://dx.doi.org/10.3390/ma9010012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5456544PMC
December 2015

Asymmetric PI3K Signaling Driving Developmental and Regenerative Cell Fate Bifurcation.

Cell Rep 2015 Dec 25;13(10):2203-18. Epub 2015 Nov 25.

Department of Microbiology and Immunology and Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA. Electronic address:

Metazoan sibling cells often diverge in activity and identity, suggesting links between growth signals and cell fate. We show that unequal transduction of nutrient-sensitive PI3K/AKT/mTOR signaling during cell division bifurcates transcriptional networks and fates of kindred cells. A sibling B lymphocyte with stronger signaling, indexed by FoxO1 inactivation and IRF4 induction, undergoes PI3K-driven Pax5 repression and plasma cell determination, while its sibling with weaker PI3K activity renews a memory or germinal center B cell fate. PI3K-driven effector T cell determination silences TCF1 in one sibling cell, while its PI3K-attenuated sibling self-renews in tandem. Prior to bifurcations achieving irreversible plasma or effector cell fate determination, asymmetric signaling during initial divisions specifies a more proliferative, differentiation-prone lymphocyte in tandem with a more quiescent memory cell sibling. By triggering cell division but transmitting unequal intensity between sibling cells, nutrient-sensitive signaling may be a frequent arbiter of cell fate bifurcations during development and repair.
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http://dx.doi.org/10.1016/j.celrep.2015.10.072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4685001PMC
December 2015

A biomimetic approach to hormone resistant prostate cancer cell isolation using inactivated Sendai virus (HVJ-E).

Biomater Sci 2016 Jan;4(1):96-103

International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan. and Graduate School of Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan.

Our study reports a versatile immobilization method of Hemagglutinating Virus of Japan Envelope (HVJ-E) for the generation of viral-mimetic surfaces for hormone resistant prostate cancer cell isolation. HVJ-E has recently attracted much attention as a new type of therapeutic material because hormone resistant prostate cancer cells such as PC-3 cells possess the HVJ-E receptors, GD1a. The HVJ-E was successfully immobilized on precursor films composed of poly-l-lysine and alginic acid via layer-by-layer assembly without changing the biological activity. The monolayer adsorption of HVJ-E particles was confirmed by quartz crystal microbalance, fluorescent and atomic force microscopy analyses. By developing the HVJ-E coating with an affinity based cell trap within a glass capillary tube, we are able to gently isolate PC-3 from LN-Cap cells that represent adenocarcinoma without compromising cell viability. We achieved approximately 100% cell separation efficiency only by 60 seconds of flowing. We believe that the proposed technique offers significant promise for the creation of a hormone resistant cancer cell trap on a broad range of materials.
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http://dx.doi.org/10.1039/c5bm00375jDOI Listing
January 2016

[Frontiers in Live Bone Imaging Researches. In vivo imaging of immune tissues].

Authors:
Takaharu Okada

Clin Calcium 2015 Jun;25(6):853-8

Lab for Tissue Dynamics, RIKEN Center for Integrative Medical Sciences, Japan.

In vivo imaging analysis of the immune tissues, especially secondary lymphoid tissues such as lymph nodes, has greatly increased our understanding of how immune responses are promoted and regulated by immune cell trafficking. Recently, in vivo tracking of follicular helper T (Tfh) cells, a vital T cell subset for B cell responses to produce antibodies, by imaging analysis and light-induced cell labeling not only revealed their migration dynamics, but also provided new insights into how Tfh cells may be involved in the generation of immunological memory.
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http://dx.doi.org/CliCa1506853858DOI Listing
June 2015

Percutaneous tapping for the treatment of sinusitis-related intracranial epidural abscess in children.

J Pediatr Neurosci 2014 Sep-Dec;9(3):286-8

Department of Neurosurgery, Tama-Hokubu Medical Center, Health and Medical Treatment Corporation, 1-7-1 Aobachou, Higashimurayamashi, Tokyo 189-8511, Japan.

A 13-year-old boy with medically intractable sinusitis-related intracranial epidural abscess in the frontal region was treated using percutaneous tapping. Drainage of pus measuring 7 ml yielded excellent postoperative course without cosmetic disadvantage on the forehead. Percutaneous tapping is considered to be the ideal treatment because of minimal invasiveness and cosmetic aspects of the wound.
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http://dx.doi.org/10.4103/1817-1745.147599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302558PMC
January 2015

Perivascular leukocyte clusters are essential for efficient activation of effector T cells in the skin.

Nat Immunol 2014 Nov 21;15(11):1064-9. Epub 2014 Sep 21.

Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan.

It remains largely unclear how antigen-presenting cells (APCs) encounter effector or memory T cells efficiently in the periphery. Here we used a mouse contact hypersensitivity (CHS) model to show that upon epicutaneous antigen challenge, dendritic cells (DCs) formed clusters with effector T cells in dermal perivascular areas to promote in situ proliferation and activation of skin T cells in a manner dependent on antigen and the integrin LFA-1. We found that DCs accumulated in perivascular areas and that DC clustering was abrogated by depletion of macrophages. Treatment with interleukin 1α (IL-1α) induced production of the chemokine CXCL2 by dermal macrophages, and DC clustering was suppressed by blockade of either the receptor for IL-1 (IL-1R) or the receptor for CXCL2 (CXCR2). Our findings suggest that the dermal leukocyte cluster is an essential structure for elicitating acquired cutaneous immunity.
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http://dx.doi.org/10.1038/ni.2992DOI Listing
November 2014

Memory B cells contribute to rapid Bcl6 expression by memory follicular helper T cells.

Proc Natl Acad Sci U S A 2014 Aug 28;111(32):11792-7. Epub 2014 Jul 28.

Laboratory of Lymphocyte Differentiation, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan;Laboratory for Lymphocyte Differentiation,

In primary humoral responses, B-cell lymphoma 6 (Bcl6) is a master regulator of follicular helper T (TFH) cell differentiation; however, its activation mechanisms and role in memory responses remain unclear. Here we demonstrate that survival of CXCR5(+) TFH memory cells, and thus subsequent recall antibody response, require Bcl6 expression. Furthermore, we show that, upon rechallenge with soluble antigen Bcl6 in memory TFH cells is rapidly induced in a dendritic cell-independent manner and that peptide:class II complexes (pMHC) on cognate memory B cells significantly contribute to this induction. Given the previous evidence that antigen-specific B cells residing in the follicles acquire antigens within minutes of injection, our results suggest that memory B cells present antigens to the cognate TFH memory cells, thereby contributing to rapid Bcl6 reexpression and differentiation of the TFH memory cells during humoral memory responses.
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http://dx.doi.org/10.1073/pnas.1404671111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136626PMC
August 2014

Foxp3(+) T cells regulate immunoglobulin a selection and facilitate diversification of bacterial species responsible for immune homeostasis.

Immunity 2014 Jul 10;41(1):152-65. Epub 2014 Jul 10.

Laboratory for Mucosal Immunity, Center for Integrative Medical Sciences (IMS-RCAI), RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Kanagawa, Japan. Electronic address:

Foxp3(+) T cells play a critical role for the maintenance of immune tolerance. Here we show that in mice, Foxp3(+) T cells contributed to diversification of gut microbiota, particularly of species belonging to Firmicutes. The control of indigenous bacteria by Foxp3(+) T cells involved regulatory functions both outside and inside germinal centers (GCs), consisting of suppression of inflammation and regulation of immunoglobulin A (IgA) selection in Peyer's patches, respectively. Diversified and selected IgAs contributed to maintenance of diversified and balanced microbiota, which in turn facilitated the expansion of Foxp3(+) T cells, induction of GCs, and IgA responses in the gut through a symbiotic regulatory loop. Thus, the adaptive immune system, through cellular and molecular components that are required for immune tolerance and through the diversification as well as selection of antibody repertoire, mediates host-microbial symbiosis by controlling the richness and balance of bacterial communities required for homeostasis.
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http://dx.doi.org/10.1016/j.immuni.2014.05.016DOI Listing
July 2014

Sphingosine-1-phosphate receptor 2 is critical for follicular helper T cell retention in germinal centers.

J Exp Med 2014 Jun 9;211(7):1297-305. Epub 2014 Jun 9.

Laboratory for Tissue Dynamics, Laboratory for Lymphocyte Differentiation, Laboratory for Immune Homeostasis, and Laboratory for Cytokine Regulation, RCAI, RIKEN Center for Integrative Medical Sciences (IMS-RCAI), Yokohama, Kanagawa 230-0045, Japan PRESTO, Japan Science and Technology Agency, Saitama, Saitama 332-0012, Japan Graduate School of Medical Life Science, Yokohama City University, Yokohama 230-0045, Japan

Follicular helper T (Tfh) cells access the B cell follicle to promote antibody responses and are particularly important for germinal center (GC) reactions. However, the molecular mechanisms of how Tfh cells are physically associated with GCs are incompletely understood. We report that the sphingosine-1-phosphate receptor 2 (S1PR2) gene is highly expressed in a subpopulation of Tfh cells that localizes in GCs. S1PR2-deficient Tfh cells exhibited reduced accumulation in GCs due to their impaired retention. T cells deficient in both S1PR2 and CXCR5 were ineffective in supporting GC responses compared with T cells deficient only in CXCR5. These results suggest that S1PR2 and CXCR5 cooperatively regulate localization of Tfh cells in GCs to support GC responses.
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http://dx.doi.org/10.1084/jem.20131666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076581PMC
June 2014

Predictors of the disappearance of triad symptoms in patients with idiopathic normal pressure hydrocephalus after shunt surgery.

J Neurol Sci 2013 May 16;328(1-2):64-9. Epub 2013 Mar 16.

Department of Psychiatry, Osaka University Graduate School of Medicine, D3 2-2, Yamadaoka, Suita, Osaka, 565-0871, Japan.

We identified factors that predict the disappearance of the triad of symptoms (gait disturbance, cognitive impairment and urinary incontinence) of idiopathic normal pressure hydrocephalus (iNPH) following shunt surgery in this study. We classified 71 patients with iNPH into those whose objective symptoms disappeared (disappearance group) or remained (residual group), for each of the triad symptoms 12 months after shunt surgery. Logistic regression analyses were used to identify the predictors of the disappearance of symptoms among 10 variables before shunt surgery (e.g., age, sex, severity of symptoms, Evans index, cerebrospinal fluid (CSF) pressure, CSF stasis on computerized tomographic cisternography, regional cerebral blood flow on single photon emission computed tomography, three kinds of prior diseases). For each of the triad symptoms, mild symptoms before shunt surgery were predictors of the disappearance of the symptom. Young age was also a predictor of the disappearance of gait disturbance. When the analysis was conducted using subscores of the Mini Mental State Examination, a successful visuoconstruction subtest and an absence of hypertension were predictors of the disappearance of cognitive impairment. None of the neuroimaging examinations predicted the disappearance of symptoms after shunt surgery in this study.
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http://dx.doi.org/10.1016/j.jns.2013.02.020DOI Listing
May 2013

Nano-decoration of the Hemagglutinating Virus of Japan envelope (HVJ-E) using a layer-by-layer assembly technique.

Langmuir 2013 Jun 7;29(24):7384-92. Epub 2013 Mar 7.

Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.

In this study, we created a nanoscale layer of hyaluronic acid (HA) on the inactivated Hemagglutinating Virus of Japan envelope (HVJ-E) via a layer-by-layer (LbL) assembly technique for CD-44 targeted delivery. HVJ-E was selected as the template virus because it has shown a tumor-suppressing ability by eliciting inflammatory cytokine production in dendritic cells. Although it has been required to increase the tumor-targeting ability and reduce nonspecific binding because HVJ-E fuses with virtually all cells and induces hemagglutination in the bloodstream, complete modifications of single-envelope-type viruses with HA have been difficult. Therefore, we studied the surface ζ potential of HVJ-E at different pH values and carefully examined the deposition conditions for the first layer using three cationic polymers: poly-L-lysine (PLL), chitosan (CH), and glycol chitosan (GC). GC-coated HVJ-E particles showed the highest disperse ability under physiological pH and salt conditions without aggregation. An HA layer was then prepared via alternating deposition of HA and GC. The successive decoration of multilayers on HVJ-E has been confirmed by dynamic light scattering (DLS), ζ potentials, and transmission electron microscopy (TEM). An enzymatic degradation assay revealed that only the outermost HA layer was selectively degraded by hyaluronidase. However, entire layers were destabilized at lower pH. Therefore, the HA/GC-coated HVJ-E describe here can be thought of as a potential bomb for cancer immunotherapy because of the ability of targeting CD44 as well as the explosion of nanodecorated HA/GC layers at endosomal pH while preventing nonspecific binding at physiological pH and salt conditions such as in the bloodstream or normal tissues.
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http://dx.doi.org/10.1021/la304572sDOI Listing
June 2013

Differentiation of germinal center B cells and follicular helper T cells as viewed by tracking Bcl6 expression dynamics.

Immunol Rev 2012 May;247(1):120-32

Research Unit for Immunodynamics, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, Japan.

Development of germinal center (GC) B cells and follicular helper T (Tfh) cells requires the transcription factor B-cell lymphoma 6 (Bcl6). Expression of Bcl6 in B cells and helper T cells is regulated by complex signals including those generated through their antigen-specific interactions, which take place in various microenvironments depending on their activation/differentiation states. In the last several years, it has become possible to detect Bcl6 protein in individual B cells and T cells by intracellular staining with the newly developed antibodies or by using the reporter mice. Experiments using these reagents have started to clarify microanatomical location of early Bcl6 upregulation in B cells and T cells, and contributed to reveal the dispensability and indispensability of B cells in the early and late phase of Tfh differentiation. They also started to reveal the diversity, plasticity, and/or instability of Tfh cells. We summarize the recent findings made by tracking Bcl6 expression together with the updated knowledge about dynamics of antigen-engaged B cells and helper T cells and discuss them in relation to possible signaling requirements for the development of GC B cells and Tfh cells.
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http://dx.doi.org/10.1111/j.1600-065X.2012.01120.xDOI Listing
May 2012

In vivo fluorescence resonance energy transfer imaging reveals differential activation of Rho-family GTPases in glioblastoma cell invasion.

J Cell Sci 2012 Feb 7;125(Pt 4):858-68. Epub 2012 Mar 7.

Department of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8501, Japan.

Two-photon excitation microscopy was used to visualized two different modes of invasion at perivascular and intraparenchymal regions of rat C6 glioblastoma cells that were orthotopically implanted into rat brains. Probes based on the principle of Förster resonance energy transfer (FRET) further revealed that glioblastoma cells penetrating the brain parenchyma showed higher Rac1 and Cdc42 activities and lower RhoA activity than those advancing in the perivascular regions. This spatial regulation of Rho-family GTPase activities was recapitulated in three-dimensional spheroid invasion assays with rat and human glioblastoma cells, in which multipod glioblastoma cells that invaded the gels and led the other glioblastoma cells exhibited higher Rac1 and Cdc42 activities than the trailing glioblastoma cells. We also studied the Cdc42-specific guanine nucleotide exchange factor Zizimin1 (also known as DOCK9) as a possible contributor to this spatially controlled activation of Rho-family GTPases, because it is known to play an essential role in the extension of neurites. We found that shRNA-mediated knockdown of Zizimin1 inhibited formation of pseudopodia and concomitant invasion of glioblastoma cells both under a 3D culture condition and in vivo. Our results suggest that the difference in the activity balance of Rac1 and Cdc42 versus RhoA determines the mode of glioblastoma invasion and that Zizimin1 contributes to the invasiveness of glioblastoma cells with high Rac1 and Cdc42 activities.
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http://dx.doi.org/10.1242/jcs.089995DOI Listing
February 2012

Four-dimensional tracking of lymphocyte migration and interactions in lymph nodes by two-photon microscopy.

Methods Enzymol 2012 ;506:437-54

Research Unit for Immunodynamics, RIKEN, Research Center for Allergy and Immunology, Yokohama, Japan.

Two-photon microscopy of live tissue imaging provides insightful information about the four-dimensional dynamics of cell behavior and has contributed to the discoveries of new biological mechanisms including those in the immunology field. In recent years, it has become easier for many researchers to perform the tissue imaging experiments, due to the refinement of the commercially available microscope systems. However, it is still crucial for the efficient visualization of biological events to optimize the sample preparation by using the best available reagents. Further, it is equally important to elicit key information from the large quantity of imaging data, whose handling is often complex and laborious. Here, by taking as an example the two-photon analysis of lymphocyte migration and interaction in explanted lymph nodes, we describe the key points that need to be considered for the successful experimental design, sample preparation, image acquisition, and data analysis.
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http://dx.doi.org/10.1016/B978-0-12-391856-7.00047-0DOI Listing
August 2012