The Impact of Age- and Sex-Specific Reference Ranges for Serum Thyrotropin and Free Thyroxine on the Diagnosis of Subclinical Thyroid Dysfunction: A Multicenter Study from Japan.

Background: Reference ranges for serum thyrotropin (TSH), free thyroxine (fT4), and free triiodothyronine (fT3) established without considering age- and sex-based differences are currently used to evaluate thyroid function. Therefore, we investigated age- and sex-based differences in serum TSH and thyroid hormone levels in euthyroid individuals. Methods: We performed cross-sectional analyses of retrospective data collected from two Japanese institutions. We estimated sex-specific 95% reference ranges for TSH and fT4 according to age strata. Results: We included data from 14,860 participants undergoing screening with a Siemens thyroid testing kit and 8,132 participants undergoing screening with an Abbott kit during annual health check-ups at Takasaki Hidaka Hospital. In addition, 515 participants visiting a specialized thyroid-focused hospital were evaluated using Tosoh kits. The median TSH level of women in their 30s was 1.5 mIU/L (2.5th percentile, 0.5; 97.5th percentile, 4.6) using the Siemens kit, while that of women in their 60s was 1.9 (0.7-7.8) mIU/L. The corresponding levels were lower in men; the age-associated increase was small. The median serum fT4 level of men in their 30s was 1.3 (1.0-1.7) ng/dL and that of men in their 60s was 1.2 (1.0-1.6) ng/dL. These levels gradually but significantly decreased with age. fT4 levels in women were lower than those in men and remained consistent with age. Serum fT3 levels were significantly higher in men than in women and gradually but significantly decreased with age. The Abbott and Tosoh kits showed similar results. When using the Siemens kit, ∼60% (216/358) of women diagnosed with subclinical hypothyroidism using manufacturer-recommended reference ranges had normal results when age- and sex-specific reference ranges were applied, demonstrating the high percentage of overdiagnosis, especially in those aged ≥60 years. Conversely, some middle-aged individuals with normal thyroid function were reassessed and classified as having subclinical hyperthyroidism by age- and sex-specific reference ranges. Conclusions: Age- and sex-specific reference ranges should be used to avoid over- and underdiagnosis of subclinical thyroid dysfunction and appropriate therapies.

Relevance of gene mutations and methylation to the growth of pancreatic intraductal papillary mucinous neoplasms based on pyrosequencing.

We aimed to assess some of the potential genetic pathways for cancer development from non-malignant intraductal papillary mucinous neoplasm (IPMN) by evaluating genetic mutations and methylation. In total, 46 dissected regions in 33 IPMN cases were analyzed and compared between malignant-potential and benign cases, or between malignant-potential and benign tissue dissected regions including low-grade IPMN dissected regions accompanied by malignant-potential regions. Several gene mutations, gene methylations, and proteins were assessed by pyrosequencing and immunohistochemical analysis. RASSF1A methylation was more frequent in malignant-potential dissected regions (p = 0.0329). LINE-1 methylation was inversely correlated with GNAS mutation (r = - 0.3739, p = 0.0105). In cases with malignant-potential dissected regions, GNAS mutation was associated with less frequent perivascular invasion (p = 0.0128), perineural invasion (p = 0.0377), and lymph node metastasis (p = 0.0377) but significantly longer overall survival, compared to malignant-potential cases without GNAS mutation (p = 0.0419). The presence of concordant KRAS and GNAS mutations in the malignant-potential and benign dissected regions were more frequent among branch-duct IPMN cases than among the other types (p = 0.0319). Methylation of RASSF1A, CDKN2A, and LINE-1 and GNAS mutation may be relevant to cancer development, IPMN subtypes, and cancer prognosis.

Long-term outcomes of nasogastric tube with Gastrografin for adhesive small bowel obstruction.

BACKGROUND: We had previously reported that the administration of Gastrografin through a nasogastric tube (NGT-G) followed by long tube (LT) strategy could be a novel standard treatment for adhesive small bowel obstruction (ASBO); however, the long-term outcomes after initial improvement remain unknown. This study aimed to analyze the long-term outcomes of first-line NGT-G. METHODS: Enrolled patients with ASBO were randomly assigned to receive LT or NGT-G between July 2016 and November 2018. Thereafter, the cumulative surgery rate, cumulative recurrence rate, and overall survival (OS) rate were analyzed. In addition, subset analysis was conducted to determine the cumulative recurrence rate according to colonic contrast with Gastrografin at 24 h. RESULTS: A total of 223 patients (LT group, n = 111; NGT-G group, n = 112) were analyzed over a median follow-up duration of 550 days. The cumulative 1-year surgery rates, cumulative 1-year recurrence rates, and 1-year OS rates in the LT and NGT-G groups were 18.8% and 18.1%, 30.0% and 31.7%, and 99.1% and 96.6%, respectively; no significant differences were observed between both groups. In the NGT-G group, a negative colonic contrast at 24 h demonstrated a higher tendency for future recurrence compared with a positive colonic contrast at 24 h (1-year recurrence rate: negative contrast, 46.9% vs positive contrast, 27.6%). CONCLUSIONS: Gastrografin through a nasogastric tube followed by LT can be a promising treatment strategy for ASBO, with long-term efficacies equivalent to initial LT placement.

Urinary microRNA biomarkers for detecting the presence of esophageal cancer.

Esophageal cancer (EC) including esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC) generally exhibits poor prognosis; hence, a noninvasive biomarker enabling early detection is necessary. Age- and sex-matched 150 healthy controls (HCs) and 43 patients with ESCC were randomly divided into two groups: 9 individuals in the discovery cohort for microarray analysis and 184 individuals in the training/test cohort with cross-validation for qRT-PCR analysis. Using 152 urine samples (144 HCs and 8 EACs), we validated the urinary miRNA biomarkers for EAC diagnosis. Among eight miRNAs selected in the discovery cohort, urinary levels of five miRNAs (miR-1273f, miR-619-5p, miR-150-3p, miR-4327, and miR-3135b) were significantly higher in the ESCC group than in the HC group, in the training/test cohort. Consistently, these five urinary miRNAs were significantly different between HC and ESCC in both training and test sets. Especially, urinary miR-1273f and miR-619-5p showed excellent values of area under the curve (AUC) ≥ 0.80 for diagnosing stage I ESCC. Similarly, the EAC group had significantly higher urinary levels of these five miRNAs than the HC group, with AUC values of approximately 0.80. The present study established novel urinary miRNA biomarkers that can early detect ESCC and EAC.

The first management using intubation of a nasogastric tube with Gastrografin enterography or long tube for non-strangulated acute small bowel obstruction: a multicenter, randomized controlled trial.

BACKGROUND: Gastrointestinal decompression is generally applied to a non-strangulated acute small bowel obstruction (NSASBO). Although long tube (LT) placement and administration of Gastrografin through a nasogastric tube (NGT-G) have shown advantages over NGT alone in previous studies, no studies appear to have compared LT and NGT-G. METHODS: In this multicenter, randomized controlled trial, patients with NSASBO were randomly assigned to receive LT or NGT-G between July 2016 and November 2018 at 11 Japanese institutions. The primary endpoint was non-inferiority of NGT-G compared to LT for non-surgery rate, and the lower limit of the 95% confidence interval for the non-surgery rate (-15%) was set as the lower margin for inferiority of NGT-G compared to LT. RESULTS: In total, 223 patients (LT group, n = 111; NGT-G group, n = 112) were analyzed in the present trial. The non-surgery rate was 87.4% in the LT group and 91.1% in the NGT-G group, with a 3.7% difference between NGT-G and LT (95.3%CI - 5.55 to 12.91; non-inferiority P = 0.00002923). On the other hand, the non-surgery rate with pure NGT-G alone (76.8%) that represents non-cross-over NGT-G without subsequent LT was significantly lower than that with LT (P = 0.039). Median procedure time was significantly shorter with NGT-G (1 min) than with LT (25 min; P < 0.001), whereas no significant differences in mortality or hospital stay were noted between groups. CONCLUSION: NGT-G is an effective alternative to LT as a first-line treatment for NSASBO. A sequential strategy comprising NGT-G followed by LT might offer a new standard for NSASBO. CLINICAL TRIALS REGISTRATION: This trial was registered with the University Hospital Medical Information Network Clinical Trials Registry (umin.ac.jp/ctr Identifier: UMIN000022669) prior to the start of this trial.

Placental growth factor is a predictive biomarker for ramucirumab treatment in advanced gastric cancer.

PURPOSE: Ramucirumab (RAM) has been used as the second-line standard chemotherapy for advanced gastric cancer (AGC) either alone or combination with paclitaxel (PTX). However, no predictive biomarkers have been identified for RAM treatment in AGC. METHODS: We retrospectively identified 26 patients who received either RAM monotherapy or RAM + PTX therapy for AGC refractory to fluoropyrimidine and platinum agents from 2015 to 2018 at Nagoya City University Hospital. First, we extracted RNA using gastric cancer (GC) tissues from two responders and two non-responders, and then analyzed 24 VEGFR-related angiogenic genes. Subsequently, we examined the relationship between the expression of each angiogenic gene and RAM clinical activity in the entire cohort. Finally, we validated using in vitro angiogenesis assays using GC cells and microvascular endothelial cells. RESULTS: We identified five angiogenic genes with aberrant expression between RAM responders and non-responders and placental growth factor (PlGF) was the most significant gene among them. Overall survival (P = 0.046) and progression-free survival (P = 0.016) were significantly shorter in the PlGF-high group than in the PlGF-low group. Overall response rates were 50% in the PlGF-low group and 0% in the PlGF-high group. In GC cells co-cultured with endothelial cells, PlGF gene silencing from GC cells significantly reinforced the inhibitory effect of RAM in the in vitro angiogenesis assay (tube formation assay and endothelial migration) through the inactivation of ERK, in comparison to the control GC cells. CONCLUSIONS: PlGF gene expression in gastric cancer tissues could be a predictive indicator of AGC treatment by RAM.

Urinary Cysteine-Rich Protein 61 and Trefoil Factor 3 as Diagnostic Biomarkers for Colorectal Cancer.

Since a fecal occult blood test for colorectal cancer (CRC) does not offer sufficient diagnostic power for CRC, novel non-invasive biomarkers are hopeful for CRC screening. We conducted the current study to discover non-invasive urinary biomarkers for diagnosing CRC. Among urine samples from 258 patients (CRC, n = 148; healthy controls, n = 110), a cohort of 176 patients composed of 88 patients with GC and 88 healthy controls was selected after age- and sex-matching using propensity score. This cohort was then randomly divided into 2 groups: 53 pairs (106 patients) in the training cohort, and 35 pairs (70 patients) in the validation cohort. No significant differences were found for baseline characteristics between the CRC and healthy control groups in both training and validation cohorts. On multivariate analysis in the training cohort, urinary levels of cysteine-rich protein 61 (uCyr61) and trefoil factor 3 (uTFF3) were identified as independent significant diagnostic markers for CRC. Moreover, uCyr61 alone and the combination of uCyr61 and uTFF3 allowed significant differentiation between healthy controls and CRC groups in the training set (uCyr61: area under the curve (AUC) = 0.745 [95% CI, 0.653-0.838]; uCyr61 + uTFF3: AUC = 0.753 [95% CI, 0.659-0.847]). In the validation cohort, uCyr61 and uTFF3 were significantly higher in the CRC group than in the healthy control group, and they also allowed significant differentiation between healthy control and CRC groups (uCyr61: AUC = 0.696 [95% CI, 0.571-0.822]; uTFF3: AUC = 0.639 [95% CI, 0.508-0.770]; uCyr61 + uTFF3: AUC = 0.720 [95% CI, 0.599-0.841]), as in the training cohort. A panel combining uCyr61 and uTFF3 offers a promising non-invasive biomarker for diagnosing CRC.

Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Combination Therapy With Intensive Granulocyte and Monocyte Adsorptive Apheresis Plus Ustekinumab in Patients With Refractory Crohn's Disease.

Ustekinumab is applied to induce clinical remission in patients with Crohn's disease. Granulocyte and monocyte absorptive apheresis depletes activated myeloid lineage leukocytes and has been applied for active Crohn's disease. This study retrospectively examined the efficacy and safety of combining intensive granulocyte and monocyte absorptive apheresis and ustekinumab for remission induction therapy in refractory Crohn's disease. Between June and September 2017, three consecutive cases (two females) with refractory Crohn's disease were treated with intensive granulocyte and monocyte absorptive apheresis plus ustekinumab. Crohn's disease activity index, and simple endoscopic score for Crohn's disease at baseline and 10 weeks were applied as treatment efficacy outcomes. In all three cases, at week 10, clinical remission was achieved, while simple endoscopic score for Crohn's disease reflected no improvement. Thus, combination therapy with intensive granulocyte and monocyte absorptive apheresis plus ustekinumab appeared to represent a safe and effective intervention for inducing clinical remission.

Refractory gastric antral ulcers without Helicobacter pylori infection and non-steroidal anti-inflammatory drugs.

Herein, we describe a rare case of refractory gastric antral ulcers. A woman in her 50 s was admitted to Nagoya City University Hospital with epigastric pain after being diagnosed with gastric antral submucosal tumor at another hospital. Findings from esophagogastroduodenoscopy and endoscopic ultrasound examination revealed that the lesion was a gastric ulcer. The patient had no Helicobacter pylori infection and no recent history of using non-steroidal anti-inflammatory drugs. On the basis of these findings, we diagnosed this as a case of refractory gastric antral ulcer (RGAU). RGAU is considered a new disease concept and detailed analyses are expected in the future.

The Mutant Thyroid Hormone Receptor Beta R320P Causes Syndrome of Resistance to Thyroid Hormone.

A 31-year-old Japanese male patient with a history of atrial fibrillation showed elevated serum levels of free thyroxine and triiodothyronine and a normal level of thyrotropin. The same abnormal hormone pattern was also found in his son. These data indicated that the index patient and the son have thyroid hormone resistance syndrome. Exon sequencing using DNA from these two patients revealed that both patients harbored a heterozygous mutation in the THRB gene: G1244C in exon 9, which results in R320P substitution. Therefore, thyroid hormone resistance syndrome caused by THRB mutation (RTHß) was diagnosed. The mutation of the 320th arginine to proline has not been found to date. In conclusion, herein, we have described the first case of RTHß that is associated with R320P mutation.

Expression and subcellular localization of AT motif binding factor 1 in colon tumours.

AT motif binding factor 1 (ATBF1) is a transcriptional regulator that functions as a tumour suppressor to negatively affect cancer cell growth. In the present study four specific polyclonal antibodies against ATBF1 were generated, and the expression and intracellular localization of ATBF1 in colonic mucosae, polyps, adenoma and adenocarcinoma tissue samples were investigated. The four polyclonal antibodies produced were as follows: MB34 and MB49, which recognize the N­ and C­terminal fragments of ATBF1, respectively; and D1­120 and MB44, which recognize the middle fragments of ATBF1 that contain three nuclear localization signals (NLS). In total, 191 colon samples were examined by immunohistochemical analysis. In addition, colon cancer cells were transfected with four ATBF1 expression vectors, and the subcellular localization of each fragment was examined. Normal colon mucosal cells were not observed to express ATBF1. However, a small number of hyperplastic polyps, serrated adenomas and tubular adenomas expressed ATBF1. Colon cancer cells were observed to express D1­120­ and MB44­reactive middle fragments of ATBF1 in their cell nuclei. However, the N­ and C­terminal fragments of ATBF1 did not translocate to the nucleus. Transfection of ATBF1 fragments revealed cleavage of the ATBF1 protein and nuclear translocation of the cleaved middle portion containing the NLS. A positive correlation between the cytoplasmic localization of the N­ and C­termini of ATBF1, nuclear localization of the middle portion of ATBF1 and malignant cancer cell invasion was observed. In conclusion, the results of the present study suggest that alterations in the expression and subcellular localization of ATBF1, as a result of post­transcriptional modifications, are associated with malignant features of colon tumours.

Adalimumab therapy in a patient with Crohn's disease with a giant pelvic paraganglioma after chemotherapy.

A 23-year-old man was diagnosed with a giant pelvic paraganglioma in September 2013, and a 6-month chemotherapy course was performed. The chemotherapy resulted in stable disease of the tumor for about 1 year. However, in April 2015, the patient complained of fever and diarrhea of more than ten times a day. Endoscopy showed serpiginous (snake-like) ulcers in the cecum, ascending, descending, and sigmoid colons, with granulomas without caseation histologically. The patient was diagnosed with the active stage of Crohn's disease (CD) in June 2015. Oral mesalazine (3000 mg/day) and an elemental diet (900 kcal/day) led to temporary clinical remission. At the beginning of January in 2016, an abdominal abscess and fistula were detected by computed tomography, which needed surgical treatment. Adalimumab administration was started at the beginning of February, since active lesions were detected endoscopically. A second endoscopy showed improvement of the inflammatory lesions 3 months after induction therapy with adalimumab. Clinical remission has been maintained with adalimumab administration, with stable disease of the tumor and no adverse events. To the best of our knowledge, this is the first report of a patient with a paraganglioma who developed CD after chemotherapy. The patient was successfully treated with adalimumab after surgery for his CD.

Transcriptional Selectivity of Epigenetic Therapy in Cancer.

A central challenge in the development of epigenetic cancer therapy is the ability to direct selectivity in modulating gene expression for disease-selective efficacy. To address this issue, we characterized by RNA-seq, DNA methylation, and ChIP-seq analyses the epigenetic response of a set of colon, breast, and leukemia cancer cell lines to small-molecule inhibitors against DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343). We also characterized the effects of DAC as combined with the other compounds. Averaged over the cancer cell models used, we found that DAC affected 8.6% of the transcriptome and that 95.4% of the genes affected were upregulated. DAC preferentially regulated genes that were silenced in cancer and that were methylated at their promoters. In contrast, Depsi affected the expression of 30.4% of the transcriptome but showed little selectivity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 affected only 2% of the transcriptome, with UNC0638 and GSK343 preferentially targeting genes marked with H3K9me2 or H3K27me3, respectively. When combined with histone methylase inhibitors, the extent of gene upregulation by DAC was extended while still maintaining selectivity for DNA-methylated genes and silenced genes. However, the genes upregulated by combination treatment exhibited limited overlap, indicating the possibility of targeting distinct sets of genes based on different epigenetic therapy combinations. Overall, our results demonstrated that DNA methyltransferase inhibitors preferentially target cancer-relevant genes and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining selectivity. Cancer Res; 77(2); 470-81. ©2016 AACR.

Ectopic Gastric and Intestinal Phenotypes, Neuroendocrine Cell Differentiation, and SOX2 Expression Correlated With Early Tumor Progression in Colorectal Laterally Spreading Tumors.

INTRODUCTION: The significance of the ectopic gastric phenotype remains unclear in patients with colorectal laterally spreading tumors (LSTs). We investigated clinicopathologic differences among LST subtypes, aiming to identify factors indicative of malignant transformation and invasion that are linked to ectopic gastric phenotype and tumor progression. MATERIALS AND METHODS: We analyzed the morphologic characteristics of 105 colorectal LSTs resected by endoscopic submucosal dissection. LSTs were classified into 2 subtypes: granular (G-LST) and nongranular (NG-LST). Resected LSTs were analyzed histologically and were immunohistochemically stained for MUC5AC, MUC6, chromogranin A, CD10, and SOX2. RESULTS: The 105 LSTs included 60 G-LSTs and 45 NG-LSTs. By histology, G-LSTs comprised 5 adenomas with low-grade dysplasia (LAs), 45 adenomas with high-grade dysplasia (HAs), and 10 adenocarcinomas invading the submucosa (SMs). NG-LSTs comprised 8 LAs, 25 HAs, and 12 SMs. MUC5AC positivity was significantly higher in G-LSTs compared to NG-LSTs (P = .002), and MUC5AC positivity in HA lesions was significantly higher than in LA lesions (P = .01). MUC6 and SOX2 positivity in SM G-LSTs, and chromogranin A positivity in SM NG-LSTs were significantly higher than in HAs (P = .01, .01, and .03, respectively). CD10 positivity in SM NG-LSTs was significantly higher than in HAs and LAs (P = .02 and .01, respectively). CONCLUSION: Ectopic gastric and intestinal phenotypes, neuroendocrine cell differentiation, and SOX2 expression differ according to tumor grade in colorectal LSTs, and these markers are correlated with early tumor progression in each LST subtype.

Mutation spectrum of TP53 gene predicts clinicopathological features and survival of gastric cancer.

BACKGROUND AND AIM: TP53 gene is frequently mutated in gastric cancer (GC), but the relationship with clinicopathological features and prognosis is conflicting. Here, we screened TP53 mutation spectrum of 214 GC patients in relation to their clinicopathological features and prognosis. RESULTS: TP53 nonsilent mutations were detected in 80 cases (37.4%), being frequently occurred as C:G to T:A single nucleotide transitions at 5'-CpG-3' sites. TP53 mutations occurred more frequently in differentiated histologic type than in undifferentiated type in the early stage (48.6% vs. 7%, P=0.0006), while the mutations correlated with venous invasion among advanced stage (47.7% vs. 20.7%, P=0.04). Subset of GC with TP53 hot spot mutations (R175, G245, R248, R273, R282) presented significantly worse overall survival and recurrence free survival compared to others (both P=0.001). METHODS: Matched biopsies from GC and adjacent tissues from 214 patients were used for the experiment. All coding regions of TP53 gene (exon2 to exon11) were examined using Sanger sequencing. CONCLUSION: Our data suggest that GC with TP53 mutations seems to develop as differentiated histologic type and show aggressive biological behavior such as venous invasion. Moreover, our data emphasizes the importance of discriminating TP53 hot spot mutations (R175, G245, R248, R273, R282) to predict worse overall survival and recurrence free survival of GC patients.

Autonomic nervous responses in colorectal polypectomy: Randomized controlled trial comparing air and carbon dioxide insufflation.

BACKGROUND AND AIM: Carbon dioxide (CO2) insufflation devices are commonly used for endoscopic examination and treatment. In this prospective randomized controlled trial (RCT), we compared patient acceptance, cardiovascular tolerance,and autonomic nervous responses between patients receiving air insufflation and CO2 insufflation. METHODS: We initially enrolled 170 patients and, of these, 158 patients in total were analyzed (air group, 83; CO2 group, 75). Autonomic nervous responses were evaluated by analysis of heart rate variability (HRV). Primary end point was superiority in the effects of CO2 insufflation on the autonomic nervous system by HRV analysis. RESULTS: Visual analog scale disclosed significantly less abdominal pain and abdominal fullness with CO2. Percentage heart rate change rate at 1 h and 4 h after the procedure was also significantly lower in the CO2 group than in the air group (1 h after: P < 0.01, 4 h after: P < 0.05). Comparison based on age showed that % heart rate change was significantly lower in the younger CO2 patients (just after colonoscopy and 1 h after: P < 0.01, 4 h after: P < 0.05), but this difference was not apparent in an older group of patients. CONCLUSIONS: This is the first RCT showing that colorectal polypectomy using CO2 insufflation significantly decreases abdominal pain and abdominal fullness common in such patients with lowered stress to the autonomous nervous system. The effects using CO2 insufflation on the sympathetic nervous system also seemed to be more prominent among younger patients.

Hepatitis virus infection affects DNA methylation in mice with humanized livers.

BACKGROUND & AIMS: Cells of tumors associated with chronic inflammation frequently have altered patterns of DNA methylation, including hepatocellular carcinomas. Chronic hepatitis has also been associated with aberrant DNA methylation, but little is known about their relationship. METHODS: Pyrosequencing was used to determine the methylation status of cultured Huh7.5.1 hepatoma cells after hepatitis C virus (HCV) infection. We also studied mice with severe combined immunodeficiency carrying the urokinase-type plasminogen activator transgene controlled by an albumin promoter (urokinase-type plasminogen activator/severe combined immunodeficient mice), in which up to 85% of hepatocytes were replaced by human hepatocytes (chimeric mice). Mice were given intravenous injections of hepatitis B virus (HBV) or HCV, liver tissues were collected, and DNA methylation profiles were determined at different time points after infection. We also compared methylation patterns between paired samples of hepatocellular carcinomas and adjacent nontumor liver tissues from patients. RESULTS: No reproducible changes in DNA methylation were observed after infection of Huh7.5.1 cells with HCV. Livers from HBV- and HCV-infected mice had genome-wide, time-dependent changes in DNA methylation, compared with uninfected urokinase-type plasminogen activator/severe combined immunodeficient mice. There were changes in 160 ± 63 genes in HBV-infected and 237 ± 110 genes in HCV-infected mice. Methylation of 149 common genes increased in HBV- and HCV-infected mice; methylation of some of these genes also increased in hepatocellular carcinoma samples from patients compared with nontumor tissues. Expression of Ifng, which is expressed by natural killer cells, increased significantly in chimeric livers, in concordance with induction of DNA methylation, after infection with HBV or HCV. Induction of Ifng was reduced after administration of an inhibitor of natural killer cell function (anti-asialo GM1). CONCLUSIONS: In chimeric mice with humanized livers, infection with HBV and HCV appears to activate a natural kill cell-dependent innate immune response. This contributes to the induction and accumulation of aberrant DNA methylation in human hepatocytes.

Detection of latent anthracycline-induced cardiotoxicity using left ventricular end-systolic wall stress-velocity of circumferential fiber-shortening relationship.

Although cardiotoxicity is a well-known side effect of anthracycline, detection of subclinical impairment of myocardial contractility at the latent stage is difficult. The left ventricular end-systolic wall stress (WS)-velocity of circumferential fiber-shortening (VCF) relationship reflects the load-independent myocardial contractility and can detect sensitively intrinsic abnormalities in myocardial contractility. Usefulness of this relationship in detecting subclinical anthracycline-induced cardiotoxicity has not yet been established. We investigated whether latent anthracycline-induced cardiotoxicity at the subclinical state can be detected by using the WS-VCF relationship in patients receiving anthracycline therapy. We studied 45 patients who had received anthracycline therapy and 40 healthy controls. All patients had preserved left ventricular ejection fraction (LVEF). WS and VCF were measured using echocardiography. VCF was corrected by heart rate. The WS-VCF relationship was derived by linear regression. Patients with data points lying below -2 SD derived from controls were regarded as having impaired intrinsic myocardial contractility. Although VCF was within normal limits in all patients, it was significantly reduced in the patient group overall compared with the control group. On the other hand, WS was significantly increased in the patient group overall compared with the control group. The WS-VCF relationship demonstrated impaired intrinsic myocardial contractility in 24 patients (53.3 %). In more than half of patients with preserved LVEF, impairment of intrinsic myocardial contractility was detected using the WS-VCF relationship, suggesting the presence of latent anthracycline-induced cardiotoxicity. The WS-VCF relationship may be able to detect sensitively latent anthracycline-induced cardiotoxicity at the subclinical stage.