Clinical features and signaling effects of RET D631Y variant multiple endocrine neoplasia type 2 (MEN2).

BACKGROUND/AIMS: Germline mutations of the rearranged during transfection (RET) gene cause multiple endocrine neoplasia type 2 (MEN2). About 85% of RET mutations in MEN2 occur in codon Cys634. The RET D631Y mutation has recently been discovered, and we have studied its molecular expression and clinical consequences. METHODS: We analyzed the clinical characteristics of a total of 34 D631Y variant MEN2 individuals from seven families. We also constructed wild-type and mutant C630Y, D631Y, and C634R/W expression vectors and investigated their effects on signaling pathways and ability to correct the phenotypes of RET mutant cells. RESULTS: The median ages at diagnosis of pheochromocytoma and medullary thyroid carcinoma (MTC) were higher in patients with RET D631Y variant MEN2 than in those with the C634R/W variant (49:53.5 years vs. 33.5:27 years, respectively), and the penetration of the D631Y mutation with respect to MTC was lower than that of the C634R/W mutation (32.3% vs. 90%). The effects of the mutant vectors on phosphorylation of RET signaling molecules and focus formation were significantly different from those of wild type, but there were no significant differences between the mutants. D631Y scored significantly higher for chemotaxis and wound healing than C630Y, but lower than C634R and C634W. CONCLUSION: We suggest that the tumorigenic potential conferred by the D631Y mutation is lower than that conferred by the C634R/W mutation, but higher than that conferred by C630Y. Thus, the risk level of the RET D631Y variant appears to be higher than that of C630Y and lower than that of C634R/W.

Serum Wisteria floribunda agglutinin-positive human Mac-2 binding protein level predicts recurrence of hepatitis B virus-related hepatocellular carcinoma after curative resection.

BACKGROUND/AIMS: To investigate whether serum Wisteria floribunda agglutinin-positive human Mac-2-binding protein (WFA+-M2BP) can predict the recurrence of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) after curative resection. METHODS: Patients with chronic hepatitis B (CHB) who underwent curative resection for HCC between 2004 and 2015 were eligible for the study. Recurrence was sub-classified as early (<2 years) or late (≥2 years). RESULTS: A total of 170 patients with CHB were selected. During the follow-up period (median, 22.6 months), 64 (37.6%) patients developed recurrence. In multivariate analyses, WFA+-M2BP level was an independent predictor of overall (hazard ratio [HR]=1.490), early (HR=1.667), and late recurrence (HR=1.416), together with male sex, des-gamma carboxyprothrombin level, maximal tumor size, portal vein invasion, and satellite nodules (all P<0.05). However, WFA+- M2BP level was not predictive of grade B-C posthepatectomy liver failure. The cutoff value that maximized the sum of sensitivity (30.2%) and specificity (90.6%) was 2.14 (area under receiver operating characteristic curve=0.632, P=0.010). Patients with a WFA+-M2BP level >2.14 experienced recurrence more frequently than those with a WFA+-M2BP level ≤2.14 (P=0.011 by log-rank test), and had poorer postoperative outcomes than those with a WFA+-M2BP level ≤2.14 in terms of overall recurrence (56.0 vs. 34.5%, P=0.047) and early recurrence (52.0 vs. 20.7%, P=0.001). CONCLUSION: WFA+-M2BP level is an independent predictive factor of HBV-related HCC recurrence after curative resection. Further studies should investigate incorporation of WFA+-M2BP level into tailored postoperative surveillance strategies for patients with CHB.

Refractoriness to transarterial chemoembolization in patients with recurrent hepatocellular carcinoma after curative resection.

BACKGROUND/AIMS: It is important to identify patients who are refractory to transarterial chemoembolization (TACE), which is performed for the treatment of hepatocellular carcinoma (HCC). We investigated the predictors of poor treatment outcomes in patients with recurrent HCC treated who were treated with TACE after curative resection. METHODS: 428 patients with recurrent HCC after curative resection who were treated with TACE were enrolled. RESULTS: The median age of the study population was 59.2 years. On multivariate analysis, ≥2 TACE procedures within 6 months (hazard ratio [HR] = 1.898), and the des-gamma carboxyprothrombin level (HR = 1.000) independently predicted the progression to Barcelona Clinic Liver Cancer (BCLC) stage C in patients with BCLC stage 0-B HCC (both P<0.05). In addition, ≥2 and ≥3 TACE procedures within 6 months independently predicted mortality in the entire study population (HR = 1.863 and 1.620, respectively). The probability of progression to BCLC stage C in patients with BCLC stage 0-B HCC and the mortality rate in the entire study population were significantly higher in patients treated with ≥2 TACE within 6 months than in those who underwent fewer procedures (P = 0.002 and P<0.001, respectively). CONCLUSIONS: More than 2 TACE procedures within 6 months might be associated with the refractoriness to TACE in patients with recurrent HCC after curative resection.

Feasibility of dynamic risk assessment for patients with repeated trans-arterial chemoembolization for hepatocellular carcinoma.

BACKGROUND: Hepatoma arterial-embolization prognostic (HAP) score and its modifications (modified HAP [mHAP] and mHAP-II), consisting of some or all of the following factors of tumor size, number, alpha-fetoprotein, bilirubin, and serum albumin, have been found to predict outcomes after trans-arterial chemoembolization (TACE) for hepatocellular carcinoma (HCC). We investigated the feasibility of using HAP-related risk scores for dynamic risk assessment during repeated TACE. METHODS: A total of 619 HCC patients treated with TACE from two institutions between 2003 and 2010 were included. RESULTS: Patients with A-B class risk scores showed significantly better survival than those with C-D class risk scores at the first (median 43.7 vs. 21.5 months for mHAP-II, 35.2 vs. 10.2 months for mHAP, and 39.8 vs. 18.6 months for HAP; all P < 0.001) and the second rounds of TACE (38.6 vs. 17.2 months for mHAP-II, 30.0 vs. 8.5 months for mHAP, and 32.6 vs. 17.3 months for HAP; all P < 0.001). Sequential assessment of risk scores at the second TACE round was applied for patients with A-B class risk scores at the first TACE round, which further identified two subgroups of A-B and C-D class risk scores with different outcomes (median survival 40.6 vs. 19.6 months for mHAP-II, 31.2 vs. 16.9 months for mHAP, and 35.8 vs. 21.0 months for HAP; all P < 0.001). Compared with mHAP and HAP, mHAP-II showed the highest likelihood ratio (22.61 vs. 14.67 and 13.97, respectively), highest linear trend (24.43 vs. 19.67 and 14.19, respectively), and lowest Akaike information criteria value (1432.51 vs. 3412.29 and 2296.98, respectively). CONCLUSIONS: All HAP-related risk scores dynamically predicted outcomes during repeated TACE. Sequential risk assessment using mHAP-II best identified optimal candidates for repeated TACE.

Fibrosis-matched outcomes between chronic hepatitis B patients with drug-induced virological response and inactive carriers.

BACKGROUND & AIMS: We compared the risk of hepatocellular carcinoma (HCC) development between patients with chronic hepatitis B (CHB) who achieved virological response (VR; HBV-DNA < 2000 IU/mL) with nucleos(t)ide analogues (NUCs) treatment (NUC-VR group) and patients with inactive CHB phase (ICHBP group). METHODS: To adjust for imbalances between NUC-VR and ICHBP groups, propensity score matching (PSM) models with 1:1 ratios were performed. RESULTS: This study included 2032 patients (n = 1291 in NUC-VR group and n = 741 in ICHBP group). Before PSM, NUC-VR group was at higher risk of HCC development than ICHBP group at 7 years (9.4% in NUC-VR group vs 3.3% in ICHBP group; P < 0.001). However, after PSM, the cumulative HCC development rates at 7 years were similar in NUC-VR and ICHBP groups using the three PSM models [2.0% vs 4.3%, PSM model-1 (612 pairs); 3.7% vs 4.4%, PSM model-2 (618 pairs); and 2.4% vs 4.3%, PSM model-3 (610 pairs)] (all P > 0.05). CONCLUSIONS: After adjusting heavier hepatic fibrosis burden in NUC-VR group, overall clinical outcomes between 2 groups had become comparable. Therefore, if appropriate, NUCs to prevent viral replication and hepatic inflammation are required for achieving better prognosis.

Experimental Investigation on 3D Graphene-CNT Hybrid Foams with Different Interactions.

Due to the exceptional properties of graphene, numerous possibilities for real applications in various fields have been provided. However, it is a challenge to fabricate bulk graphene materials with properties arising from the nature of individual graphene sheets, and which assemble into monolithic three-dimensional structures. If 3D structured graphene foam were made instead of 2D structured graphene, it is expected that it would be a facile fabrication, with relatively low cost with the possibility of scale-up, and would maintain the intrinsic properties of graphene. To solve the weaknesses of 2D structured graphene, this study aimed to fabricate a 3D graphene-carbon nanotubes (CNT) hybrid foam. In this study, CNT was used to reinforce the graphene foams. In addition, two different surfactants, known as sodium dodecylbenzene sulphonate (SDBS) and cetyltrimethylammonium bromide (CTAB), were applied to help CNT dispersion. The π⁻π interaction was induced by SDBS/CNT, while ionic interaction was derived from CTAB/CNT. To confirm the charge effect with different surfactants, SEM, Zeta-potential, FT-IR, Raman spectroscopy, and compression tests were performed. When using a cationic surfactant, CTAB, compressive modulus, and strength increased due to the formation of relatively strong ionic bonding.

Reproducibility of European Association for the Study of the Liver criteria and modified Response Evaluation Criteria in Solid Tumors in patients treated with sorafenib.

BACKGROUND & AIMS: The European Association for the Study of the Liver criteria and the modified Response Evaluation Criteria in Solid Tumors are used for assessing the treatment outcomes of hepatocellular carcinoma. We investigated the inter- and intra-observer reproducibility of the European Association for the Study of the Liver criteria and modified Response Evaluation Criteria in Solid Tumors in patients with advanced hepatocellular carcinoma treated with sorafenib. METHODS: A total of 99 patients with treatment-naive advanced hepatocellular carcinoma receiving sorafenib were included. The κ-values for the inter- and intra-observer agreement of the treatment response were calculated. RESULTS: Inter-observer agreement for baseline tumour number was excellent, as reflected by the high κ-value. The κ-statistics showed "excellent" concordance between the 2 sets of measurements by observer A regarding the overall responses using the European Association for the Study of the Liver criteria (κ = .948, agreement rate = 84.8%) and modified Response Evaluation Criteria in Solid Tumors (κ = .944, agreement rate = 83.8%; all P < .001). In addition, high κ-values indicated concordance between the first sets of measurements by observers A and B (κ = .991 by the European Association for the Study of the Liver criteria and .988 by modified Response Evaluation Criteria in Solid Tumors, all P < .001). When agreements in radiological overall responses between the 2 sets of measurements by observer B and between the second sets of measurements by observers A and B were calculated, similar results regarding high κ-values (>.8) were obtained. CONCLUSIONS: The reproducibility of the European Association for the Study of the Liver criteria and modified Response Evaluation Criteria in Solid Tumors in assessing treatment outcomes was high in patients with advanced hepatocellular carcinoma treated with sorafenib.

Evaluation of estrogenic potential by herbal formula, HPC 03 for in vitro and in vivo.

HPC 03 is herbal formula that consists of extracts from Angelica gigas, Cnidium officinale Makino and Cinnamomum cassia Presl. The present study evaluated the estrogenic potential of HPC 03 by using in vitro and in vivo models. The regulatory mechanisms of HPC 03 in estrogen-dependent MCF-7 cells were assessed. HPC 03 induced the proliferation of estrogen receptor-positive MCF-7 cells, and the proliferation was blocked by the addition of the estrogen antagonist tamoxifen. The estrogen receptorα/ß luciferase activities were significantly increased by HPC 03 treatment, which also increased the mRNA expression of the estrogen-responsive genes Psen2, Pgr and Ctsd Also, we evaluated the ameliorative effects of HPC 03 on menopausal symptoms in ovariectomized rats. HPC 03 treatment in OVX rats significantly affected the uterine weight, increased the expression of estrogen-responsive genes Pgr and Psen2 in uterus, increased bone mineral density loss in the femur and inhibited body weight increase. Serum E2, collagen type 1 and osteocalcin were significantly increased, while serum LH, FSH and ALP were decreased compared with OVX rats. HPC 03 may be a promising candidate for the treatment of menopause, but further research is necessary to determine whether the observed effects also occur in humans.

Scopoletin Stimulates Melanogenesis via cAMP/PKA Pathway and Partially p38 Activation.

Scopoletin was recently shown to stimulate melanogenesis through cAMP-response element-binding protein (CREB) phosphorylation. In this study, we investigated the molecular events of melanogenesis-induced by scopoletin. After exposure to scopoletin, the protein levels of tyrosinase and tyrosianse related protein-1 (TRP-1) were significantly increased in B16F10 cells. The mRNA levels of tyrosinase and microphthalmia-associated transcription factor (MITF) were also enhanced by scopoletin. cAMP production and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were increased by scopoletin treatment. Scopoletin-mediated increase of intracellular melanin and tyrosinase expression were significantly attenuated by protein kinase A (PKA) inhibitors (H-89 and KT5720), while a protein kinase C (PKC) inhibitor (Ro-32-0432) had no effect and a p38 MAPK inhibitor (SB203580) partially blocked the scopoletin-induced intracellular melanin and tyrosinase expression. Moreover, scopoletin synergistically with cell-permeable cAMP analog (dibutyryl cAMP) significantly induced tyrosinase activity and melanin content in B16F10 cells. The silencing of p38 MAPK by small interfering RNA (siRNA) decreased the scopoletin-induced tyrosinase expression in B16F10 cells. These results suggest that scopoletin could induce melanin synthesis through the cAMP/PKA pathway and partially p38 MAPK activation in B16F10 cells.

Useful Endoscopic Ultrasonography Parameters and a Predictive Model for the Recurrence of Esophageal Varices and Bleeding after Variceal Ligation.

BACKGROUND/AIMS: To identify the usefulness of endoscopic ultrasonography with a mini-probe (EUM) and to create a predictive model for esophageal variceal (EV) recurrence and bleeding following esophageal variceal ligation (EVL). METHODS: A total of 144 patients who received EUM prior to prophylactic EVL and met the inclusion criteria were enrolled. EUM findings, EV diameter, paraesophageal vein diameter, and the number of perforating veins were assessed. RESULTS: EV recurrence was observed in 42 patients (29.2%), 10 of whom experienced EV bleeding. Larger diameter of the paraesophageal vein (odds ratio [OR], 1.51; 95% confidence interval [CI], 1.17 to 1.96; p=0.002) and perforating vein (OR, 3.27; 95% CI, 1.11 to 9.65; p=0.032) were significant predictive factors for EV recurrence. However, the diameter of the paraesophageal vein was the only significant risk factor for EV bleeding (adjusted OR, 1.51; 95% CI, 1.06 to 2.16; p=0.022). The areas under the curves of the predictive model for EV recurrence and bleeding were 0.872 (95% CI, 0.811 to 0.934) and 0.811 (95% CI, 0.630 to 0.992), respectively. CONCLUSIONS: The diameter of the paraesophageal vein was a significant predictive factor for EV recurrence and bleeding. The predictive model constructed based on the significant EUM findings exhibited good performance.

Association Between Level of Fibrosis, Rather Than Antiviral Regimen, and Outcomes of Patients With Chronic Hepatitis B.

BACKGROUND & AIMS: We performed a propensity-score matched analysis to investigate whether entecavir, compared with lamivudine, can reduce risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B after adjusting for level of fibrosis. METHODS: We performed a retrospective study of 1079 patients with chronic hepatitis B who received first-line therapy with lamivudine (n = 435) or entecavir (n = 644) from 2006 through 2013. Only patients with available liver stiffness value measured by transient elastography were recruited. Liver cirrhosis was diagnosed by ultrasonography. To adjust for the imbalance of patients treated with lamivudine versus entecavir, we performed propensity-score matching (PSM), at a ratio of 1:1, using 7 factors (age, sex, hepatitis B e antigen, alanine aminotransferase, serum albumin, platelet count, and liver stiffness; PSM1) or 8 factors (variables of PSM1 plus ultrasonography measurements of cirrhosis; PSM2). Patients with virologic breakthrough or resistance mutations received rescue therapy. RESULTS: Over the 7-year period, 91 patients developed HCC and 104 had liver-related events in the entire cohort. In multivariate analyses, level of fibrosis, but not antiviral regimen, was independently associated with risk of HCC (P < .05). The PSM1 group included 342 pairs of patients and the PSM2 group included 338 pairs. Similar proportions of patients given lamivudine versus entecavir developed HCC in each model (10.5% given lamivudine vs 9.9% given entecavir in PSM1 and 11.9% vs 12.6% in PSM2; all P > .05). When PSM was applied to patients with liver stiffness value ≤13 kPa or >13 kPa, patients given lamivudine versus entecavir still had similar cumulative rates of HCC development (all P > .05). CONCLUSIONS: In a PSM analysis, we associated level of fibrosis, rather than antiviral regimen, with risk of HCC, when patients received appropriate rescue therapy in case of virologic breakthrough or resistance mutations.

Exenatide reverses dysregulated microRNAs in high-fat diet-induced obese mice.

Exenatide has beneficial effects on insulin sensitivity in several animal models; however, its mechanism of action remains unclear. Furthermore, the relationship between the effect of exenatide on the changes in the relative abundance of microRNAs (miRNAs), which play a role in regulating glucose and lipid metabolism, is not fully understood. Therefore, we assessed the effect of exenatide on miRNA expression in a high-fat diet (HFD)-induced mouse model of obesity. Both HFD control and exenatide-treated HFD mice showed similar body weight gain and increase in ß-cell mass. Insulin levels were significantly lower in exenatide-treated mice than in HFD control mice. The levels of miRNA-15a, 29c, 124a, and 375 in the pancreas were significantly increased in HFD control mice. Furthermore, the levels of miRNA-29c, 124a, and 146a in the liver and miRNA-15a, 29c, 124a, and 146a in the muscle were significantly increased. In contrast, the levels of miRNA-15a, 29c, 124a, and 375 in the serum were significantly decreased. These effects were reversed by treatment with exenatide. Our results provide experimental evidence that exenatide-mediated amelioration of insulin sensitivity is associated with antagonistic changes in the relative abundance of miRNA-15a, 29c, 124a, and 375 in tissues and serum, thus highlighting their usefulness as biomarkers for monitoring insulin sensitivity and response to exenatide treatment in experimental diabetes.

Human umbilical cord blood-derived mesenchymal stem cells improve functional recovery through thrombospondin1, pantraxin3, and vascular endothelial growth factor in the ischemic rat brain.

Cell therapy is a potential therapeutic method for cerebral ischemia, which remains a serious problem. In the search for more effective therapeutic methods, many kinds of stem cells from various tissues have been developed and tested as candidate therapeutic agents. Among them, human umbilical cord blood (hUCB)-derived mesenchymal stem cells (MSCs) are widely used for cell therapy because of their genetic flexibility. To confirm that they are effective and understand how they affect ischemic neural cells, hUCB-MSCs were directly administered ipsilaterally into an ischemic zone induced by middle cerebral artery occlusion (MCAO). We found that the neurobehavioral performance of the hUCB-MSC group was significantly improved compared with that of the vehicle-injected control group. The infarct was also remarkably smaller in the hUCB-MSC group. Additionally, hUCB-MSC transplantation resulted in a greater number of newly generated cells and angiogenic and tissue repair factors and a lower number of inflammatory events in the penumbra zone. To determine why these events occurred, hUCB-MSCs were assayed under hypoxic and normoxic conditions in vitro. The results showed that hUCB-MSCs exhibit higher expression levels of thrombospondin1, pantraxin3, and vascular endothelial growth factor under hypoxic conditions than under normoxic conditions. These results were found to be correlated with our in vivo immunofluorescent staining results. On the basis of these findings, we suggest that hUCB-MSCs may have a beneficial effect on cerebral ischemia, especially through angiogenesis, neurogenesis, and anti-inflammatory effects, and thus could be used as a therapeutic agent to treat neurological disorders such as cerebral ischemia.

Association of vitamin B12 deficiency and metformin use in patients with type 2 diabetes.

We evaluated the prevalence of vitamin B12 deficiency and associated factors in type 2 diabetes patients using metformin. A total of 799 type 2 diabetes patients using metformin was enrolled. Vitamin B12 and folate levels were quantified by chemiluminescent enzyme immunoassay. Vitamin B12 deficiency was defined as vitamin B12 ≤ 300 pg/mL without folate deficiency (folate > 4 ng/mL). The prevalence of vitamin B12 deficiency in metformin-treated type 2 diabetes patients was 9.5% (n = 76), and the mean vitamin B12 level was 662.5 ± 246.7 pg/mL. Vitamin B12 deficient patients had longer duration of metformin use (P < 0.001) and higher daily metformin dose (P < 0.001) than non-deficient patients. Compared with daily metformin dose of ≤ 1,000 mg, the adjusted odds ratio for 1,000-2,000 mg, and ≥ 2,000 mg were 2.52 (95% CI, 1.27-4.99, P = 0.008) and 3.80 (95% CI, 1.82-7.92, P < 0.001). Compared with metformin use of < 4 yr, the adjusted odds ratios for 4-10 yr, and ≥ 10 yr were 4.65 (95% CI, 2.36-9.16, P < 0.001) and 9.21 (95% CI, 3.38-25.11, P < 0.001), respectively. In conclusion, our study indicates that patients with type 2 diabetes treated with metformin should be screened for vitamin B12 deficiency, especially at higher dosages (> 1,000 mg) and longer durations (≥ 4 yr) of treatment.

Unresolved subclinical hypothyroidism is independently associated with progression of chronic kidney disease.

BACKGROUND AND AIM: Patients with chronic kidney disease (CKD) often have subclinical hypothyroidism. However, few reports have investigated changes in the status of subclinical hypothyroidism in CKD patients and its clinical significance in CKD progression. METHODS: We included 168 patients with nondialysis-dependent CKD stages 2-4. The normalization of subclinical hypothyroidism during follow-up was assessed, and the association between transitions in subclinical hypothyroid status and the rate of decline of the estimated glomerular filtration rate (eGFR) was investigated. RESULTS: At baseline, 127 patients were euthyroid and 41 (24.4%) patients were diagnosed with subclinical hypothyroidism. Of these 41 patients, 21 (51.2%) spontaneously resolved to euthyroid during follow-up. The rate of eGFR decline of patients with resolved subclinical hypothyroidism was similar to that of euthyroid patients. The patients with unresolved subclinical hypothyroidism showed a steeper renal function decline than patients with euthyroidism or resolved subclinical hypothyroidism (all p < 0.05). The progression to end-stage renal disease was more frequent in those with unresolved subclinical hypothyroidism than in those who were euthyroid (p = 0.006). In multivariate linear regression for rate of eGFR decrease, unresolved subclinical hypothyroidism (ß = -5.77, p = 0.001), baseline renal function (ß = -0.12, p < 0.001) and level of proteinuria (ß = -2.36, p = 0.015) were independently associated with the rate of renal function decline. CONCLUSIONS: Half of the CKD patients with subclinical hypothyroidism did not resolve to euthyroidism, and this lack of resolution was independently associated with rapid renal function decline.