Proteomic analysis of fatty liver induced by starvation of medaka fish larvae

When medaka fish (Oryzias latipes) larvae are grown in the absence of exogenous nutrition, the liver becomes dark and positive to Oil Red O staining from 7 days post-hatch (dph). We determined the mechanism of this starvation-induced development of fatty liver by proteomic analysis using livers obtained from larvae grown in the presence or absence of 2% glucose at 5 dph. Results showed that changes in the expression levels of enzymes involved in glycolysis or the tricarboxylic acid cycle were modest, whereas the expression levels of enzymes involved in amino acid catabolism or β-oxidation of fatty acids were significantly elevated, suggesting that they become major energy sources under starvation conditions. Expression levels of enzymes for the uptake and β-oxidation of fatty acids as well as synthesis of triacylglycerol were elevated, whereas those for the synthesis of cholesterol as well as export of cholesterol and triacylglycerol were decreased under starvation conditions, which explains the accumulation of triacylglycerol in the liver. Our results provide the basis for future research to understand how gene malfunction(s) affects the development of fatty liver, which can lead to nonalcoholic steatohepatitis and then to liver cirrhosis.Key words: amino acid catabolism, β-oxidation, triacylglycerol, cholesterol, export

乳癌術前化学療法において腋窩リンパ節転移が陰性化するための効果予測因子の検討

Purpose: We investigated the role of tumor-infiltrating lymphocytes (TILs) in pretreatment primary breast cancer to predict pathological response to neoadjuvant chemotherapy (NAC) in patients with clinical node-positive disease (cN +). Methods: The subjects of this study were 60 patients with cN + , who received NAC followed by breast surgery with axillary lymph node dissection (ALND). We conducted a semi-quantitative assessment of TILs in pretreatment primary tumors and their association with clinicopathological factors and axillary lymph node metastasis. Results: We observed a higher number of TILs in tumors with negative hormone receptors, positive human epidermal growth factor receptor 2, or high Ki67. TILs were associated with a favorable response to NAC in primary tumors. The rate of axillary pathologic complete response (Ax-pCR) was significantly higher in patients with a high number of TILs than in patients with a low number of TILs (72.0% versus 17.1%, p < 0.001). In multivariable analysis, a high number of TILs was a significant predictor of Ax-pCR as well as of pCR of the primary tumor after NAC. Importantly, all patients with HER2-positive tumors in the high TILs group showed Ax-pCR on ALND. Conclusion: TILs in pretreatment primary breast cancer had the potential to predict therapeutic efficacy of NAC in patients with clinical node-positive disease.博士（医学）・乙第1498号・令和3年3月15日© Springer Nature Singapore Pte Ltd. 2020This is a post-peer-review, pre-copyedit version of an article published in Surgery today. The final authenticated version is available online at: https://doi.org/10.1007/s00595-020-02157-6

The involvement of the vasa vasorum in the development of vasculitis in animal model of Kawasaki disease

BACKGROUND: Kawasaki Disease (KD) involves a diffuse and systemic vasculitis of unknown etiology that mainly affects infants and children. Although a considerable number of analyses of the clinical, histopathological and molecular biological details underlying the mechanism responsible for the development of coronary arterial lesions, it is still poorly understood. The purpose of this study was to analyze the state of angiogenesis, vasculogenesis and the distribution of blood vessels using an animal model of KD like vasculitis. We investigated the involvement of the vasa vasorum from the adventitia in the vascular involvement and the development of the disease state by performing sequential histopathology, scanning electron microscopy (SEM) and micro computed tomography (CT) studies using a murine model of vasculitis induced by the Candida albicans water-soluble fraction (CAWS). METHODS: To prepare the animal model of KD like vasculitis, CAWS was intraperitoneally injected into C57BL/6N mice for five consecutive days as reported by Ohno et al. We observed the changes of the vasa vasorum at the aorta and the orifices of the coronary arteries by SEM and micro CT, and also compared the neovascularization at the media and adventitia of the aorta by an immunohistochemical analysis. RESULTS: As previously reported, obvious inflammation was detected two weeks after the injection of CAWS, and also intimal thickening was observed three weeks after the injection. We found that the vasa vasorum in the adventitia of the aorta was increased in the model mice. The vasa vasorum started increasing one week after the injection of CAWS, before any obvious vasculitis was microscopically detected. CONCLUSION: The present results indicate that the vasculitis in Kawasaki disease starts as a disorder of the vasa vasorum

Patient-reported dyspnea and health predict waitlist mortality in patients waiting for lung transplantation in Japan

Background: Waitlist mortality due to donor shortage for lung transplantation is a serious problem worldwide. Currently, the selection of recipients in Japan is mainly based on the registration order. Hence, scientific evidence for risk stratification regarding waitlist mortality is urgently needed. We hypothesized that patient-reported dyspnea and health would predict mortality in patients waitlisted for lung transplantation. Methods: We analyzed factors related to waitlist mortality using data of 203 patients who were registered as candidates for lung transplantation from deceased donors. Dyspnea was evaluated using the modified Medical Research Council (mMRC) dyspnea scale, and the health status was determined with St. George's Respiratory Questionnaire (SGRQ). Results: Among 197 patients who met the inclusion criteria, the main underlying disease was interstitial lung disease (99 patients). During the median follow-up period of 572 days, 72 patients died and 96 received lung transplantation (69 from deceased donors). Univariable competing risk analyses revealed that both mMRC dyspnea and SGRQ Total score were significantly associated with waitlist mortality (p = 0.003 and p < 0.001, respectively) as well as age, interstitial lung disease, arterial partial pressure of carbon dioxide, and forced vital capacity. Multivariable competing risk analyses revealed that the mMRC and SGRQ score were associated with waitlist mortality in addition to age and interstitial lung disease. Conclusions: Both mMRC dyspnea and SGRQ score were significantly associated with waitlist mortality, in addition to other clinical variables such as patients' background, underlying disease, and pulmonary function. Patient-reported dyspnea and health may be measured through multi-dimensional analysis (including subjective perceptions) and for risk stratification regarding waitlist mortality

Heparin cofactor II reduces albuminuria

Aims/Introduction: Thrombin exerts various pathophysiological functions by activating protease-activated receptors (PARs). Recent data have shown that PARs influence the development of glomerular diseases including diabetic kidney disease (DKD) by regulating inflammation. Heparin cofactor II (HCII) specifically inactivates thrombin; thus, we hypothesized that low plasma HCII activity correlates with DKD development, as represented by albuminuria. Materials and Methods: Plasma HCII activity and spot urine biomarkers, including albumin and liver-type fatty acid-binding protein (L-FABP), were determined as the urine albumin-to-creatinine ratio (uACR) and the urine L-FABP-to-creatinine ratio (uL-FABPCR) in 310 Japanese patients with diabetes mellitus (176 males and 134 females). The relationships between plasma HCII activities and those DKD urine biomarkers were statistically evaluated. In addition, the relationship between plasma HCII activities and annual uACR changes was statistically evaluated for 201/310 patients (115 males and 86 females). Results: The mean plasma HCII activity of all participants was 93.8 ± 17.7%. Multivariate-regression analysis including confounding factors showed that plasma HCII activity independently contributed to the suppression of the uACR and log-transformed uACR values (P = 0.036 and P = 0.006, respectively) but not uL-FABPCR (P = 0.541). In addition, plasma HCII activity significantly and inversely correlated with annual uACR and log-transformed uACR increments after adjusting for confounding factors (P = 0.001 and P = 0.014, respectively). Conclusions: The plasma HCII activity was inversely and specifically associated with glomerular injury in patients with diabetes. The results suggest that HCII can serve as a novel predictive factor for early-stage DKD development, as represented by albuminuria

Heparin Cofactor II and NAFLD in T2DM

Aims: Thrombin exerts various pathophysiological functions by activating protease-activated receptors (PARs), and thrombin-induced activation of PARs promotes the development of non-alcoholic fatty liver disease (NAFLD). Since heparin cofactor II (HCII) specifically inactivates thrombin action, we hypothesized that plasma HCII activity correlates with the severity of NAFLD. Methods: A cross-sectional study was conducted. Plasma HCII activity and noninvasive clinical markers of hepatic fibrosis including fibrosis-4 (FIB-4) index, NAFLD fibrosis score (NFS) and aspartate aminotransferase-to-platelet ratio index (APRI) were determined in 305 Japanese patients with type 2 diabetes mellitus (T2DM). The relationships between plasma HCII activity and the clinical markers were statistically evaluated. Results: Multiple regression analysis including confounding factors showed that plasma HCII activity independently contributed to decreases in FIB-4 index (p＜0.001), NFS (p＜0.001) and APRI (p=0.004). In addition, logistic regression analysis for the prevalence of advanced hepatic fibrosis defined by the cutoff points of the clinical scores showed that plasma HCII activity was the sole and common negative factor for prevalence of advanced hepatic fibrosis (FIB-4 index: p=0.002, NFS: p=0.026 and APRI: p=0.012). Conclusions: Plasma HCII activity was inversely associated with clinical hepatic fibrosis indices including FIB-4 index, NFS and APRI and with the prevalence of advanced hepatic fibrosis in patients with T2DM. The results suggest that HCII can serve as a novel biomarker for assessment of hepatic fibrosis of NAFLD in patients with T2DM

A Patient with Type 3 Autoimmune Polyglandular Syndrome who Developed Systemic Lupus Erythematosus 8 years after the Diagnosis of Autoimmune Hepatitis

Eight years prior to her present admission, a 61-year-old Japanese woman was diagnosed with autoimmune hepatitis, slowly progressive insulin-dependent diabetes mellitus, and chronic thyroiditis; she had been treated with oral prednisolone (PSL). After she suddenly discontinued PSL, she newly developed systemic lupus erythematosus. A combination therapy of oral PSL and intravenous cyclophosphamide resulted in remission. She was finally diagnosed with autoimmune polyglandular syndrome (APS) type 3 (3A ,3B, 3D), complicated with four different autoimmune diseases. Since patients with type 3 APS may present many manifestations over a long period of time, they should be carefully monitored

Anti-Tumor Effect against Human Cancer Xenografts by a Fully Human Monoclonal Antibody to a Variant 8-Epitope of CD44R1 Expressed on Cancer Stem Cells

BACKGROUND: CD44 is a major cellular receptor for hyaluronic acids. The stem structure of CD44 encoded by ten normal exons can be enlarged by ten variant exons (v1-v10) by alternative splicing. We have succeeded in preparing MV5 fully human IgM and its class-switched GV5 IgG monoclonal antibody (mAb) recognizing the extracellular domain of a CD44R1 isoform that contains the inserted region coded by variant (v8, v9 and v10) exons and is expressed on the surface of various human epithelial cancer cells. METHODS AND PRINCIPAL FINDINGS: We demonstrated the growth inhibition of human cancer xenografts by a GV5 IgG mAb reshaped from an MV5 IgM. The epitope recognized by MV5 and GV5 was identified to a v8-coding region by the analysis of mAb binding to various recombinant CD44 proteins by enzyme-linked immunosorbent assay. GV5 showed preferential reactivity against various malignant human cells versus normal human cells assessed by flow cytometry and immunohistological analysis. When ME180 human uterine cervix carcinoma cells were subcutaneously inoculated to athymic mice with GV5, significant inhibition of tumor formation was observed. Furthermore, intraperitoneal injections of GV5markedly inhibited the growth of visible established tumors from HSC-3 human larynx carcinoma cells that had been subcutaneously transplanted one week before the first treatment with GV5. From in vitro experiments, antibody-dependent cellular cytotoxicity and internalization of CD44R1 seemed to be possible mechanisms for in vivo anti-tumor activity by GV5. CONCLUSIONS: CD44R1 is an excellent molecular target for mAb therapy of cancer, possibly superior to molecules targeted by existing therapeutic mAb, such as Trastuzumab and Cetuximab recognizing human epidermal growth factor receptor family

Matrix metalloproteinase haplotypes associated with coronary artery aneurysm formation in patients with Kawasaki disease.

Aneurysms of the vascular wall represent a final common pathway for a number of inflammatory processes, including atherosclerosis and idiopathic vasculitis syndromes. Kawasaki disease (KD) is an acute, self-limited vasculitis in children and the leading cause of acquired coronary artery aneurysms. We sought to identify shared molecular mechanisms of aneurysm formation by genotyping eight polymorphisms in matrix metalloproteinase (MMP)-1, 3, 7, 12 and 13 in the gene cluster on Chr.11q22, whose gene products have been implicated in aneurysm formation or are known to have elastase activity. We genotyped 482 US-UK KD patients (aneurysm+: n=111, aneurysm-: n=371) and tested our findings in an independent cohort of 200 Japanese KD patients (aneurysm+: n=58, aneurysm-: n=142). Analysis of the five MMP genes identified modest trends in allele and genotype frequencies for MMP-3 rs3025058 (-/T) and haplotypes containing MMP-3 rs3025058 (-/T) and MMP-12 rs2276109 (A/G) (nominal P=2 to 4 × 10(-5)) that conferred increased risk of aneurysm formation in US-UK subjects. This finding was validated in Japanese subjects and suggests the importance of this locus in aneurysm formation in children with KD. The region encompassing these risk haplotypes is a prime candidate for resequencing to look for rare genetic variation that may influence aneurysm formation