How can we design a curriculum for a resilient medical student? - A blueprint for resiliency programs for med students in Japan

Recent research around the world has consistently reported that medical students experience a high rate of psychological morbidity, depersonalization, and low personal accomplishment. Resilience-enhancing programs have been proposed and implemented even in Japan. However, most of them remain extracurricular programs that are not specifically tailored to medical students. Additionally, they mostly mimic resiliency programs in North America, although studies have indicated that cultural perspective to the self, others, and context contribute to the capacity to respond to a stressful situation.In this context, the presenters investigated what factors might affect the similarities or differences in the perceptions of resilience among experienced palliative care physicians in Canada and Japan in 2017-2018 in order to propose a theory for a resiliency curriculum from a different cultural perspective. This study showed that Japanese physicians are more likely to rely on “Relationships” with other persons such as mentors, family, friends, or colleagues; in contrast, Canadian physicians tended to be more focused on individual factors such as “Autonomy” and “Confidence”.As a result, Showa University School of Medicine in Japan has developed a progressively advancing resiliency program for first through fourth year medical students as part of a new curriculum, implementation of which will begin in the spring of 2020. This represents one of the largest revisions in the school’s history. In this presentation, a blueprint for resiliency programs in a new curriculum will be presented, including course description, course content, educational objectives, learning resources, timetables, and instructional strategies.

The Synergistic Antitumor Effect of Combined Anti-Human Epidermal Growth Factor Receptor 2 Antibody and Gamma Interferon Therapy on Antibody-resistant Breast Cancer Cells

The anti-human epidermal growth factor receptor 2 （HER2） antibody （Ab） is a molecularly targeted Ab for cancer therapy. In the field of breast cancer, approximately 20％ overexpress HER2 protein. However, the recurrence rate is 30％ and the metastasis rate is 18％ one year after treatment of anti-HER2 Ab for HER2 positive breast cancer. The resistance to Ab treatment is a major problem for patients. We previously reported that anti-HER2 Ab and Gamma Interferon （IFN-γ） combined therapy show a higher anti-tumor effect than typical therapy in in vitro and in vivo mouse experiments. In this study, we evaluated whether anti-HER2 Ab and IFN-γ combined therapy shows a good synergistic effect against drug-resistant HER2 positive breast cancer cells and a higher antitumor effect than chemotherapy as a conventional clinical treatment. Further, we evaluated a synergy effect with the PD-L1 as a new check point inhibitor. The resistant cell lines were made under the continuous presence of Ab until cell growth was not affected by the drug. The resistant cells were divided into the appropriate number of groups, and then treated with anti-cancer therapy. We evaluated the antitumor effect for both the in vitro study and in vivo mouse xenograft model prepared with the same immunogenicity. The differences of immunofluorescence staining of CD8, Gr-1 and PDL-1 in tissues were investigated, especially in relation to the immune system. The combined therapy showed a significantly higher anti-tumor effect than other groups in in vitro and in vivo experiments. The combined therapy affected anti-tumor immunity in this immunofluorescence experiment. Taken together, we showed the possibility that combined therapy could be an effective treatment option for anti-HER2 Ab resistant breast cancer, thus helping patients suffering from cancer progression after developing treatment resistance

Pleiotropic Effects of Linagliptin Monotherapy on Levels of Nitric Oxide, Nitric Oxide Synthase, and Superoxide Dismutase in Hemodialysis Patients with Diabetes

Linagliptin is an anti-diabetic drug and the only bile-excreted dipeptidyl peptidase-4 inhibitor. Malnutrition-inflammation-atherosclerosis syndrome is an important prognostic factor for hemodialysis patients, and we previously reported anti-inflammatory effects of linagliptin in hemodialysis patients with diabetes. Inflammation can accelerate oxidative stress, vasoconstriction, and platelet aggregation. However, few studies have investigated the pleiotropic effects of linagliptin treatment on inflammation in hemodialysis patients. In this study, we have extended our previous investigations of these effects in a longer and more thorough follow-up of hemodialysis patients with diabetes. We examined 20 hemodialysis patients with diabetes who were not receiving oral diabetes drugs or insulin therapy and who exhibited inadequate glycemic control (glycated albumin levels＞20%). Linagliptin (5mg) was administered daily, and we evaluated the patients’ superoxide dismutase, 8-hydroxydeoxyguanosine, nitric oxide, nitric oxide synthase, and asymmetric dimethylarginine levels in serum at baseline and after 1, 3, and 6 months of treatment. After 6 months of treatment, superoxide dismutase levels had significantly decreased from 8.8±0.5U/ml to 7.0±0.5U/ml. Nitric oxide synthase levels were significantly increased at 3 and 6 months (maximum, 94.2±13.2µg/ml; baseline, 31.6±5.5µg/ml). After 3 months of treatment, nitric oxide levels had significantly increased from 64.5±6.6µmol/l to 104±15.4µmol/l, and remained significantly elevated at 6 months. Asymmetric dimethylarginine and 8-hydroxydeoxyguanosine levels did not change during the 6-month treatment course, and no patients exhibited hypoglycemia or other significant adverse effects. Linagliptin treatment significantly changed various markers of inflammation relevant to the atherosclerosis in malnutrition-inflammation-atherosclerosis syndrome. Therefore, linagliptin monotherapy has pleiotropic effects on inflammation in hemodialysis patients with diabetes, and may improve their prognosis

Recombinant methioninase combined with doxorubicin (DOX) regresses a DOX-resistant synovial sarcoma in a patient-derived orthotopic xenograft (PDOX) mouse model.

Synovial sarcoma (SS) is a recalcitrant subgroup of soft tissue sarcoma (STS). A tumor from a patient with high grade SS from a lower extremity was grown orthotopically in the right biceps femoris muscle of nude mice to establish a patient-derived orthotopic xenograft (PDOX) mouse model. The PDOX mice were randomized into the following groups when tumor volume reached approximately 100 mm3: G1, control without treatment; G2, doxorubicin (DOX) (3 mg/kg, intraperitoneal [i.p.] injection, weekly, for 2 weeks; G3, rMETase (100 unit/mouse, i.p., daily, for 2 weeks); G4 DOX (3mg/kg), i.p. weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks). On day 14 after treatment initiation, all therapies significantly inhibited tumor growth compared to untreated control, except DOX: (DOX: p = 0.48; rMETase: p < 0.005; DOX combined with rMETase < 0.0001). DOX combined with rMETase was significantly more effective than both DOX alone (p < 0.001) and rMETase alone (p < 0.05). The relative body weight on day 14 compared with day 0 did not significantly differ between any treatment group or untreated control. The results indicate that r-METase can overcome DOX-resistance in this recalcitrant disease

Proton pump inhibitors block iron absorption through direct regulation of hepcidin via the aryl hydrocarbon receptor-mediated pathway

Proton pump inhibitors (PPIs) have been used worldwide to treat gastrointestinal disorders. A recent study showed that long-term use of PPIs caused iron deficiency; however, it is unclear whether PPIs affect iron metabolism directly. We investigated the effect of PPIs on the peptide hepcidin, an important iron regulatory hormone. First, we used the FDA Adverse Event Reporting System database and analyzed the influence of PPIs. We found that PPIs, as well as H2 blockers, increased the odds ratio of iron-deficient anemia. Next, HepG2 cells were used to examine the action of PPIs and H2 blockers on hepcidin. PPIs augmented hepcidin expression, while H2 blockers did not. In fact, the PPI omeprazole increased hepcidin secretion, and omeprazole-induced hepcidin upregulation was inhibited by gene silencing or the pharmacological inhibition of the aryl hydrocarbon receptor. In mouse experiments, omeprazole also increased hepatic hepcidin mRNA expression and blood hepcidin levels. In mice treated with omeprazole, protein levels of duodenal and splenic ferroportin decreased. Taken together, PPIs directly affect iron metabolism by suppressing iron absorption through the inhibition of duodenal ferroportin via hepcidin upregulation. These findings provide a new insight into the molecular mechanism of PPI-induced iron deficiency

CA9 and PRELID2; hypoxia-responsive potential therapeutic targets for pancreatic ductal adenocarcinoma as per bioinformatics analyses

A strong hypoxic environment has been observed in pancreatic ductal adenocarcinoma (PDAC) cells, which contributes to drug resistance, tumor progression, and metastasis. Therefore, we performed bioinformatics analyses to investigate potential targets for the treatment of PDAC. To identify potential genes as effective PDAC treatment targets, we selected all genes whose expression level was related to worse overall survival (OS) in The Cancer Genome Atlas (TCGA) database and selected only the genes that matched with the genes upregulated due to hypoxia in pancreatic cancer cells in the dataset obtained from the Gene Expression Omnibus (GEO) database. Although the extracted 107 hypoxia-responsive genes included the genes that were slightly enriched in angiogenic factors, TCGA data analysis revealed that the expression level of endothelial cell (EC) markers did not affect OS. Finally, we selected CA9 and PRELID2 as potential targets for PDAC treatment and elucidated that a CA9 inhibitor, U-104, suppressed pancreatic cancer cell growth more effectively than 5-fluorouracil (5-FU) and PRELID2 siRNA treatment suppressed the cell growth stronger than CA9 siRNA treatment. Thus, we elucidated that specific inhibition of PRELID2 as well as CA9, extracted via exhaustive bioinformatic analyses of clinical datasets, could be a more effective strategy for PDAC treatment

Construction of an All-in-one Double-conditional shRNA Expression Vector

Gene silencing by RNA interference （RNAi） is widely used for assessing gene function. An important advance in the RNAi field was the discovery that plasmid-based RNAi can substitute for synthetic small interfering RNA in vitro and in vivo. However, constitutive and ubiquitous knockdown of gene expression by RNAi in mice can limit the scope of experiments because this process can lead to embryonic lethality, or result in compensatory overexpression of other genes such that no phenotypic abnormalities occur. Either way, analyses of the physiological roles of the gene of interest in adult mice are not possible. To overcome these limitations, we previously constructed a double-conditional short-hairpin RNA （shRNA） expression vector that can regulate shRNA expression in a spatio-temporal manner with a tetracycline-inducible floxed stuffer sequence selectively excised by application of Cre recombinase. In this study, we aimed to modify this vector to create an all-in-one vector that produces double-conditional transgenic mice through a single round of gene transfer to fertilized eggs. We added a coding region for nuclear localizing Cre （NCre） recombinase with a multi-cloning site for a cell-specific promoter into the double-conditional short-hairpin RNA （shRNA） expression vector that we previously constructed. Using Escherichia coli, we confirmed successful construction of the vector. First, we confirmed isopropyl-β-D-thiogalactopyranoside-induced expression of NCre recombinase through the lac operon as a specific promoter by western blotting. Second, we confirmed functional recombination of the floxed sequence of loxP-like TATA-lox by analysing restriction enzyme-digested fragments. This all-in-one double-conditional shRNA expression vector will be useful for reversible in vitro and in vivo knockdown of target gene expression, in target cells via promoter-specific expression of NCre, and at specific times by tetracycline application

Apoptosis-induced Proliferation in UV-Irradiated Human Conjunctival Epithelial Cells

A pterygium is a benign growth that develops on the conjunctiva and, in some cases, extends to the cornea and interferes with vision. Excessive exposure to ultraviolet (UV) light is one of the causes of pterygium development. We previously reported that UV-induced apoptosis is led by production of reactive oxygen species (ROS) that activate p38 mitogen-activated protein kinase (MAPK) in human conjunctival epithelial (HCE) cells. Also, ROS-dependent induction of interleukin-11 (IL-11) has been reported to upregulate MAPK pathways, which results in compensatory proliferation. In this study, we examined the effect of UV exposure on HCE cells, in terms of change in apoptosis, ROS generation, phosphorylation of c-Jun N-terminal kinase (JNK), levels of IL-11 (a key cytokine in tissue repair and compensatory proliferation), production of activator protein 1 (AP-1), and expression of c-myc, c-fos and c-jun (which provides evidence of healthy cell proliferation). Apoptosis in HCE cells was induced by UV light irradiation (312nm, 4.94mW/cm2). Apoptosis was measured using the Muse Annexin V and Dead Cell Assay Kit. ROS generation was measured by using 5-(and 6-) chloromethyl-2\u277\u27-dichlorodihydrofluorescein diacetate, acetyl ester. JNK phosphorylation, IL-11 levels and AP-1 production were measured by enzyme-linked immunosorbent assays (ELISAs). Imnunocytochemical staining was used to measure c-myc, c-fos and c-jun expression. UV irradiation increased ROS generation, phosphorylation of JNK, and apoptotic cell count. IL-11 levels and AP-1 production were significantly increased by UV irradiation. The irradiated cells had increased expression of c-myc, c-fos and c-jun, and treatment of the cells with IL-11 significantly increased expression of c-myc, c-fos and c-jun. These results suggest that the release of IL-11 from UV-induced apoptotic HCE cells and surrounding healthy cells could promote proliferation to maintain homeostasis

Diphenhydramine against cisplatin nephrotoxicity

Cisplatin is widely used as an anti-tumor drug for the treatment of solid tumors. Unfortunately, it causes nephrotoxicity as a critical side effect, limiting its use, given that no preventive drug against cisplatin-induced nephrotoxicity is currently available. This study identified that a previously developed drug, diphenhydramine, may provide a novel treatment for cisplatin-induced nephrotoxicity based on the results of the analysis of medical big data. We evaluated the actual efficacy of diphenhydramine via in vitro and in vivo experiments in a mouse model. Diphenhydramine inhibited cisplatin-induced cell death in renal proximal tubular cells. Mice administered cisplatin developed kidney injury with renal dysfunction (plasma creatinine: 0.43 ± 0.04 mg/dl vs 0.15 ± 0.01 mg/dl, p<0.01) and showed augmented oxidative stress, increased apoptosis, elevated inflammatory cytokines, and mitogen-activated protein kinases activation; however, most of these symptoms were suppressed by treatment with diphenhydramine. Further, the renal concentration of cisplatin was attenuated in diphenhydramine-treated mice (platinum content: 70.0 ± 3.3 µg/g dry kidney weight vs 53.4 ± 3.6 µg/g dry kidney weight, p<0.05). Importantly, diphenhydramine did not influence or interfere with the anti-tumor effect of cisplatin in any of the in vitro or in vivo experiments. Moreover, a retrospective clinical study of 1467 cancer patients treated with cisplatin showed that patients who had used diphenhydramine exhibited less acute kidney injury than patients who had not used diphenhydramine (6.1 % vs 22.4 %, p<0.05). Thus, diphenhydramine demonstrated efficacy as a novel preventive medicine against cisplatin-induced nephrotoxicity

Increase in Matrix Metalloproteinase-2 and 9 in the Liver of Nonalcoholic Steatohepatitis (NASH) Model Rats

Nonalcoholic steatohepatitis (NASH) is regarded as a hepatic manifestation of the metabolic syndrome which can progress to hepatic cirrhosis and hepatocellular carcinoma. It is thought that matrix metalloproteinases (MMPs) play an important role in hepatic fibrosis and we previously reported a correlation between oxidative stress and MMP-9 expression. However, the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in the progression of NASH is unclear. In this study we used spontaneously hypertensive and hyperlipidemic rats (SHHR) fed a high-fat diet and 30% sucrose solution (HFDS) as a model for NASH, in order to clarify the relationships between oxidative stress, liver weight (LW), MMPs and TIMPs at various time-points in the progression of NASH. Male SHHR and Sprague-Dawley (SD) rats were divided into four groups: SHHR-normal diet (ND), SHHR-HFDS, SD-ND and SD-HFDS. Hepatic fibrosis was clearly increased at 13 months in SHHR-HFDS, resembpling NASH. LW and oxidative stress markers in plasma were increased in SHHR-HFDS compared to the other groups. Oxidative stress was correlated with LW in all rats. Expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNA, measured by real-time polymerase chain reaction, was increased in the liver of SHHR-HFDS at 13 months. This study suggests that oxidative stress, MMPs and TIMPs may play an important role in the progression of NASH