Activities and progress in research of MICD

The 5-year project "Systematic elucidation of pathogenesis and development of diagnosis and treatment by molecular imaging" was carried out at Takii campus in 2011-2012 and Hirakata campus in 2013-2015. On the occasion of consolidating the campus in Hirakata in April 2013, to promote collaborative research and translational research from basic to clinical medicine such as early diagnosis and treatment, we started three activities: a cancer-oriented consortium, a regenerative medicine consortium, and lunch-time research talks among basic medical departments. At that time we established Molecular Imaging Center of Diseases (MICD) on the 5th floor of the north wing of the Hirakata campus and assigned a lab assistant in it. As the result of promoting collaborative research with other laboratories in Japan and abroad, the project has progressed satisfactorily, as evidenced by the publication of more than 250 papers in foreign journals including Cell, Nature, Nat. Cell Biol., Nat. Neurosci., Nat. Commun., Neuron, Proc. Natl. Acad. Sci. USA, Sci. Rep., J. Biol. Chem., Neuron, J. Neurosci., Eur. J. Neurosci., J. Immunol., Blood, and J. Gastroenterol.  Our research activities have been three times evaluated in situ at the 1st, 2nd and final annual meetings and documantary evaluation of the 5-year research activities by advisory board members of MICD. This section picks up 6 topics of research activities by all 36 core members in MICD and the excellent achievements matched to the objectives to MICD have been introduced with figures in the front. Detailed achievements by individual core members at the Hirakata campus are included in Supplementary Information 10 and 11 of this report. Please refer those conducted at the Takii campus to MICD Interim Report 2011-2012.

1. Identification of tissue stem cells
 In this project, aiming the application of cell transplantation and regenerative medicine and the development of cancer therapy, we attempted to identify tissue stem cells in various tissues in mice and humans and elucidate a mechanism of transition to cancer stem cells at multiple aspects. The filiform papillae of the tongue are one of the tissues with most rapid turnover rates in the mammalian body and lingual keratinized epithelial cells are thought be the source of squamous cell carcinoma of the tongue. Ueno (Stem Cell Pathology) showed that stem cells positive for Bmi1, keratin 14 and keratin 5 are present in the base but not at the very bottom of the interpapillary pit and that one stem cell per interpapillary pit survives for a long term. While the cells are usually in a slow-growing or resting state, they entered the cell cycle upon irradiation-induced injury and regenerated tongue epithelium. The elimination of Bmi1-positive stem cells significantly suppressed the regeneration. Taken together, Ueno reported that the stem cells identified by using a multicolor lineage tracing method established by himself are important for tissue maintenance and regeneration of the lingual epithelium (Fig. 1, Nature 464:549–553, 2010; Nat. Cell Biol. 15:511-518, 2013). Furthermore a novel lingual epithelial organoid culture system was established from lingual epithelial cells and Bmi1-positive stem cells using a three-dimensional matrix. Ueno observed that organoids in culture could be engrafted and maturate in the tongue of recipient mice and that the organoids generated from carcinogen-treated mice had an abnormal configuration (Fig. 2, Sci. Rep. 3:3224, 2013). Thus Ueno succeeded in the establishment of multicolor lineage tracing method applicable to identification of intestinal stem cell such as tongue and colon, generation and maintenance of testis mentioned below and analysis of cancer development in live tissues in vivo.

 A mouse homologue of Drosophila ovo, Ovol2 identified by Ito's group (Molecular and Functional Biology) is a zinc finger transcriptional factor and expressed in embryonic stem cells. Although Ovol2 knockout mice are lethal as a result of defects in extraembryonic and embryonic vascularization, neural tube and heart formation, epidermis-specific conditional Ovol2 knockout mice revealed its exact in vivo roles in the skin. Ovol2 as well as Ovol1 is expressed in progenitor cells of the epidermis and hair follicles. Simultaneous ablation of transcriptional repressor-encoding Ovol1 and Ovol2 (DKO) blocked terminal differentiation of embryonic epidermal progenitor cells and resulted in expansion of the basal/spinous compartments. DKO embryos lacked a functional permeability barrier due to immature cornified layer, as illustrated by persistent dye penetration (Fig. 3, Dev. Cell 29:47-58, 2014).

 In adult mice, Ovol2 is strongly expressed in the testis. Ito prepared Ovol2-specific antibody and revealed that Ovol2 expression is restricted to the XY body in mouse and human spermatocytes at the pachytene stage. These results suggested that Ovol2 plays an important role in the XY body during spermatogenesis, possibly in the processes of XY body formation and meiotic sex chromosome inactivation (Fig. 4, J. Androl. 33:277-286, 2012; Andrologia, in press, 2016). Ueno reported that Bmi1 is a seminiferous stage-dependent marker for long-term germ stem cells and that Bmi1-positive cells play important roles in maintaining germ stem cells and in regenerating spermatogenic progenitors after injury by the multicolor lineage tracing method (Sci. Rep. 4:6175, 2014).

 Azoospermia or severe oligozoospermia is a major cause of male infertility. Testosterone is one of the androgens synthesized from cholesterol as a precursor in the Leydig cells of the testis could be visualized in the testis after derivatization with Girald’s T reagent by imaging mass spectrometry. One hour after i.p. injection of human chorionic gonadotropin, the biosynthesis of testosterone was observed in Leydig cells and testosterone released from Leydig cells was distributed in the inside of the seminiferous tubules (Fig. 5, Anal. Bioanal. Chem. in press, 2016).

 Lamins are thought to be implicated in normal morphogenesis and cell-differentiation. In the nervous system, their abnormality causes neurodegenerative diseases such as Hutchinson-Gilford Progeria syndrome and leukodystrophy. Yamada (Regenerative Neural Medicine) investigated the expression patterns of lamin isotypes in the adult neural tissues. In the neurogenic regions of adult rat brain, the lamins are involved in cell differentiation by changing the composition of lamin isotypes from neural stem cells, progenitor cells to mature neurons. Yamada also showed that neuronal precursors in the subventricular zone (SVZ) are tightly regulated and required stimuli stronger than those in the subgranular zone (SGZ).

 Nishiyama and Kanda (Public Health) focused on the identification of tissue stem cells in the pituitary and auditory pathway. The population of side population (SP) cells possessing sphere formation was about 1% of cochlea nuclei prepared from 3-week-old mice and decreased by aging. SP cells were characterized by gene expression. Next, Kanda identified and characterized tissue stem cells in the inferior colliculi which integrates sound information in the central auditory neural pathway and demonstrated that Promin-1 is a marker of neural stem/progenitor cells present in the inferior colliculi which can be differentiated into both neurons and glia by its induction.

 Sonoda (Stem Cell Biology) developed a highly effective isolation method of mouse bone-derived very small embryonic-like (VSELs) phenotypic stem cells, named bone-derived small cells (BDSCs), using enzymatic treatment of murine bone and FACS. Then he succeeded in isolation of BDSCs with 50-100 efficiency as compared with the conventional method. BDSCs were small (6~7 µm) and possessed a relatively large nucleus surrounded by a narrow rim of cytoplasm. Moreover, these BDSCs could differentiate to hematopoietic cells in vitro. Very interestingly, they transdifferentiated to hepatocytes in vivo after transplantation to syngeneic mice with massive liver injury (Stem Cells Dev. 25:27-42, 2016). Sonoda also developed a highly effective isolation method of human BDSCs from human bone for future possible clinical application. In parallel, Sonoda established human bone marrow-derived CD271+SSEA-4+ mesenchymal stromal cells (DP-MSCs) and characterized their functions by co-culture with CD34-negative (CD34-) very primitive hematopoietic stem cells (HSCs). DP-MSCs could support CD34- HSCs in the co-culture system and CD34+ HSCs were efficiently produced (Stem Cells 33:1554-1565, 2015). Based on these data, Sonoda is now developing an efficient ex vivo expansion system for human CD34+ HSCs.

 Shiojima (Internal Medicine 2) developed a method to effectively mobilize cardiac stem cells into peripheral blood via upregulation of hepatic growth factor. Human cardiac stem cell-like cells that are capable of differentiating into multiple cell types in the heart was previously shown to be mobilized into circulation during open heart surgery. In this project, Shiojima found that intravenous infusion of heparin mobilized cardiac stem cells in peripheral circulation and demonstrated that heparin released hepatocyte growth factor bound to extracellular matrix into circulation, which induced the mobilization of cardiac stem cells.

 Okazaki (Internal Medicine 3) determined the phosphorylated Smad2/3 at Thr220 /Thr179 (pSmad2/3L-Thr) as a novel and useful marker of epithelial stem (or progenitor) cells around crypt bases in small intestine and colon. pSmad2/3L-Thr- positive cells were located adjacent to actively proliferating cells and not immunostained with Ki67, a proliferating marker of cell cycle in various animal models. pSmad2/3L-Thr-explessing cells were slow-cycling cells and increased concurrent with hyperplastic changes in the regeneration phase of DSS-induced colitis. Associated with the regeneration and recovery, pSmad2/3L-Thr-positive cells markedly increased in crypto bases of the digestive tract and tubular complex of the pancreas.

2. Analyses of molecular dynamics, cellular function and pathogenesis by two-photon microscopy
 With two-photon imaging using the lymph node tissue slice method, Kinashi (Molecular Genetics) revealed that high-speed interstitial migration of T cells requires adhesion through LFA-1 and ICAM-1 (Fig. 6). Small GTP-binding protein Rap1 regulates adhesive processes during lymphocyte trafficking mediated by integrins through its receptor RapL and its downstream molecule Ste20-like kinase Mst1. Since antigen-specific suppressive functions of regulatory T cells were impaired in Mst1-/- mice, Kinashi demonstrated that Mst1 has a key role in regulating self-antigen recognition by thymocytes in the medulla (Nat. Commun. 3:1098, 2012). Kinashi confirmed that Mst1-/- naive T cells showed defective antigen-specific adhesive structure, i.e. immunological synapse on the lipid bilayer displaying peptide-MHC and ICAM-1 and demonstrated that Mst1 is required for high-affinity LFA-1 through kindlin3. Thus from two-photon microscopic analyses using in vitro and tissue slice system, fibroblastic reticular cells that express ICAM-1 and constitute the T cell zone play a critical role in rapid, directional migration of T cells. Autoimmune-like symptoms became evident with aging in Mst1-/- mice due to impaired thymocyte selection (J. Immunol. 191:1188-1199, 2013; Sci. Signal. 7:ra72, 2014).

 Noxious and non-noxious stimuli are conveyed by primary afferent fibers which form synapses with spinal neurons, processed in network of dorsal horn of the spinal cord, transmitted to the cortex, and recognized as pain and touch, respectively. To clarify how sensory reception from the periphery is processed in the spinal cord, Ito and Matsumura (Molecular and Functional Biology) have analyzed spinal responses morphologically and functionally using two-photon microscopy and fluorescent protein-expressing mice. Morphological alterations of spines in the neurites of the spinal dorsal horn rapidly occurred after inflammation in the foot and leg via activation of AMPA and NMDA receptors by using thy1-YFP mice expressing fluorescent protein specific to the nervous system (Fig. 7, Eur. J. Neurosci. 41:989-997, 2015). Ito and Nishida expanded the two-photon microscopic research of in vivo structural imaging to in vivo functional imaging in the spinal cord by the intrauterine gene transfection method. While in vivo patch clamp can analyze a cellular response in only one spinal neuron, two-photon microscopy could analyze it in around 100 cells simultaneously and for more than 6 h in vivo. Ito applied thermal, mechanical and touch stimuli to the skin of mice expressing the Ca2+ indicator protein YCnano50 in the spinal cord and succeeded in making a temporal and spatial map of the stimulus-response of spinal neurons to different stimuli (PLoS One 9:e103321, 2014).

3. Molecular imaging methods of candidate molecules related to diseases by imaging mass spectrometry
 Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis are caused by loss of function due to neuronal death. Pathological changes were examined in postmortem brain of patients or tissue samples of model animals. Imaging mass spectrometry (IMS) is a gradually prevalent tool for analysis of small molecule distribution in the field of neurochemistry. Yao (Molecular and Functional Biology) optimized analytical conditions such as matrix, ionization, quick sampling of materials, and laser intensity and first succeeded in imaging of ACh by IMS showing that its distribution was coincident with that of the degrading enzyme ACh esterase in rat brain (Fig. 8, Anal. Bioanal. Chem. 403:1851-1861, 2012). Since IMS measures the ACh content of synaptic vesicles in the axon terminals, different from measurement of a metabolite of released ACh from synapses, its detection of ACh will be useful to analyze animal models and postmortem brain samples of patients with neurodegenerative disorders such Alzheimer's disease.

 To establish molecular imaging of pathological samples on glass slides by IMS, Kwon (Surgery) and Yao applied the IMS technique to formalin-fixed paraffin-embedded samples to identify a molecule responsible for the intestinal obstruction. After the deparaffinization and enzymatic digestion, they identified it as amylopectin by the IMS. This result was confirmed by PAS staining, iodine, and MS and MS/MS analysis of authentic amylopectin. The IMS imaging of paraffin-embedded tissue samples will expand its application to pathological diagnosis.

 Yamada (Regenerative Neural Medicine) identified the monoclonal antibody O4 used for an oligodendrocyte marker for a long time, as sulfatide by IMS and confirmed by mice lacking cerebroside sulfotransferase, an enzyme of sulfatide biosynthesis. The sulfatide with short chain fatty acids was shown to regulate early oligodendrocyte development.

 There are two types of melanin in the skin: photoreactive pheomelanin and photoprotective eumelanin. While melanocytes of mice are present in the dermis, human melanocytes are present in the epidermis. Okamoto (Dermatology) produced photoreactive mice derived from stem cell factor transgenic mice, which are suitable for photo-biological tests in humans. Neither melanin, eumelanin nor pheomelanin was detected in the skin of the mice by mass microscope, because melanin is a polymer of small molecules and could not be ionized by pretreatment and matrices used.

 It is well known that endogenous digitalis-like substances (EDLS) are produced upon sodium loading in rats and humans and it is assumed to be a cause of hypertension related to excess intake of sodium for many years. Since EDLS are structurally similar with the similar molecular weight, it was difficult to identify them immunohistochemically with specific antibodies. Takahashi (Clinical Sciences and Laboratory Medicine) found marinobufotoxin and marinobufagenin as candidates of EDLD and detected the authentic samples at as low as a few pg by IMS. However, EDLS could not be detected in cells and tissues such as adrenal and hypothalamus.

 Although the IMS enables us to analyze small target molecules including lipids, neurotransmitters, metabolites, it took much time to optimize the analytical conditions such as ionization efficiency, increasing signal/noise ratio, and identification of MS fragments and specialized knowledge was required for obtaining reliable data.

 Atomic force microscopy (AFM) has gained recognition as an imaging tool for surfaces of various materials ranging from solids to soft biological materials. Kihara, Kusumoto and Kageshima (Physics) have tried to analyze structure and dynamics of solid-liquid interfaces and water response, conformation change of peptides and proteins and organelles by soft X-ray microscopy and AFM.

4. Analysis of brain functions in mice and monkeys and clinical research
 The striatum is innervated from various regions of cerebral cortex including frontal cortex and regulates the order of behaviors, evaluation, and integration of information. Sugimoto (Anatomy and Brain Science) characterized the lateral region of the striatum by GRP155 immunoreactivity and in situ hybridization of glutamate decarboxylase (GAD) 1 isoforms. GRP155 was predominantly expressed in the medium spiny neurons that contain D1 dopamine receptors and GRP155-expressing neurons were in charge of the direct pathway by which information from sensory-motor cortex transmits to the output portion of the basal ganglia.

 The role of the basal ganglia in decision making in face of both appetitive and aversive options has been clarified focusing on the dopaminergic neurons of the striatum. Nakamura and Isoda (Cognitive Neuroscience) analyzed cognitive processes in the monkey (Nat. Neurosci. 15:1307-1312, 2012; J. Neurosci. 35:6195-6208, 2015). Nakamura demonstrated that the stress impaired the reward-based decision making process by the neuronal mechanisms involving the basal ganglia. The value of one’s own reward is influenced by a reward to others. Electrophysiological recordings in monkey brains showed that medial prefrontal neurons coded self-reward information and other-reward information, whereas midbrain dopamine neurons coded a subjective value of one’s own reward. These studies using monkeys are steady for analyzing various contexts of behaviors and open a new way to clarify the connection between prefrontal cortex and the limbic system that has been now revealed by fMRI.

 Parkinson's disease is a neurodegenerative disease presenting progressive motor dysfunction such as tremor, akinesia and rigidity and is caused by dopaminergic neuronal loss in the substantia nigra pars compacta. Kusaka (Neurology) focused on the analysis of inclusion body in the postmortem brain of neurodegenerative diseases in the first half of the project and elucidation of pathogenesis of Parkinson’s model rats and levodopa-induced dyskinesia rats in the second half of the project.

 It remains unknown how more than 1000 odorant receptors reported by the Nobel Prize laureate R. Axel bind to 100000 odorants and these signals are transmitted and integrated in the brain. Since the sensitivity of smell molecules is varied among individuals, emotion and behaviors induced by smell have been considered to be controlled by learned mechanism. Kobayakawa (Functional Neuroscience) revealed that fear induced by odorant molecules are separated into inert and learned information and transmitted to the brain via separate pathways (Proc. Natl. Acad. Sci. USA 112:E311-E320, 2015). Furthermore, by using optogenetics, pharmacological genetics, and in vivo fiber photometry system, Kobayakawa revealed that serotonin receptor 2A-expressing cells in the central amygdala control the hierarchy of the innate and learned fear, prioritizing innate fear over learned fear (Fig. 9, Cell 163:1153-1164, 2015). These results suggest that smell is induced by the binding of odorant molecules to their receptors like hormone and that it is not always recognized by a pattern of many odorants. Since there are many drugs for mental disorders which serve as odorants, a possibility that administration of a drug that alleviates learned fear may worsen innate fear. It is important to dissect and analyze the contribution of innate and learned systems in mental disorders and prescribe appropriate drugs for the treatment.

 In humans, noninvasive MRI is a powerful tool to analyze brain functions. Mirror neurons are known to contribute to creating a sense of intimacy and their dysfunction is considered to be one of the core deficits of socially isolating disorders. Kinoshita and Saito (Neuropsychiatry) succeeded in the fiber tracts containing mirror neurons of patients and healthy control by diffusion tensor imaging in collaboration with Psychiatry Neuroimaging Laboratory of Harvard University and aimed to evaluate their disorders before and after psychoanalytical treatment.

5. Establishment of disease animal models toward elucidation of pathogenesis and development of diagnosis and treatment
 Different from stiff and strong collagen fibers, elastic fibers, one of extracellular matrix, are ones with flexibility and elasticity. Although latent TGFβ binding protein 4 (LTBP4) is one of the LTBP family proteins, which bind to inactivate and store it in the extracellular matrix, Nakamura and Akamai (Molecular Pharmacology) found that amount of LTBP4 protein was greatly decreased in the skin of old donor. LTBP4 mutant mice had stiff, tortuous artery with inelastic property, indicative of artery sclerosis. By fiber formation assay on primary cultured human dermal fibroblasts, recombinant LTBP4 protein in the culture medium of LTBP4-deficient fibroblast cells restored elastic fiber meshwork formation in the cells in a dose-dependent manner. Nakamura concluded that LTBP4 protein is a rate-limiting factor for elastic fiber formation in the cultured cells and suggested that LTBP4 is a target molecule of elastic fiber restoration (Fig. 10, Proc. Natl. Acad. Sci. USA 110: 2852-2857, 2013). While micro fibrils, an extracellular matrix, provide a scaffold for elastic tissues such as lung, aorta and skin, they themselves connect lens with ciliary muscle in the ciliary body of the eye. Nakamura also found that LTBP2 knockout mice developed lens luxation caused by the impaired formation of ciliary body, suggesting that they are an ideal animal model for congenital lens luxation, ectopia lentis and glaucoma in human (Fig. 11, Hum. Mol. Genet. 23:5672-5682, 2014).

 Matsuda (Cellular and Molecular Physiology) analyzed the role of adenosine in CFTR Cl- channel of exocytosis of the pancreas and demonstrated that adenosine receptor control the secretion of pancreatic juice by forming the complex of the A-kinase anchoring protein ezrin and CFTR Cl- channel.

 Although there was no definitive treatment for this intractable burning pain 'causalgia' over 150 years since its discovery, it was reported to be treated by surgery with in situ tissue engineering. To clarify the mechanism of peripheral nerve regeneration and promote translational research, Ito (Molecular and Functional Biology) established a mouse model of nerve regeneration in thy1-YFP mice expressing YFP specific to the nervous system. This nerve regeneration model has an advantage of chasing the process of nerve regeneration in vivo using the same operated mice for a long period. Unexpectedly, we found that nerve regeneration was markedly delayed in mice deficient in Nax, a sodium concentration-dependent Na+ channel and the delay was restored by the continuous supply of lactate, a metabolite of glucose. Nax was expressed in Schwann cells. Schwann cells in the peripheral nerve play two roles of astrocytes and oligodendrocytes in the central nervous system. Ito demonstrated that Schwann cells accelerated nerve regeneration by energy supply to regenerated axons via a lactate shuttle in addition to the supply of growth factors in the nerve tract (Fig. 12, Eur. J. Neurosci. 39720-729, 2014). Furthermore, endothelin and blood supply were shown to need nerve regeneration.

 Fujisawa (Viral Oncology) demonstrated that HTLV-1infected humanized mouse model (hu-NOG) is useful for the evaluation of the efficacy of immunization against HYLV-1 infection and leukemogenesis. Fujisawa succeeded in HTLV-1 infection in hu-NOG generated by intra-bone marrow-bone marrow transplantation of human stem cells (Blood 123:346-355, 2014). In infected cells, abnormal proliferation of infected T cell with highly lobulated or flower-shaped nuclei, splenomegaly and infiltration to the liver were observed. Fujisawa clearly showed that Tax-peptide vaccine was effective in the suppression of leukemic growth of HTLV-1 infected T cells in hu-NOG. In this humanized model, host immune system against HYLV-1-infected lymphocyte plays a crucial role in maintaining the asymptomatic state of HHTLV-1-infected carrier people. Furthermore for the development of the prevention of HTLV-1, intranasal vaccination of Tax–peptide vaccine was found as effective as the subcutaneous injection.

 The phosphoinositide 3-kinase (PI3K)-mTOR pathway is important for the differentiation, proliferation and survival of a variety of cells and is shown to be abnormal in various tumor cells. Matsuda (Cell Signaling) has focused on mTORC1 pathway and established mTORC1 signal-deficient mice lacking Raptor specifically in cell lineage. While the decrease in number of thymocytes ware not observed, Th17 differentiation was impaired in T cell-specific deletion. In dendritic cell-specific deletion, IL10 production from a certain type of dendritic cells in the gastrointestinal tract was impaired, leading to uncontrolled inflammation in the intestine (J. Immunol. 188:4736-4740, 2012). In 2014, Matsuda demonstrated that the development of earl T cell progenitors was impaired at the DN2 stage in Raptor deficiency in all tissues including hematopoietic cells and suggested that mTORC1 is a target molecule for acute T cell leukemia (Proc. Natl. Acad. Sci. USA 111:3805-3810, 2014). In these mice, the number of B cells was markedly reduced but the number of common lymphoid progenitors was not decreased. B-cell specific deleted mice revealed that mTORC1 signal plays an essential role in B cell development at pro-B cell stage in the bone marrow. Thus, Matsuda established Raptor deficient mice specific to cell lineage serially and revealed its roles in vivo.

 Takahashi (Ophthalmology) prepared type 1 choroidal neovascularization animal model and investigated the effect of microparticles on neovascularization in the model. Lee (Model Animals) tried to establish the technology for generation of genetically modified mice using the CRISPR/Cas9-mediated genome editing to support researchers of MICD.

6. Development of diagnosis and treatment and clinical application
 Ikehara (Stem Cell Disorders) proposed that incurable diseases with aging-associated symptoms are linked to abnormalities in the immune system, and suggested that transplantation of both intra-bone marrow-bone marrow (IBM-BM) and thymus can be effective interventions in the treatment of autoimmune diseases and aging-associated symptoms. Ikehara also showed that these diseases were caused by bone marrow mesenchymal cells, but not by hematopoietic cells. For clinical application of IBM-BM transplantation to patients, Ikehara collaborated with Nomura (Internal Medicine 1) and Iida (Orthopedic Surgery).

 Nakatani (Spinal Cord Regeneration) summarized clinical research for the treatment of spinal cord injury by intrathecal administration of cultured autologous bone marrow stromal cells into the cerebrospinal fluid perfomed in 5 patients with complete tetraplegia in the previous project. The patients were closely observed for 6 months and further followed up for 1 to 4 years with no adverse responses in biochemical and radiographic examinations. Functional recovery was evaluated as remarkable for two patients, gradual or limited for 2 and in vain for 1. This clinical study confirmed that intrathecal administration of cultured autologous bone marrow stromal cells was safe and feasible for treatment of spinal cord injury.

 It is quite important for surgical treatment of cancers to evaluate invasion of blood vessels into tumor, remnant of tumor tissue after resection and metastasis of lymph nodes accurately during operation. Indocyanine green (ICG) and the porphyrin precursor 5-aminolevulinic acid (5-ALA) have been approved as fluorescence imaging agents in the clinical setting. Kwon and Kaibori (Surgery) evaluated the usefulness of ICG and 5-ALA for intraoperative identification of latent small liver tumors with 48 patients. The sensitivity, specificity, and accuracy for detecting the preoperatively identified main tumors were 96%, 50%, and 94%, respectively with ICG and 57%, 100%, and 58%, respectively, with 5-ALA. Five new tumors were detected by both ICG and 5-ALA fluorescence imaging during operation. Although the sensitivity for main tumor detection was relatively high by ICG fluorescence imaging, the specificity was high by 5-ALA imaging. Intraoperative fluorescence navigation by ICG and 5-ALA compensates the sensitivity and specificity for the detection of latent small liver tumors and its usefulness can be expected.

 Surgical insults may cause inflammatory stress. Shingu (Medical Control and Stress Response) found that the intravenous anesthetic propofol and the selective α2-adrenergic receptor antagonist dexmedetomide possessed anti-inflammatory properties. Which anesthetic is selected in various clinical situations is important for the improvement of prognosis of patients.

 Orthopedic implants are removed because of aseptic loosening and prosthetic joint infections of hip and knee. It was challenging, but difficult and inaccurate, to identify microorganisms growing in biofilms for the diagnosis of implant-associated infections. Since it takes at least 5 days to 10 days to identify microorganisms by conventional culture, rapid examination with high sensitivity is required for early treatment following diagnosis of implant-associated infections. Iida (Orthopedic Surgery) improved the extraction of microorganisms in biofilm by sonication and is now constructing a rapid diagnostic system with next generation sequencer in a combination with PCR.

 Next generation sequencer is useful for comprehensive analysis of microRNAs, because they are involved in a variety of life phenomena and pathogenesis and rather stable. Akane (Legal Medicine) examined the possibility of microRNAs as postmortem biomarkers with next generation sequencer.

 A molecular target drug has recently come to be used for the medical treatment of malignant tumors. Tsuta (Pathology and Laboratory Medicine) developed a diagnosis kit for malignant tumors using the phosphor integrated dot with high illuminance and high dynamic range, superior to conventional fluorescent dye.

 Shimojo (Molecular and Functional Biology) found that expression of sREST, an isoform of RE-1 silencing transcription factor (REST/NRSF) was related to the pathogenesis of small cell lung cancer and that nSR100 involved in alternative splicing of REST was also expressed in a cell line of human small cell lung cancer. In collaboration with clinical departments in cancer-oriented consortium, it was shown that miRNAs that inhibit nSR100 expression were present in patients with small cell lung cancer. Shimojo established an in vitro assay system for the evaluation of nSR100 expression and applied it for the development of diagnosis and drugs. "Search for oligonucleotides toward the treatment of small cell lung cancer targeting nSR100" was adopted by a grant from Japan Agency for Medical Research and Development (AMED) in 2015.

 Sawada, Utsunomiya, and Tanigawa (Radiology) were engaged in basic research on developing radioisotope-labeled anti-cancer drugs for early diagnosis and treatment with SPECT/CT instrument installed in Radioisotope (RI) imaging room of MICD. RI-labeled anti-Her2 antibody, CD38 antibody, anti-ferritin antibody were examined for early diagnosis for breast cancer, multiple myeloma, and all kinds of tumors, respectively, and 90Y-labeled CD38 antibody was shown to be accumulated in transplanted tumors. 90Y-labeled lipiodol was also shown to be effective in liver metastasis of solid tumors in rabbits.

 Dendritic cells (DCs) are master cells in activating immune responses through enhancing by cytokine production and priming naive CD4+ T cells and implicate in the processes of various inflammatory responses. Nomura and Ito (Internal Medicine 1) provided new insights of the cross-talk between DC and platelets in enhancement of immune inflammatory responses. Interaction of thymic stromal lymphoprotein (TSLP) and DCs plays an essential role in evoking Th2 responses in allergy. Activated platelets enhanced chemokine production by TSLP-activated DCs. Nomura also demonstrated that IL33 produced by epithelial cells works as a positive regulator of TSLP-DC axis that initiates and maintains the Th2 cell-mediated inflammatory responses and suggested that platelets and IL33 would be new targets for the treatment of allergic disorders. Furthermore, Nomura suggested a role of statins, lipid lowering drugs, in controlling the TSLP-DC axis and their therapeutic potentials for allergic diseases.

 Kusumoto (Plastic and Reconstructive Surgery) has been engaged in basic research on wound healing and cytoskeletal changes of adipocyte-derived stem cells and focused on its clinical application to breast reconstruction using adipocyte-derived stem cells after resecting breast cancer.

Research Activities
2011 Research Activities PDF
2012 Research Activities PDF
2012-13 Interim Report PDF
2011-15 Final Report PDF