Astrocyte-neuron lactate shuttle (ANLS) signaling contributes to learning or memory and induces long-lasting nociceptive behavior. Monocarboxylate transporters (MCTs) have been reported to transport L-lactate. While MCT1, MCT2, and MCT4 are expressed in the rodent brain, it remains unclear which MCT isoform is functionally expressed by human astrocytes. Furthermore, there have been only a few reports on MCT1- or MCT4-selective inhibitors. First, we established the contribution of each MCT isoform to L-lactate transport in human astrocytes. The cellular uptake of L-lactate was found to be pH- and concentration-dependent with a Km value of 0.64 mM for L-lactate uptake. This Km value was similar to that previously established for MCT1-mediated L-lactate uptake. Next, we tried to identify a selective MCT1 or MCT4 inhibitor. While inhibitory effects were observed for MCT1 with 5-oxoproline, no inhibitory effect was observed for MCT4. Bindarit was demonstrated to exhibit selective inhibitory effect against MCT4, but not against MCT1. These findings provide novel insights into the mechanism of L-lactate transport by astrocytes, and contribute to developing novel targets for ANLS.

Nitric oxide (NO) is a highly diffusible molecule generated from a family of NO syntheses (NOSs) that convert L-arginine to L-citrulline using oxygen and NADPH. Under physiological conditions, NO is produced at low levels in response to endogenous stimuli such as acetylcholine and acts as critical messenger of intracellular signaling pathways. NO also has a variety of physiological functions such as neurotransmission, inflammatory reaction, vasodilation, and stimulates both angiogenesis and cell proliferation.

Protein S-nitrosylation is an NO-dependent reversible post-translational modification of cysteine that regulates both protein structure and function in bacteria, plants, and mammals. Over the past couple of decades, advanced proteomic approaches have led to the identification of more than a thousand S-nitrosylated (SNO-) proteins with diverse cellular functions. So far, we demonstrated that PDI, PTEN, and HDAC6 are targets of NO and regulate the enzymatic activity via S-nitrosylation.

Recently, we found a novel substrate of NO that is involved in the regulation of gene expression. In addition, we attempted to develop a specific inhibitor of S-nitrosylation of this substrate by the hierarchical virtual screening approaches. We will discuss those findings in this session.

H₂S is produced by enzymes and has various physiological roles including neuromodulation, vascular tone regulation, cytoprotection against oxidative stress. We previously demonstrated that H₂S relaxes vascular smooth muscle in synergy with NO. In the process of the study about the effect of H₂S on transient receptor potential (TRP) channels, we found that H₂S_n activates TRP ankyrine 1 (TRPA1) channels much more potently than does H₂S and that 3-mercaptopyruvate sulfurtransferase (3MST) produces H₂S₂ and H₂S₃. The chemical interaction of H₂S with nitric oxide (NO) has been reported to generate several products including nitroxyl (HNO), nitrosopersulfide (HSSNO) and H₂S_n. The effect of H₂S_n on TRPA1 channels is suppressed by reduction and by cyanide, while that of HNO is resistant to reduction and that of HSSNO to cyanide. Based on these observations we concluded that H₂S_n are chemical entities generated by the interaction of H₂S with NO. I will focus on the production of H₂S_n and a potential mechanism of the synergistic effect of H₂S and NO.

In this symposium, we introduce our recent studies regarding new actions of diet-derived nitric oxide (NO) and bone marrow-derived NO. 

A new pathway in which NO is synthesized from NO metabolites; nitrite and nitrate (NOx), is recently discovered. However, whether dietary NOx deficiency causes diseases remains to be clarified. We demonstrated that long-term low NOx diet caused metabolic syndrome, vascular dysfunction, and cardiovascular death in mice. These results indicate that dietary NOx plays a pivotal role in the prevention of those disorders (Diabetologia 2017).

NO, synthesized by NOSs (nNOS, iNOS, and eNOS), plays a role in the development of pulmonary hypertension. However, the role of NO/NOSs in bone marrow cells in pulmonary hypertension remains elusive. We showed that transplantation of n/i/eNOSs-deficient bone marrow significantly aggravated hypoxia-induced pulmonary hypertension in wild-type mice, and transplantation of wild-type bone marrow significantly ameliorated hypoxia-induced pulmonary hypertension in n/i/eNOSs-deficient mice. These results show that myelocytic n/i/eNOSs play a crucial protective role in the pathogenesis of pulmonary hypertension (Am J Respir Crit Care Med 2018).

Nitric oxide(NO) bioavailability is limited in senescence. We studied it in human umbilical venous endothelial cells(HUVECs). NO donor and transfection with endothelial NO synthase(eNOS) into HUVECs each decreased SA-b-gal positive cells and increased telomerase activity. 17b-estradiol decreased SA-b-gal-positive cells and caused cell proliferation. L-arginine(L-Arg) or L-citrulline(L-Cit) partially inhibited, and combination of L-arg and L-cit(LALC) strongly prevented, high glucose-induced senescence. Following 3-day stimulation of HUVECs under high-glucose with L-Arg or L-Cit or LALC, endothelial senescence and function were evaluated.  These amino acids were also administered to dyslipidemic type 2 diabetic(ZDFM) rats fed a high-cholesterol diet for 4 weeks.  L-Cit and LALC retarded HG-induced endothelial senescence, and restored telomerase activity.  p22-phox was not altered, but L-Cit decreased ROS.  Under HG, L-Cit and LALC increased NO. and eNOS and phosphorylated eNOS were decreased. In ZDFM rats, SA-b-gal on the aortic surface was reduced by L-Cit and LALC. LALC for 4 weeks increased plasma NO. Thus, L-Cit and LCLA inhibited HG-induced endothelial senescence and NO-cGMP pathway. The delay in endothelial senescence through NO and eNOS may have clinical utility in the treatment of atherosclerosis in elderly.

Cancer patients nowadays can choose a wide variety of cancer therapies based on new anti-cancer drugs and state-of-the art operational technology. Also, cancer has been recognized as curable diseases with increased cancer survivors. On the other hand, cancer survivors who have various problems during and after treatment of cancer therapy are increasing. Recently, as an increase in the number of cancer survivors, emphasis has been placed on the importance of development of survivors' QOL such as cardiovascular disorders occurred in cancer survivors. Indeed, certain anticancer drugs and molecular targeted therapies induce cardiotoxicity, which limit the widespread implementation of cancer treatment and significant decrease of QOL in cancer patients. In addition, cardiac dysfunction induced by cancer cachexia has also been reported. In view of these backgrounds, it is necessary to clarify the dynamics of cardiovascular system of cancer survivors and patients with cancer cachexia, to prevent cardiac dysfunction accompanying with cancer therapy and cachexia. Quite recently, interdisciplinary research area namely Cardio-oncology has been established to try to solve these issues. In the present study, we will present anticancer drug-induced cardiotoxicity and cardiac dysfunction occurring in cancer cachexia, and discuss our research approaches and data to understand such pathophysiology and possible prevention and therapies.

As cancer patients live longer, it is important to recognize cardiotoxicity that induce electrophysiological or structural changes by many oncology drugs, such as anthracyclines and tyrosine kinase inhibitors. Thus, understanding of cardio-oncology (also known as onco-cardiology) is more critical for the effective care of cancer patients. Huma induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used to evaluate cardiotoxicity as highly physiologically relevant human cells. We all-japan consortium have proposed a new proarrhythmia risk assessment method using hiPSC-CMs by our large-scale validation study. In addition to proarrhythmia risk, it is necessary to detect other type of side effects, such as chronic effects and impaired contractility from the viewpoint of cardio-oncology. To investigate how to use hiPSC-CMs for oncology drugs, we have attempted to make standardized protocols for long-term effects of drugs using motion vector system. In the symposium, I would like to share our recent data and discuss future perspectives for the usage of hiPSC-CMs in the cardio-oncology field.

Genomic DNA, which contains all of the genetic information, is damaged by a variety of endogenous and environmental factors such as genotoxic chemicals, ionizing radiation and UV light. Consequently, the DNA repair process is constantly active as it responds to damage in the DNA structure. Not only cardiotoxicity of anticancer drug treatment but also ischemic heart disease and heart failure associated with overloaded pressure interfere with DNA damage response and DNA repair regulation in cardiomyocytes. DNA methylation, catalyzed by the DNMTs, plays an important role in maintaining genome stability, but the molecular mechanism is not clear. In this study, we examine and outline the links between DNA methylation and the DNA damage repair systems and discuss the possible mechanisms of how they are orchestrated, with a focus on cardiotoxicity of anticancer drugs.

Advances in cancer treatment have led to dramatic increase in cancer survivors.  

Although cardiotoxicity resulting from anthracyclines and radiation therapies has been known for decades, the emergence of novel cancer treatment-related cardiovascular diseases (CTRCD) have been recognized to be associated with molecularly targeted therapies as well as immune checkpoint inhibitors.

Cardio-Oncology is a new interdisciplinary research opportunity at the intersection of cardiovascular disease and cancer.

Research priorities need to be identified to diagnose, treat, and prevent the previously unknown CTRCD(s), including (1) myocardial dysfunction and heart failure, (2) coronary artery disease, (3) valvular disease, (4) arrhythmias and QT-prolongation, (5) arterial hypertension, (6) thromboembolic disease and others.

For example, understanding fundamental mechanisms underlying CTRCD is essential to the development of new methods to manage these toxicities. The application of more suitable disease models and more effective methods for toxicity screening will serve to advance our understanding of CTRCD. Animal models have been successfully employed to predict potential problems in some cases, but more highly predictive models are also needed. Biobanks and other specimens with patient registries would facilitate the validation of biomarkers, genome analysis, and imaging methods.

Group 2 innate lymphoid cells (ILC2s), a new type of innate lymphocyte that we originally reported as natural helper cells in 2010, are known to regulate type 2 immune responses in an antigen-independent manner. In contrast to Th2 cells, ILC2s lack rearranged antigen receptors and are directly stimulated by epithelial cell-derived cytokines such as IL-33 and IL-25. Activated ILC2s are capable of producing a variety of cytokines, chemokines, and peptides including IL-5, IL-6, IL-9, IL-13, GM-CSF, amphiregulin, eotaxin, and methionine-enkephalin. ILC2s play a vital role in protection against parasite infections and induction of eosinophilic inflammation, which is involved in asthma, atopic dermatitis, eosinophilic esophagitis, allergic rhinitis, and chronic rhinosinusitis with nasal polyps. ILC2s are now considered to be associated with a wide range of diseases including allergic diseases, infection, obesity, cancer, and fibrosis.

IL-4, a key type 2 cytokine, is involved in multiple immune reactions including Th2 cell development, IgG1 and IgE production by B cells and M2 macrophage differentiation. While Th2 cells produce IL-4 together with IL-5 and IL-13 under antigen-induced TCR stimulation, ILC2s fail to produce IL-4 under IL-33 stimulation, which induces a large amount of IL-5 and IL-13. Based on this fact, ILC2s are not thought to contribute to IL-4-mediated immune responses, even though they express high levels of the IL-4 gene.

Recently, we identified the physiological condition that induces IL-4 production from ILC2. The mechanisms for IL-4 production from ILC2 were more intricate than that in Th2 cells and differ widely from those in IL-5 and IL-13 production. Furthermore, IL-4 from ILC2s elevates polyclonal IgE levels in steady state, helminth infection and allergy, and supports survival and expansion of FcεR⁺ cells such as basophils and mast cells. These findings provide evidence for factors involved in susceptibility to allergic diseases, which is still not understood, but is an important issue in the treatment or prevention of allergic disorders.

Pulmonary arterial hypertension (PAH) is a serious disease characterized by arteriopathy in the small to medium-sized distal pulmonary arteries, that is associated with arterial muscularization, concentric intimal thickening, and the formation of plexiform lesions. Inflammation and autoimmunity are currently thought of as critical factors to the pathogenesis of PAH. Interleukin-6 (IL-6), a multifunctional pro-inflammatory cytokine, is elevated in the serum of pulmonary arterial hypertension (PAH) patients and can predict the survival of idiopathic (I)PAH patients. Previous animal experiments and clinical human studies indicate that IL-6 is important in the pathogenesis of PAH. We recently found that IL-6/IL-21 signaling axis plays a critical role in the pathogenesis of PAH (PNAS. 112(20): E2677, 2015). First, we found that IL-6 blockade by the monoclonal anti-IL-6 receptor antibody, MR16-1, ameliorated hypoxia-induced pulmonary hypertension (HPH) and prevented the hypoxia-induced accumulation of Th17 cells and M2 macrophages in the lungs. Furthermore, the hypoxia-induced upregulation of IL-17 and IL-21, which are primarily produced by Th17 cells, was also ameliorated by IL-6 blockade in mice. Whereas IL-17 blockade with an anti-IL-17 neutralizing antibody had no effect on HPH, IL-21 receptor-deficient mice were resistant to HPH and exhibited no significant accumulation of M2 macrophages in the lungs. Consistently, IL-21 indeed promoted the polarization of primary alveolar macrophages toward the M2 phenotype. Moreover, significantly enhanced expressions of IL-21 and M2 macrophage markers were detected in the lungs of IPAH patients who underwent lung transplantation. We are currently examining the effect of IL-21-blockade on the pathogenesis of severe PAH rat model (namely Sugen5416(Su)/hypoxia (Hx) PAH model). IL-21 receptor deletion significantly ameliorated the pathologies of Su/Hx PAH in rats. Taken together, these findings indicate that IL-21blockade might be a promising therapeutic option for refractory PAH. We would like to validate the therapeutic effect of IL-21 blockade for refractory patients with PAH in the near future.

Background and Purpose: Pulmonary arterial hypertension (PAH) is a multifactorial disease characterized by pulmonary arterial remodeling in which the Src family non-receptor tyrosine kinases including Fyn play non-trivial roles. In this study, we explored the therapeutic potential of eicosapentaenoic acid (EPA) and its metabolite resolvin E1 (RvE1) for PAH through inhibition of Fyn in vitroand in vivo.

Method: Cardiodynamic parameters of rat hearts were measured by the echocardiography. Contractile responses of isolated pulmonary arteries were examined by the isometric tension measurement. Proliferation of human pulmonary artery smooth muscle cells (HPASMCs) derived from PAH patients were evaluated by the MTT assay. Stress fiber formation and STAT3 phosphorylation in HPAECs and HPASMCs were examined by immunohistochemical and western blot analyses, respectively.

Results : Administration of EPA to MCT-treated rats significantly improved the pathological changes characteristic for PAH, i.e. pulmonary arterial thickening, right ventricle dysfunction and cardiovascular fibrosis. Pulmonary arteries from MCT-treated rats showed exaggerated contractile responses compared with those from vehicle-treated rats, which were greatly normalized by EPA treatment. Administration of EPA or RvE1 decelerated the enhanced proliferation of PAH patient-derived PASMCs. Immunocytochemical and western blot analyses showed that a dominant negative form of Fyn prevented TGF-β2-induced stress fiber formation and IL-6-induced STAT3 phosphorylation. EPA and RvE1 suppressed Src family activity by modulating it's autophosphorylation level.

Summary: EPA significantly improved PAH-associated pathophysiology and cardiac dysfunction, which is likely mediated at least in part via Fyn inhibition. These results also point to the therapeutic significance of targeting this molecule in PAH treatment.

Cardiac fibrosis is a hallmark of various heart diseases including hypertrophy, heart failure, and arrhythmia. Cardiac fibroblasts play an important role in fibrogenesis because they differentiate to myofibroblasts under various pathological conditions. Thus, targeting cardiac fibroblast differentiation to attenuate fibrosis represents a new therapeutic strategy for fibrosis associated heart diseases. We have previously used fibroblasts from atrial fibrillation (AF) patients and demonstrated that the Transient Receptor Potential Melastatin 7 (TRPM7) plays an essential role in fibroblast differentiation to myofibroblasts. Here we propose that TRPM7 plays a key role in fibrosis-associated arrhythmia. We used transverse aortic restriction (TAC) induced hypertrophy/heart failure mouse model to generate fibrosis in the hearts. We found that deletion of Trpm7(TRPM7-KO) significantly increased survival rate after TAC, and improved heart performance. Moreover, TRPM7 deletion drastically reduced fibrosis in both atria and ventricles. The reduced fibrosis in TRPM7-KO-TAC mice significantly decreased the vulnerability of AF and the duration of induced AF. Thus, TRPM7 plays an important role in fibrosis associated AF, and may serve as a therapeutic target for fibrosis associated arrhythmia.

Sleep/wakefulness state change is regulated by neurons, however, its regulatory mechanism is still unclear. Here we found that GABAergic neurons in the ventral tegmental area (VTA) have an important role in the regulation of sleep/wakefulness. Adeno-associate virus (AAV) vectors were injected into VTA of glutamic acid decarboxylase (GAD)-Cre mice in which GABAergic neurons are exclusively express Cre. To manipulate GABAergic neurons in the VTA, channelrhodopsin2 (ChR2), anion-channelrhodopsin2 (ACR2) or hM3Dq was expressed by AAV. Chemogenetical activation of these neurons significantly increased time in NREM sleep. To reveal neural mechanism, slice patch clamp was performed. AAV was injected to VTA to express ChR2 in the GABAergic neurons in the VTA. Orexin neurons expressing fluorescent protein were identified and recorded. Then, GABAergic nerve terminals from VTA were stimulated by blue light. Blue light significantly inhibited activity of orexin neurons. On the other hand, optogenetical inhibition of these neurons using ACR2 immediately induced wakefulness. To evaluate physiological importance of this response, these neurons were inhibited during recovery sleep after sleep deprivation for 4 hr. Inhibition of GABAergic neurons induced wakefulness even in the very sleepy condition. These results suggest that activity of GABAergic neurons in the VTA is critical to change sleep/wakefulness state especially in the regulation of NREM sleep and wakefulness.

Peripheral organ functions such as cardiovascular and respiratory activity are controlled by the nervous system. Though most early studies have mainly focused on physiological events within a single organ, it remains largely unknown how the central nervous system and peripheral organs interact with each other. To address this issue, we hereby developed a recording method that comprehensively monitors electrical biosignals representing cardiac rhythm, breathing rhythm, awake/sleep-related muscle contraction, and collective neuronal activity of multiple brain regions. Using this novel technique, we examined physiological changes in central-peripheral activity in rats that were subject to social defeat stress. Rats were classified into stress-susceptible and stress-resilient groups, based on cardiac and respiratory signals. Multi-dimensional discriminant analysis revealed that certain activity patterns of cortical oscillations could predict future animal's susceptibility against stress. Furthermore, stress-susceptible animals exhibited decreases in activity levels in multiple brain regions, including the hippocampus, the somatosensory cortex, and the thalamus. Such dynamic changes in cortical activity is a possible mechanism to cause abnormal activity of the peripheral organs in response to mental stress episodes.

Although previous studies have indicated that various brain areas involve in the acquisition of associative learning, it still remains unclear how the neural circuitry system regulates the learning processes. In order to clarify these mechanisms, we have developed retrograde vectors which are applicable as an approach for the study of neural circuits based on brain functions by combining with cell targeting, opto/chemogenetics in selective neural pathways. By using these methods, we addressed the roles of the pathways originating from the parafascicular nucleus (PF) and central lateral nucleus (CL) in the intralaminar thalamic nuclei to the striatum in mice. Interestingly, these two pathways control both the acquisition and performance of visual associated discrimination. In addition, the elimination of the CL-derived thalamostriatal neurons impaired the behavioral switching flexibility through the reversal and the set shifting tasks. Our data suggest that the PF and CL thalamostriatal systems are involved in cognitive function of basal ganglia circuitry, and that these two circuits possess distinct roles in the control of behavioral selection and flexibility.

Our understanding of brain function under the drug treatment and pathology has been promoted by genetically encoded tools for labeling neuronal subpopulation and manipulating neuronal function. Recent studies have indicated that ventral tegmental area (VTA) dopamine (DA) neurons are heterogeneous in their properties. In the present study, we demonstrated that morphine (MRP)-responsive neurons were isolated from the VTA of c-Fos-EGFP/Rpl10a transgenic mice expressing an EGFP fused to Rpl10a, under the control of the c-fos promoter and purified using MACS and FACS. These experiences showed that systemic administration of MRP activated a subset of VTA neurons, including TH-positive DA neurons. Furthermore, using c-Fos-eNpHR mice, optical suppression of MRP-responsive VTA neurons significantly inhibited analgesic responses induced by systemic administration of MRP. These results suggest that the activation of MRP-responsive mesolimbic DA neurons partly modulates MRP-induced analgesia. The present findings provide evidence that labeling neuronal subpopulation and manipulating neuronal function using genetic engineering technology is very powerful tool for detecting functional neural circuits under drug treatment and disease conditions.

Lower urinary tract symptoms (LUTS) are categorized as storage, voiding, and post-micturition symptoms of the lower urinary tract. Recent clinical evidence have shown that hypertension and atherosclerosis resulting in disturbed blood flow are linked to the development of LUTS. Spontaneously hypertensive rat (SHR) is genetically hypertensive rat and shows various urinary disorder model such as detrusor overactivity/overactive bladder and benign prostatic hyperplasia (BPH) with a decrease in pelvic blood flow. Alpha1 adrenoceptor antagonists are widely used as the treatment of BPH/LUTS. Moreover, there is increasing evidence that alpha1 adrenoceptor antagonists improve the voiding symptoms as well as storage symptoms in BPH/LUTS patients. We reported that chronic daily treatment with silodosin (a selective alpha1A adrenoceptor antagonist) ameliorated the decreased bladder blood flow and urodynamic parameter in SHR. Moreover, chronic treatment with silodosin improved the prostatic morphological changes and decreased prostatic blood flow in SHR. These data suggest that silodosin might improve the LUTS via a recovery of pelvic blood flow.

Bladder afferent nerves are composed by myelinated Aδ- and unmyelinated C-fibers. During the storage phase of urine, distention of the bladder has long been considered to evoke afferent activity via Aδ-fibers connected in series with the smooth muscle fibers. In contrast, a previous study in cats revealed that more than 90% of C-fibers do not respond to normal bladder distension, being so called "silent" fibers. However, at least in rats, C-fibers can respond to normal bladder distension like Aδ-fibers, although they may also fulfill a potentially different role in the bladder sensory function in response to abnormal stimuli.

The symptoms of overactive bladder (OAB) or interstitial cystitis (IC) are believed to be commonly related to the sensory (afferent) function. In our laboratory, a direct measuring technique of mechanosensitive single-unit afferent activities of the primary bladder nerves in the rat has been established, and we have investigated the direct effects of drugs (anticholinergics, β3-adrenoceptor agonists, α1-adrenoceptor antagonists, PDE type5 inhibitors, etc.) on the bladder afferent function.

In this symposium, we will show some of our results and propose a possible additional action on sensory pathway of drugs as therapeutic agents for OAB or IC.

Tadalafil has recently been reported to improve the International Prostatic Symptom Score and total National Institutes of Health Chronic Prostatitis Symptom Index score of patients with benign prostatic hyperplasia with chronic pelvic pain syndrome (Nishino et al., Hinyokika Kiyo 2017 63:101-105). We therefore investigated the effect of tadalafil on chronic pelvic pain, which is a hallmark of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), and the inflammatory changes in two types of rat CP/CPPS model: experimental autoimmune prostatitis (EAP) and prostatitis induced by 17β-estradiol treatment combined with castration (hormone/castration-induced prostatitis; HCP) (Okamoto et al., The Prostate 2018 78:707-713; Kurita et al., The Prostate 2018 78:1157-1165; Yamaguchi et al., The Prostate in press). In the EAP model, we observed inflammation in the ventral prostate, while in the HCP model we observed inflammation in the lateral prostate. Consistent with previous studies, pelvic pain was observed in the EAP model. In addition, we found for the first time that HCP led to a significant increase in pelvic pain. Repeated treatment with tadalafil attenuated the pelvic pain and prostatic inflammation in both models. We observed that tadalafil would exhibit its pain attenuation effect through its anti-inflammatory action in the inflamed prostate.

Current guidelines for the treatment of benign prostatic hyperplasia (BPH) in several countries recommend the use of α₁-adrenoceptor antagonists (α1-blockers) or 5-alpha-reductase inhibitors (5ARIs), or phosphodiesterase 5 (PDE5) inhibitors for patients with lower urinary tract symptoms (LUTS) suggestive of BPH (LUTS/BPH). α1-blockers, such as tamsulosin and silodosin which are popular and frequently prescribed α1-blockers, improve LUTS/BPH by relaxing the smooth muscle tone of the prostate and bladder neck. 5ARIs, such as dutasteride and finasteride, were reported to improve LUTS by decreasing prostate size through inhibition of 5-AR. Meanwhile, tadalafil is the only PDE5-inhibitor approved for patients with LUTS/BPH. Smooth muscle relaxation in the bladder, urethra, and prostate due to increased nitric oxide/cGMP pathway activity via inhibition of PDE5 isoenzymes was reported to be the mechanism of LUTS improvement with tadalafil. Additionally, The use of an anticholinergic agent or a β3-adrenergic receptor agonist should be considered in patients with overactive bladder symptoms (overactive bladder symptom score ≥6).

In this symposium, I would like to introduce most recent findings of these curative drugs for LUTS in clinical practice.

The peel of Citrus kawachiensis (Kawachi Bankan), a citrus species grown in Ehime, Japan, is abundant in auraptene. Hyperglycemia and brain ischemia induce inflammation and oxidative stress and cause massive damage in the brain; therefore, we examined the anti-inflammatory and other effects of the dried peel powder of C. kawachiensis in the type 2 diabetic db/db mice model and global cerebral ischemia mice. The C. kawachiensis treatment inhibited astroglial activation in the hippocampus and the hyperphosphorylation of tau protein in hippocampal neurons, and also relieved the suppression of neurogenesis in the dentate gyrus of the hippocampus in the db/db mice. The C. kawachiensis treatment inhibited microglial and astroglial activation, and neuronal cell death in the hippocampus of transient global cerebral ischemia mice. It was suggested that the dried peel powder of C. kawachiensis exerts anti-inflammatory and neuroprotective effects in the brain. Auraptene, a coumarin compound, have been shown to exert anti-inflammatory effects in peripheral tissues; therefore, we attempted to demonstrate the effect in the brains in streptozotocin-induced hyperglycemic mice and transient global cerebral ischemia mice. Auraptene administration showed the similar effects as the peel of C. kawachiensis in the hippocampus of these mice models. These results suggested that auraptene have potential effects as a neuroprotective agent in the peel of C. kawachiensis.

Extract of Cyclolepis genistoides D. Don (vernacular name palo azul; palo) is a traditional medicine used in Paraguay for diabetes and is sold in Japan as dietary supplement. This study aimed to elucidate the mechanism of anti-diabetes activity of palo, especially focused on insulin resistance. Palo promoted adipocytes differentiation and regulated adipokine profiles in 3T3-L1 adipocytes by modulation of PPARγ, a major regulator of adipose differentiation. While palo didn't affect insulin signaling molecules, it promoted GLUT4 (glucose uptake transporter) translocation from cytoplasm to plasma membrane and increased 2-DG uptake under insulin stimuli. Human adipocyte showed almost similar profile with 3T3-L1 against palo treatment. In addition, as the other insulin targeted cell, effect on muscle differentiation was examined. Palo increased differentiation of C2C12 mouse muscle myoblasts and increased 5'-AMP-activated protein kinase (AMPK) activation. Finally palo treatment (250 or 1000 mg/kg) was performed with C57BL/6J mice for 14 weeks, being fed high-fat-diet (HFD60) simultaneously. Palo 250 mg/kg exhibited a tendency to decrease adipose volume but with increase of PPARγ mRNA expression, and decreased blood glucose level (P=0.058). Put it all together, palo has a potential to have antidiabetic effect by modulating insulin resistance via PPARγ pathway.

To promote β-cell function and survival would provide therapeutic approaches to prevent the onset and development of type 2 diabetes. Nobiletin, a citrus flavonoid, is known to improve hyperglycemia and insulin resistance in type 2 diabetic model mice. However, little is known about the effect of these citrus flavonoids on β-cells. In the present study, we investigated the effects of citrus flavonoids on insulin secretion from pancreatic β-cells and β-cell apoptosis. Nobiletin, a polymethoxylated flavone found in the peels of citrus fruits, significantly increased glucose-induced insulin secretion (GSIS) at 10 and 100 μM. In addition, nobiletin at 10 μM significantly inhibited thapsigargin-induced apoptosis of INS-1 cells. At this concentration, nobiletin significantly potentiated forskolin-induced cAMP accumulation in INS-1 cells. Sudachitin, a 5,7,40-tridesmethyl nobiletin derivative, also significantly increased GSIS at 100 μM and slightly inhibited β-cell apoptosis, although the effects of sudachitin were less potent than those of nobiletin. These findings suggest that citrus flavonoids facilitates GSIS and prevents ER stress-mediated β-cell apoptosis. Thus, citrus flavonoids may be used as a novel agent for the prevention of type 2 diabetes by promoting both survival and function of β-cells.

Sudachi (Citrus sudachi) is a small sour citrus. It grows exclusively in Tokushima region of Shikoku island, but is a typical seasoning for Japanese fish cuisine. We have demonstrated that repetitive administration of crude sudachi peel extends life span in diabetic model rats, and successfully identified a couple of molecules and fractions which are beneficial for our health in terms of energy metabolism from peels of sudachi. A limonene-derivative, which turned out to facilitate expression of a longevity gene, sirt1, exhibited lipid-lowering effects in HFD-fed mice as well as in cultured cells, for instance. Our recent results on the metabolic effects of such components in sudachi peel will be shared in the current symposium.

Allergen specific immunotherapy is the only curative treatment for IgE-mediated allergic diseases in contrast to symptomatic treatment such as anti-histamine agents. Subcutaneous immunotherapy (SCIT) has been introduced in Japan for treatment of allergic rhinitis and/or asthma caused by pollens and/or house dust mites (HDM) in early 1960s, and the clinical efficacy has been well-known. However, the major drawbacks of SCIT are necessity of repeated painful injections as well as the risk of severe systemic adverse reactions. Sublingual immunotherapy (SLIT) was developed to resolve these issues. In Japan, Japanese cedar (JC) pollen SLIT-drop was developed initially for treatment of JC pollinosis, and approved for patients of 12 years of age and older in 2014. For adolescent and adult patients with HDM-allergic rhinitis, HDM SLIT-tablet was launched in 2015 and subsequently approved to be also available for pediatric patients (<12 years of age) in 2018. Moreover, JC pollen SLIT-tablet for JC pollinosis was approved in 2018 for all patients with no age limit. In this symposium, we present the development of SLIT drop/tablets including the current understanding on the mechanism of action.

Allergen immunotherapy (AIT) is an effective treatment for allergic rhinitis, although a substantial proportion of the patients is refractory. We aimed to elucidate the mechanisms underlying the effectiveness of AIT. A 2-year clinical study was performed in adult patients with Japanese cedar pollinosis with sublingual administration of cedar pollen extract. After dividing high-responder (HR) patients with improved severity scores and non-responder (NR) patients with unchanged or exacerbated symptoms, differences in HR and NR patients were evaluated by analyzing peripheral blood cellular, serum, and genetic profiles before and after the AIT. This treatment was highly effective for rhinitis symptoms, though unimproved clinical responses were seen in _～30% of the treated patients. Serum cytokine bead array analysis failed to distinguish NR from HR patients, though cluster analysis of the serum parameters revealed a positive correlation between Th1/Th2 cytokines in HR patients before and after the AIT. In the expression of a taste receptor in CD4⁺ T cells, a copy number variation-related difference was observed between HR and NR patients. Through the pathway analysis of CD4⁺ T cell-expressing genes, an apoptosis pathway was implicated in the efficacy of AIT. CD4⁺ T cell is a predominant target of AIT to exhibit its efficacy on allergic rhinitis.

Th2 cells are well known to play important roles in allergic diseases including allergic rhinitis (AR). Meanwhile, the factors that induce and sustain the pathogenesis of AR remain unclear. The recent development of sublingual Immunotherapy (SLIT) is expected to allow changes to the underlying pathogenesis of AR. However, the phenotype of the pathogenic Th2 cells (Tpath2) cells in house dust mite-induced AR (HDM-AR) and the relation between Tpath2 and SLIT efficacy have not been clarified. Therefore we analyzed the cytokine production and frequency of HDM-reactive T-cell subsets in peripheral blood mononuclear cells (PBMCs) using flow cytometry in 89 HDM-AR patients (placebo; n = 43 and HDM 300 IR; n = 46) who participated in a placebo-controlled study of SLIT with HDM tablets. All patients provided samples both before treatment as a baseline and at the end of the 52-week study. HDM-reactive IL-5⁺IL-13⁺CD27^-CD161⁺CD4⁺ cells and ST2⁺CD45RO⁺CD4⁺ cells were observed in the PBMCs from each patient with HDM-AR; these cells significantly decreased after SLIT in the group treated with active tablets. HDM-reactive ST2⁺CD45RO⁺CD4⁺ cells were significantly lower in active-responders.

In conclusion, HDM-reactive ST2⁺CD45RO⁺CD4⁺ cells or those combined with IL-5⁺IL-13⁺CD27^-CD161⁺CD4⁺ cells may be useful as markers indicating the successful treatment of SLIT. These cells may play a crucial role in the pathogenesis of HDM-AR as Tpath2.

Mechanisms of allergen immunotherapy have not been fully elucidated. We have analyzed whether numbers of Foxp3⁺ Treg cells and Tr1 cells (IL-10-producing Foxp3^- CD4⁺ T cells) are increased in SCIT-treated Japanese cedar pollinosis patients and allergic mice. Peripheral blood mononuclear cells (PBMCs) were collected from the patients treated with or without SCIT. Ovalbumin (OVA)-sensitized mice received s.c. dosages of OVA for SCIT, followed by intratracheal challenges with OVA. The lungs were collected from the allergic mice treated with or without SCIT. The human PBMCs and murine lung cells were stimulated, and analyzed by flow cytometer. In both strains, numbers of Tr1 cells but not Foxp3⁺ Treg cells in SCIT-treated individuals were significantly larger than those in the non-SCIT-treated. In mice, SCIT treatment ameliorated allergic airway inflammation such as eosinophilia, hyperresponsiveness and histological changes. In another experiment of mice, Tr1 cells were induced in vitro by culture of splenocytes of sensitized mice with OVA and cytokines, and adoptively transferred to sensitized mice, resulting in effective suppression of the allergic airway inflammation. In conclusion, Tr1 cells could play roles in clinical effectiveness of SCIT.

A microRNA (miRNA) is a kind of a small non-coding RNA functioning RNA silencing and post-transcriptional regulation of gene expression. The changes in expression of various miRNAs are reported to be associated with diseases, such as cancer, neurodegenerative diseases, and so on.

Glaucoma and retinitis pigmentosa (RP) are the leading causes of blindness in adults. Excitotoxicity caused by excess glutamate in the retinal extracellular space is thought to be one of the mechanisms of retinal ganglion cell death induced by glaucoma. RP is characterized by progressive photoreceptor-selective degeneration, and caused by mutation of the genes related to the function of photoreceptor and retinal pigment epithelium.

Recently, we demonstrated that the expression levels of some miRNAs were changed in the murine retina challenged excitotoxicity and that of a hereditary RP model animal. Retinal neurodegeneration in the NMDA-injected eye and the hereditary RP model animal could be reduced by intravitreal or subretinal injection of the molecules that modulate miRNA function, such as miRNA mimics and miRNA inhibitors. These results suggest that the development of retinal degenerative diseases could be inhibited by regulation of microRNA function.

Glaucoma is a leading cause of blindness worldwide. Although an elevated intraocular pressure (IOP) is considered to damages retinal ganglion cells (RGCs) thereby causing blindness, it has become apparent that many risk factors other than IOP are involved in the etiology of glaucoma. Recent genome wide association studies (GWAS) have identified that single nucleotide polymorphism (SNP) of ABCA1 gene is the highest risk for glaucoma. However, its pathogenic mechanisms are totally unclear. To address this issue, we analyzed molecular mechanisms using conventional ABCA1 knockout (KO) mice. We found that deficiency of ABCA did not increase IOP levels regardless of their age. Importantly, ABCA1KO mice at middle-age (12 months old) showed significant increases in the number of apoptotic RGCs. We also found that ABCA1 was enriched in astrocytes. To further clarifying the role of astrocytic ABCA1, we generated astrocyte-specific ABCA1 knockout (cKO) mice. The cKO mice had no IOP elevation and increased the number of apoptotic RGCs. The cKO mice also showed impaired visual functions at middle-age. Taken together, our data showed that (1) ABCA1 has no impact on IOP; (2) loss-of-function of ABCA1 is involved in glaucoma; and (3) ABCA1 in glial cells contributes to pathogenesis of glaucoma.

Retinitis Pigmentosa is a group of hereditary diseases with rod photoreceptors degeneration, which is followed by central cone degeneration in the advanced stage. The patients experience night blindness and the progressive loss of visual field. Since the retinal bipolar cells that receive signals from the dying photoreceptors remain for a while, one therapeutic approach is to supply the photoreceptors to reconstruct initial visual transmission to these bipolar cells. We previously showed that mouse ES or iPS-derived retinas can develop structured photoreceptor layers after transplantation and the proximal presence of pre- and post-synaptic markers of the host photoreceptors and the graft bipolar cells were confirmed either by immunohistochemistry or genetic labeling. Evident light responsive activities of host retinal ganglion cells (RGC) were recorded by multiple electrode array system (MEA) over the grafted area in the retinas of end-stage retinal degeneration mice (rd1) that has only few remaining activities. Behavior test results also suggested the recovery of light perception in these mice after transplantation. Human ES/iPS retinas can also develop photoreceptor layers after transplantation in immune-deficient end stage retinal degeneration models such as NOG-rd1 or rhodopsin mutant SD-Foxn1 Tg(S334ter)3LavRrrc nude rats with some synaptic contact between the host and the graft by immunohistochemistry. We are preparing clinical trials using iPS-derived retinas.

Vision is the sense we most depend on in our daily lives to interpret our surroundings. Visual information from the world enters the eye and reaches the retina. Ganglion cells in the retina send the information via the lateral geniculate nucleus to the primary visual cortex (V1). Neurons in V1 extract simple local features such as oriented lines and edges, by responding selectively to lines or edges with a particular slope (orientation selectivity). The V1 sends its output to a hierarchical series of higher visual areas, which represent a variety of higher order visual features, including motion, image segmentation, and object recognition. Impairment of the visual pathway at any stage will potentially cause blindness. Thus, modulating the visual pathway could be a therapeutic target of visual impairment. Acetylcholine modulates neuronal activities in various brain regions to control brain functions including attention, memory, and cognition. Here we determined the effect of donepezil, an acetylcholinesterase inhibitor, on visual recognition by examining the visual detection task and V1 activity in mice.

In pathological mechanism of repeat diseases, "RNA toxicity" is observed, which repeat-derived RNA binds to various RNA binding proteins, thereby causing abnormalities in RNA metabolism. In addition to its RNA toxicity, "RAN translation" has been found, in which repeat RNA initiates translation in a start codon (AUG) independent manner. In many neurological diseases caused by abnormal elongation of repeat sequences, polypeptides derived from repeat RNA are accumulated in cells. In fragile X-related tremor/ataxia syndrome (FXTAS), which is a triplet repeat disease, elongated CGG repeats in the 5 untranslated region of FMR1 mRNA elicits RAN translation, polyglycine-containing protein (FMRpolyG). In this study, we analyzed the binding protein with FMRpolyG and investigated its involvement with FXTAS pathology. Using immunohistochemistry, FMRpolyG was observed in mouse A9 monochromosomal hybrid cells that harbor whole human X chromosome derived from FXTAS, but not in cells derived from a normal individual. In order to identify binding proteins to FMRpolyG, cell lysates of mouse A9 cells carrying the X chromosome from FXTAS were immunoprecipitated using FMRpolyG antibody, and shotgun proteomics analysis was performed. As a result, it was revealed that causative molecules of some repeat diseases and exosome-related proteins bind to FMRpolyG. These results suggest that we can clarify the mechanisms involved in the onset of other neurological diseases through research on FXTAS.

Accumulation of insoluble alpha-synuclein (aS) is a pathological hallmark of some neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, collectively termed synucleinopathies. aS is deposited in a hyperphosphorylated and ubiquitinated form with β-sheet-rich fibrillar structure in diseased brain. A growing body of evidence suggests that spreading of aS pathology occur by prion-like propagation mechanisms. Our study revealed that intracerebral injection of insoluble aS into wild-type mice can induce prion-like propagation of phosphorylated aS pathology even at 1 month after injection, while injection into aS knockout mice failed to induce any pathologies. We also have demonstrated that intracerebral injection of insoluble aS into adult common marmoset resulted in spreading of aS pathologies and neurodegeneration. These in vivo experiments clearly indicate that insoluble aS has prion-like properties and it propagates through neural networks. The underlying mechanisms of aS propagation are poorly understood, however, aS propagation model animals would be useful in elucidating pathogenetic mechanisms and developing disease-modifying drugs for synucleinopathies.

Sleep abnormality is a prevalent but under-recognized symptom affecting patients with various neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD) from the early stage of the diseases. Sleep abnormality of these patients was conventionally attributed to AD or PD pathology that affects the brain regions regulating sleep-wake or circadian rhythm. On the contrary, various epidemiological studies have demonstrated the association of sleep abnormality with an increased risk of AD or PD. However, relevant disease models to prove the causal relationship between sleep abnormality and neurodegenerative diseases were lacking.

We recently succeeded in inducing chronic sleep abnormality closely resembling that of AD patients in AD model mice and revealed that chronic sleep fragmentation, a specific subtype of sleep abnormality frequently observed in the patients of neurodegenerative diseases, indeed exacerbates AD pathology in the mice brain. Our findings are in accord with previous epidemiological studies in humans and thus would contribute to the understanding of the underlying pathomechanisms and informing the development of disease-modifying therapy for neurodegenerative diseases.

Neurodegenerative diseases are caused by progressive degeneration of specific neurons. To overcome neurodegenerative diseases, the exploitation of preventive drugs is strongly expected, since impaired neurons are not regenerated by drugs. Spinocerebellar ataxia (SCA) is a group of dominantly-inherited neurodegenerative diseases. SCA is classified into SCA1-47 by the variance of causal genes. Since SCA patients are commonly characterized by cerebellar ataxia and atrophy of cerebellum, it is possible that there are common pathogenic mechanisms in SCAs. However, we can not find any shared functions among SCA-causing proteins. We have explored the molecular pathogenesis of several SCA-causing proteins. Especially, we have constructed in vitro SCA model to express these mutant proteins in primary cultured cerebellar Purkinje cells (PCs), which are characterized highly-branched dendrites and are important for cerebellar functions. Several SCA-causing mutant proteins commonly impair dendritic development of PCs. We assume that this phenomenon is one of the common phenotypes of SCA in vitro. This SCA model would be useful for the efficient exploration of novel preventive drugs against various types of SCAs.

Tumor microenvironment has been implicated in tumor development and progression. As a three-dimensional tumor microenvironment model, air liquid interface (ALI) organoid culture system was recently produced. In our previous study, ALI organoid model from normal and tumor colorectal tissues of human patients was established. Both organoids were successfully generated and showed cystic structures containing an epithelial layer and surrounding mesenchymal stromal cells. Structures of tumor organoids closely resembled primary tumor epithelium. Tumor organoids were more resistant to toxicity of 5-fluorouracil and Irinotecan than colorectal cancer cell lines, SW480, SW620 and HCT116 (Usui et al., Stem Cells Int, 2016, Curr Protoc Toxicol, 2018). We also demonstrated that Hedgehog signals mediate anti-cancer drug resistance in colorectal tumor patient-derived ALI organoids and that the inhibitors are useful as a combinational therapeutic strategy against colorectal cancer (Usui et al., Int J Mol Sci, 2018).

Peroxisome proliferator-activated receptor gamma (PPARG) is a ligand-activated transcription factor regulating metabolic and vascular function. We previously reported that mice (S-DN) expressing dominant-negative PPARG in smooth muscle cells (SMC) are hypertensive, exhibit impaired vascular relaxation, and display reduced expression of a novel PPARG target gene, RhoBTB1. We hypothesized that RhoBTB1 may play a protective role in vascular function that is disrupted in S-DN mice and in other models of hypertension. We then generated double transgenic mice (termed S-RhoBTB1) with tamoxifen-inducible, Cre-dependent expression of RhoBTB1 in SMC. S-RhoBTB1 mice were crossed with S-DN to produce mice (S-DN/S-RhoBTB1) in which tamoxifen-treatment restored RhoBTB1 expression in aorta to normal. Increased blood pressure (BP) and impaired vasodilation in S-DN were reversed by restoration of RhoBTB1 in SMC. To test if RhoBTB1 can prevent angiotensin (Ang) II-induced hypertension, Ang II (490 ng/min/kg) was infused in tamoxifen-treated S-RhoBTB1 for two weeks. Ang II-induced increased BP and impaired vasodilation were blunted in S-RhoBTB1. We conclude that a novel PPARG target gene, RhoBTB1, functions in SMC to facilitate vasodilation and mediates a protective anti-hypertensive effect.

Nonalcoholic fatty liver disease (NAFLD) is now the major chronic liver disease in the world. Iron overload occurring in CLDs is a risk factor for the disease progression; however, it is still unclear whether iron overload contributes to the progression of NAFLD to nonalcoholic steatohepatitis (NASH). In this study, we investigated the pathological role of iron overload in a rat NASH model. Six-week-old male F344 rats were fed a control, high-fat (HF), high-fat high-iron (HFHI) and high-iron (HI) diets for 30 weeks. Rats in HF and HFHI groups showed an ALT-dominant elevation of serum transaminases, hepatic steatosis, and inflammation with upregulation of proinflammatory cytokines. In HFHI group, the number of inflammatory foci, equivalent to lobular inflammation in NASH patients, significantly increased with upregulation of inflammatory cytokines such as TNF-α, compared to HF group. Macrophages laden with iron were seen in the inflammatory lesion of HFHI group. These results suggested that dietary iron supplementation enhances experimental steatohepatitis induced by long-term HF diet feeding. Excessive activation of macrophages stimulated by iron accumulation can be involved in the exacerbation of inflammation.

Nonalcoholic fatty liver disease (NAFLD) is highly associated with the metabolic syndrome, and occurs as a more serious form of the disease, nonalcoholic steatohepatitis (NASH). NASH is diagnosed pathologically by histological evaluation of fibrosis, inflammation, and other features, such as hepatocyte ballooning. IL-19 is a member of the IL-10 family and is an anti-inflammatory cytokine produced mainly by macrophages. The last 10 years from the finding of IL-19, investigations underline the role of IL-19 in the immunological diseases such as inflammatory bowel disease, pancreatitis, and contact hypersensitivity. However, the involvement of IL-19 in liver inflammation and liver fibrosis is not well understood. We investigated the immunological role of IL-19 in NAFLD/NASH model mice fed by a choline-deficient and high-fat diet with methionine and cholesterol for 2 months. IL-19 knockout mice (KO) showed the major feature of NASH such as pericellular fibrosis, although wild-type mice showed steatohepatitis. In this symposium, we will report the recent advances in the role of IL-19 as inflammatory mediators in NASH. IL-19KO may be a valuable tool to study the NAFLD/NASH.

Lately, integrating liberal arts and basic medicine is being discussed in various fields. The discussion stresses the shift from the teaching method that attempts to stuff students with knowledge, as many of us have hitherto done, to the one that teaches students important principles repeatedly and encourages them to learn voluntarily, so-called active learning. The shift from passive learning to active learning directs us to a new medical education where students are required not just to acquire knowledge (I know xxx) but also to perform based on the knowledge (I can do xxx).In addition, medical schools are to follow the model core curriculum, which was revised in 2017. Taking into consideration the changes in international public health and medical systems, it notes that the medical school curriculum should train students to have practical clinical abilities with which they respond to citizens' needs for ethics, medical safety, team medical care, regional inclusive care system, healthy longevity society, etc. While medical schools are expected to make such a transformation from the traditional approach, they need to meet the international certification standards, which require students to engage in clinical practice for at least 1/3 of their six-year medical education. It is not an exaggeration to say that it is impossible for us to continue traditional lecture-style classes covering the same contents of liberal arts and basic medicine. Instead, we should actively integrate the curriculum.

Pharmacology is a broad science (1) representing a comprehensive summary of anatomy, physiology, and biochemistry from a viewpoint of the action mechanism of drugs, on one hand, and (2) relating various clinical practices from a viewpoint of therapeutic drugs for different diseases in clinical medicine, on the other. Benefiting from the contents of (1) and (2), it can and should integrate well with other fields of study in the curriculum.

Traditionally, discipline-based education is the most commonly applied educational method for undergraduate education. However, problem-solving in real-life settings requires skills to integrate various knowledge. To educate such skill to medical students, integrative curriculum that includes various disciplines, such as anatomy, physiology, biochemistry, pharmacology and clinical medicine, needs to be constructed. For such purpose, we use team-based learning (TBL) using clinical cases. Unlike problem-based learning (PBL), in which students should find problems by themselves, questions for TBL are prepared by moderators (teachers). TBL can be given after a set of lecture corses, such as endocrine physiology. During the TBL class, after clinical case presentation, teachers give tasks to students, (e.g., "Describe abnormal symptoms and explain why", "Why doctor prescribed this drug?"). Students should discuss within the team (4～6 students/team) to answer such questions by integrating their knowledge obtained through lectures. Then teacher randomly ask students and evaluate as a team so that all students within a team should share the common knowledge and ideas. Using this strategy, various disciplines can be integrated. Students can learn how to integrate their knowledge fo solve real-life setting questions. Furthermore, students can understand the importance to learn basic medicine through such experience.

Since prevention of prescription error with understanding drug actions should be one of pragmatic outcomes for all medical students, curriculum of pharmacology and clinical pharmacology at medical school should be integrated to achieve this purpose. There are, however, very few clinical pharmacologists in Japan. Drugs are prescribed by nearly all doctors, most would see themselves as practicing clinical pharmacology & therapeutics every day. Clinical pharmacology, even if with strong clinical background, has looked weak as a specialty without an organ or a disease. Clinical pharmacologists/pharmacologist used to have a close relationship with research into, and management of chronic common diseases such as cardiovascular diseases, which has increasingly devoted to specialists or primary care. More importantly, the shift from pharmacology(theory)-based medicine to evidence based medicine after the CAST trial has effect curriculum of medical schools.

　We should not, however, be too pessimistic. Current problem regarding drug therapy such as polypharmacy and potential inappropriate prescription may be handled by clinical pharmacologists rather than specialists. Development of functional biomarkers based on pharmacological action to describe patients who will benefit from new drugs may lead to precision medicine.

Currently, medical schools in Japan are under pressure to reform the curriculum to respond to field-specific certification, which is the quality assurance system of medical education. Among the criteria required by field certification, the curriculum of many medical schools in Japan is one of the points to overcome is curriculum integration. "Integration" here means "to relate learning matters taught in different academic systems or departments to each other, unite and organize". There are two types of "integration" required for the curriculum: "horizontal integration" and "vertical integration". "Horizontal integration" means that by integrating individual scientific systems among basic medicine or among clinical medicine, "Vertical integration" refers to the longitudinal integration of basic medicine and clinical medicine.

Looking at the curriculum integration of nine universities that have published external evaluation results out of universities that have undergone external trial by FY 2015 according to JACME (The Japan Accreditation Council for Medical Education and Evaluation Organization) standards. Although both the horizontal integration and the vertical integration have been partially accomplished at many universities, there are not many universities that have achieved complete integration, which is said to be a future task.

Here, I report on the experience that I gave a lecture on digestive system relays by three departments of anatomy, pathology, pharmacology, to the third grade of medical school.

We often receive suggestion from authorities on the importance of horizontal or vertical integration of lectures. The integration is important in a sense but must not be a purpose. Instead, we need proper reasons for the integration, i.e. benefit for both students and teachers. To make a new subject "Neuroscience" is very good reason for the horizontal integration. The progress of neuroscience is remarkable, but we had neither subject named "neuroscience" nor lecture system to teach this systematically. Therefore, we integrated some parts of lecture of anatomy, physiology, biochemistry and pharmacology horizontally for new subject neuroscience. In general, neuroscience course progresses in the order of structure, function, biochemistry and medication treatment, each of which is in charge of 2 anatomy, 2 physiology, 1 biochemistry and 1 pharmacology departments. By prior arrangements, we are trying to avoid duplication or teaching omission. After completing the neuroscience course, we take a questionnaire and continue devising to make better horizontal integration. Our horizontal integration has just started and still has a lot of problems. We would like to share its benefits and problems in this symposium.

For accreditation for medical education by JACME (Japan Accreditation Council for Medical Education), a new curriculum in my school has been started from a few years ago. Many departments adopted new integrated medical lectures. In my department, collaborative studies with many departments such as physiology, biochemistry, anesthesiology, psychiatry, or infectious diseases has been carried out. At the edge of these collaborative studies, several horizontal or vertical integration lectures have been planned (ex. Basic principles and clinical use of antibiotics, neurotransmitters and psychotropic drugs, ect …). In particular, I mention a vertical integration lecture of biochemistry, pharmacology, and anesthesiology. The advantage of the integration lectures is that the students recognize basic medical sciences are valid for medical education. However, a lot of effort is required to succeed the lectures. Advanced discussions with all teachers involved in the lectures are needed to remove overlapping contents. Visual effects using moving pictures about medical activities such as operations, anesthesia, or medical practice can cultivate a better understanding for the pre-CC (clinical clerkship) students.

In 2018, the number of nursing universities increased to 263 schools in Japan. One out of three universities have a nursing school, and nursing universities continue to increase.

Japanese nursing education in universities is facing many problems some of which are, difficulties in providing enough facilities, maintenance of the education level, estrangement from bachelor education and nursing practices after graduation, and reinforcement of abilities of evidence-based nursing practices.

Therefore, Ministry of Education, Culture, Sports, Science and Technology introduced the "Model Core Curriculum for Nursing Education in Japan" (MCCNE) in 2017. MCCNE aims at the acquirement of necessary and indispensable nursing competencies in the undergraduate course, which enumerates learning targets to be useful for making the curriculum.

MCCNE has 7 areas to develop qualities and abilities of a nurse for a lifetime. A is basic qualities and abilities required of a nurse. B is society and nursing science. C is basic knowledge for understanding people they care for, includes pharmacological science. D is basic nursing practice skills. E is basic nursing abilities to work in different areas of health care. F is clinical training, and G is nursing research. Clinical training should be integrated all of the areas.

We expect activation of the nursing education through utilizing MCCNE in Japan.

Among medical care incidents in Japan, an increase in medicine-related errors by nurses have been reported. These errors may be caused by a lack of knowledge of clinical pharmacology and drug interactions. It is important for nurses to acquire risk-management skills based on clinical pharmacology. In order to improve fundamental knowledge in clinical pharmacology for nurses, various training programs exist. We reviewed a number of educational programs in medicine and report our results.

Based on our results, it is necessary for medical education programs to include the following 5 essential elements: 1)An analysis of frequently-occurring medication errors in the field of clinical pharmacology, 2) an examination of therapeutic drugs for patient treatment and patient observation practices, 3) an emphasis on the pharmacokinetics and drug interactions between s and food/ drugs, 4) assessment of patient symptoms and risk-management and the drug efficacy, 5) the necessity of using the package inserts.

Effective methods for teaching include case studies and group discussions.

The most important role of nurses as medical staff is "information practitioner" that provide patient information to other medical staff. Nurses are the most familiar medical staff to the patients when they are cure- and care-hospitalized. It leads to the best effect for the patient by conveying the information even if it is a trivial, obtained in daily relations with the patient to other medical professionals. In other words, nurses play a major role in "awareness" and "connecting" the information to medical staff to the changes in the condition of the patient. Recently, pharmacists reside in wards and administer medication therapy for hospitalized patients. But it is difficult to properly grasp the condition of all patients. In order for all patients undergoing medication to properly complete, the nurse is aware of the knowledge for the adverse drug reactions (ADRs) of each drug that patients take. It is very much important to notice its sign of ADRs in early and to prevent the severity of the ADRs by making appropriate treatment. In this symposium, I would like to talk about the importance of pharmaceutical knowledge and the Pharmacists-Nurses coordination for patient safety from the standpoint of pharmacists.