Evaluation of antimicrobial selective pressure using the multicenter semiautomatic surveillance system Japan surveillance for infection prevention and healthcare epidemiology.

BACKGROUND: Evaluating the selective pressure of antimicrobials on bacteria is important for promoting antimicrobial stewardship programs (ASPs). The aim of this study was to assess the selective pressure of antimicrobials by evaluating their use (carbapenem [CBP] and CBP-sparing therapy) over time and the detection status of CBP-resistant organisms using multicenter data. METHODS: Among the facilities whose data were registered in the Japan Surveillance for Infection Prevention and Healthcare Epidemiology from 2017 to 2020, those that had data on the use of CBP and CBP-sparing therapy (fluoroquinolones [FQs], cefmetazole [CMZ], piperacillin-tazobactam [PIP/TAZ], ampicillin-sulbactam [ABPC/SBT], ceftriaxone/cefotaxime [CTRX/CTX], CAZ (ceftazidime), cefepime [CFPM], and aminoglycosides [AGs]) as well as on CBP-resistant Enterobacterales (CRE) and CBP-resistant Pseudomonas aeruginosa (CRPA) detection were included. Alcohol-based hand rubbing (ABHR) usage was also analyzed. Regression analyses, including multivariable regression analysis, were performed to evaluate trends. The association of antimicrobial use density (AUD) with CRE and CRPA detection rates was evaluated. RESULTS: In 28 facilities nationwide, CBP, FQ, CAZ, AG, and PIP/TAZ use decreased over the 3-year period, whereas the use of CMZ, ABPC/SBT, CTRX/CTX, CFPM, and ABHR as well as the rates of CRE and CRPA detection increased. The average AUD did not significantly correlate with CRE and CRPA detection rates. The multivariable regression analysis did not reveal any significant correlation between each AUD or ABHR and CRE or CRPA detection. CONCLUSION: CBP and ABHR use showed a decreasing and an increasing trend, respectively, while CRPA and CRE detection rates exhibited a gradual increase. The considerably low CRE and CRPA detection rates suggest that slight differences in numbers may have been observed as excessive trend changes. Further investigation is warranted to evaluate selective pressure while considering the characteristics of ASP and the mechanisms underlying resistance.

Comparison of the effects of cefmetazole and meropenem on microbiome: A pilot study.

BACKGROUND: Cefmetazole (CMZ) is a carbapenem-sparing option in the treatment of extended-spectrum beta-lactamase (ESBL)-producing bacterial infection. In this pilot study, we aimed to compare the effects of antimicrobial treatment (meropenem [MP] and CMZ) with those of no antimicrobial treatment (control group) on the microbiome. METHODS: The study was a multicenter, prospective, observational pilot study conducted from October 2020 to October 2022. Feces and saliva samples were collected for microbiome analyses at two time points (early-period: days 1-3; and late-period: days 4-30) for the antimicrobial treatment group, and at one time point for the control group. RESULTS: Five feces (MP-F and CMZ-F) and five saliva (MP-S and CMZ-S) samples were included in the MP and the CMZ groups. Ten feces (C-F) and saliva (C-S) samples were included in the control group. Group α diversity was notably lower in the late-period MP-F group than the control group as determined with the Shannon richness index. ß diversity analysis of the feces samples based on weighted and unweighted UniFrac distances revealed distinctions in both the late-period CMZ-F and MP-F groups compared with the control group. Weighted UniFrac analysis showed that only the early-period MP-F group differed from the control group. In the saliva samples, weighted and unweighted UniFrac analyses showed significant differences between the control group and the early CMZ, late CMZ, and late MP groups. CONCLUSIONS: MP treatment may cause larger impact on the feces microbiome than CMZ in Japanese patients.

Ruptured Occipital Artery Aneurysm Following Craniotomy in a Patient with Neurofibromatosis Type 1: Case Report.

Objective: Neurofibromatosis type 1 (NF1) is associated with vascular fragility, which results in aneurysms, arteriovenous fistulas, and dissections. Here, we describe a case of endovascular treatment of a ruptured occipital artery aneurysm that occurred after a craniotomy in a patient with NF1. Case Presentation: A 46-year-old man with a history of NF1 underwent a right lateral suboccipital craniotomy to remove a cavernous hemangioma in the right middle cerebellar peduncle. Severe bleeding occurred in the occipital artery during the craniotomy. Due to vessel fragility, coagulation and ligation were not possible, and pressure hemostasis was achieved using cellulose oxide and fibrin glue. On postoperative day 12, the patient developed a sudden swelling on the right side of the neck as well as tracheal compression. Contrast-enhanced CT revealed a ruptured aneurysm in the right occipital artery. Transarterial embolization was performed under general anesthesia the same day. Right external carotid angiography showed an 18-mm-diameter fusiform aneurysm in the occipital artery. The aneurysm ruptured inferiorly to form a large pseudoaneurysm with significant jet flow. An arteriovenous fistula was also observed in a nearby vein. A microcatheter was inserted into the fusiform aneurysm under proximal blood flow control, and embolization was performed using coils and N-butyl-2-cyanoacrylate. Conclusion: Compared to surgical repair of ruptured occipital artery aneurysms, endovascular treatment appears to be safe, effective, minimally invasive, and rapid. Ruptured occipital artery aneurysms in NF1 patients can cause neck swelling and airway compression and should be recognized as a potentially lethal condition.

Endoscopic Cerebellar Necrosectomy for Space-occupying Cerebellar Infarction: A Case Report.

Suboccipital decompressive craniectomy with or without resection of necrosis is the preferred treatment for space-occupying cerebellar infarctions with neurological deterioration due to brainstem compression and obstructive hydrocephalus. We herein present our experience with treating space-occupying cerebellar infarctions successfully using endoscopic necrosectomy. A total of 27 patients were admitted to our hospital due to cerebellar infarctions between April 2021 and November 2023. Four patients required surgical interventions due to a drop in consciousness level or compression of the fourth ventricle and brainstem with acute hydrocephalus confirmed by a computed tomography (CT) scan. Three patients were performed endoscopic necrosectomy through a burr hole in a supine-lateral position. Removing most of the necrotic tissue was possible, resulting in early decompression of the fourth ventricle and brainstem. Endoscopic necrosectomy is less invasive than suboccipital decompressive craniectomy. An endoscopic necrosectomy can be performed for patients with unstable health conditions in a supine-lateral position. Therefore, endoscopic necrosectomy might be an effective method for treating patients with space-occupying cerebellar infarctions and poor general condition, although an objective evaluation of the extent and degree of removal is needed.

Temporal trends in microbial detection during the COVID-19 pandemic: Analysis of the Japan surveillance for Infection Prevention and Healthcare Epidemiology (J-SIPHE) database.

The impact of the COVID-19 pandemic on the incidence of microbial infections and other metrics related to antimicrobial resistance (AMR) has not yet been fully described. Using data from Japan Surveillance for Infection Prevention and Healthcare Epidemiology (J-SIPHE), a national surveillance database system that routinely collects clinical and epidemiological data on microbial infections, infection control practices, antimicrobial use, and AMR emergence from participating institutions in Japan, we assessed the temporal changes in AMR-related metrics before and after the start of the COVID-19 pandemic. We found that an apparent decrease in the incidence of microbial infections in 2020 compared with 2019 may have been driven primarily by a reduction in bed occupancy, although the incidence showed a constant or even slightly increasing trend after adjusting for bed occupancy. Meanwhile, we found that the incidence of Streptococcus pneumoniae dramatically decreased from April 2020 onward, probably due to stringent non-pharmaceutical interventions against COVID-19. Antimicrobial use showed a weak increasing trend, while the use of hand sanitiser at the included medical institutions increased by about 50% in 2020 compared with 2019.

Rate of blood culture submissions in Japan as an indicator of bloodstream infections.

Bloodstream infections can be missed if blood cultures are not submitted properly. We therefore examined the optimal number of blood cultures submitted to provide an indicator of the incidence of bloodstream infections in Japan. We analysed the number of blood cultures submitted per 1000 patient days as an indicator of the incidence of bloodstream infections, using data on blood cultures from 117 acute care hospitals in Japan. Kruskal-Wallis and Dunn tests were used to determine plateau numbers of blood cultures submitted per 1000 patient days. The median number of blood culture sets per 1000 patient days was 26.2, the median rate of solitary blood culture submissions was 8.0%, the median contamination rate was 1.3%, the median positivity rate including contaminants was 13.4%, and the median incidence of bloodstream infections per 1000 patient days was 2.8. The incidence of detected bloodstream infections increased with increasing blood culture submissions up to plateau around 45 submissions per 1000 patient days. In acute care hospitals in Japan, the incidence of BSI increased as the rate of blood culture submissions increased, but the positivity rate may reach a plateau at about 45 submissions per 1000 patient days, and this might be an indicator for the optimal number of blood culture submission in Japan.

Factors associated with anti-SARS-CoV-2 IgG antibody production in patients convalescing from COVID-19.

INTRODUCTION: Among patients with coronavirus disease 2019 (COVID-19), the factors that affect anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody production remain unclear. This study aimed to identify such factors among patients convalescing from COVID-19. METHODS: This study comprised patients who had been diagnosed with COVID-19 between January 1 and June 30, 2020 and gave consent for anti-SARS-CoV-2 spike protein antibody measurement using enzyme-linked immunosorbent assay during their acute and/or convalescent phases. Factors related to elevated antibody titers and the relationship between the days from disease onset and the development of antibody titers were assessed. RESULTS: A total of 84 participants enrolled in the study. Nineteen participants had antibody titers measured during the convalescent phase only, and 65 participants had antibody titers measured during the acute and convalescent phases. The antibody titers peaked in weeks 5 and 6. The stepwise multivariate log-normal analysis revealed that male sex (P = 0.04), diabetes mellitus (P = 0.03), and high C-reactive protein levels during the disease course (P < 0.001) were associated with elevated IgG antibodies. Glucocorticoid use was not associated with antibody titers. CONCLUSION: The study found that high values of maximum CRP levels during the acute phase, male sex, and diabetes mellitus were associated with elevated antibody titers. Antibody titers tended to be highest in the first 5 or 6 weeks after the onset of symptoms.

No SARS-CoV-2 RNA detected in the convalescent plasma of COVID-19 patients with different disease severity.

INTRODUCTION: Convalescent plasma transfusion (CPT), a potential therapy for coronavirus disease 2019 (COVID-19), requires strict quality control of the donor blood. Whether to confirm the disappearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (RNAemia) in convalescent donor blood or not is unclear. Reports recommending the proof of viral disappearance from the blood are controversial. Foreseeing CPT in treating COVID-19 patients in Japan, we investigated RNAemia in 100 convalescent donors with mild, moderate, and severe COVID-19. METHODS: Between April 30 and July 30, 2020, we measured RNAemia in the plasma samples of donors with resolved COVID-19. Data on patients' demographics, comorbidities, pneumonia, treatment, and real-time polymerase chain reaction results for SARS-CoV-2 were collected. Date of onset of initial symptoms or date of positive testing (for asymptomatic patients) were self-reported by the patients. Disease severity was defined as: no, mild, moderate oxygen demand, or severe (requiring mechanical ventilation). RESULTS: Of 100 donors (58 males [58.0%]; median age, 47 [range 22-69] years) screened as of July 30, 2020, 77 (77.0%); 19 (19.0%); and 4 (4.0%) had mild, moderate, and severe disease, respectively. Median time between onset and testing was 68.5 (range, 21-167) days. SARS-CoV-2 RNA was not detected in any of the plasma samples. CONCLUSION: RNAemia was not found in recovered COVID-19 patients at least 21, 27, and 57 days after the onset of mild, moderate, and severe symptoms. Our study may contribute to determining a suitable time for collecting convalescent plasma from COVID-19 patients and to future CPT use.

Clinical Epidemiology of Hospitalized Patients With Coronavirus Disease 2019 (COVID-19) in Japan: Report of the COVID-19 Registry Japan.

BACKGROUND: There is limited understanding of the characteristics of patients with coronavirus disease 2019 (COVID-19) requiring hospitalization in Japan. METHODS: This study included 2638 cases enrolled from 227 healthcare facilities that participated in the COVID-19 Registry Japan (COVIREGI-JP). The inclusion criteria for enrollment of a case in COVIREGI-JP are both (1) a positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test and (2) inpatient treatment at a healthcare facility. RESULTS: The median age of hospitalized patients with COVID-19 was 56 years (interquartile range [IQR], 40-71 years). More than half of cases were male (58.9%, 1542/2619). Nearly 60% of the cases had close contact to confirmed or suspected cases of COVID-19. The median duration of symptoms before admission was 7 days (IQR, 4-10 days). The most common comorbidities were hypertension (15%, 396/2638) and diabetes without complications (14.2%, 374/2638). The number of nonsevere cases (68.2%, n = 1798) was twice the number of severe cases (31.8%, n = 840) at admission. The respiratory support during hospitalization includes those who received no oxygen support (61.6%, 1623/2636) followed by those who received supplemental oxygen (29.9%, 788/2636) and invasive mechanical ventilation/extracorporeal membrane oxygenation (8.5%, 225/2636). Overall, 66.9% (1762/2634) of patients were discharged home, while 7.5% (197/2634) died. CONCLUSIONS: We identified the clinical epidemiological features of COVID-19 in hospitalized patients in Japan. When compared with existing inpatient studies in other countries, these results demonstrated fewer comorbidities and a trend towards lower mortality.

Midazolam Intoxication in a Premature Neonate.

PURPOSE: We report the case of a male neonate with a respiratory disorder who developed adverse cardiorespiratory symptoms after the continuous infusion of midazolam. METHODS: To clarify the cause of cardiogenic shock, we performed whole exome sequencing and screened relative single-nucleotide variants of 2 cytochrome P450 (CYP) isoforms, CYP3A4 and CYP3A5, which play a dominant role in the metabolic elimination of midazolam. We measured endogenous cortisol 6ß-hydroxylation clearance to phenotypically assess CYP3A activity. FINDINGS: The CYP3A activity level in the patient was significantly lower than the mean CYP3A activity level in healthy adults. Three intronic mutations in the CYP3A4 and CYP3A5 isoforms were detected in the patient. IMPLICATIONS: Our findings suggest that the midazolam concentration in plasma was achieved at above the steady-state concentration during continuous infusion used to sedate neonates receiving mechanical ventilatory support. Evaluation of the drug-metabolizing ability based on CYP3A might be useful if adverse electrophysiologic variables or the induction of tachycardia occur because of delayed elimination.

Validation of Cefazolin as Initial Antibiotic for First Upper Urinary Tract Infection in Children.

To validate the policy of administering cefazolin (CEZ) as a first-line antibiotic to children who are hospitalized with their first febrile urinary tract infection (UTI), we evaluated microbial susceptibility to CEZ and the efficacy of CEZ. The 75 enrolled children with febrile UTI were initially treated with CEZ. Switching CEZ was not required in 84% of the patients. The median fever duration, prevalence of bacteremia, prevalence of UTI caused by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli, and median duration of hospitalization were significantly higher in the CEZ-ineffective group. The risks of vesicoureteral reflux, indication of operation, and renal scarring are not increased, even when CEZ is ineffective as a first-line antibiotic. CEZ is effective in more than 80% of pediatric patients with their first febrile UTI, but it should be switched to appropriate antibiotics considering sepsis or the ESBL-producing Enterobacteriaceae pathogen, when fever does not improve within 72 hours.

EVI1, a target gene for amplification at 3q26, antagonizes transforming growth factor-ß-mediated growth inhibition in hepatocellular carcinoma.

EVI1 (ecotropic viral integration site 1) is one of the most aggressive oncogenes associated with myeloid leukemia. We investigated DNA copy number aberrations in human hepatocellular carcinoma (HCC) cell lines using a high-density oligonucleotide microarray. We found that a novel amplification at the chromosomal region 3q26 occurs in the HCC cell line JHH-1, and that MECOM (MDS1 and EVI1 complex locus), which lies within the 3q26 region, was amplified. Quantitative PCR analysis of the three transcripts transcribed from MECOM indicated that only EVI1, but not the fusion transcript MDS1-EVI1 or MDS1, was overexpressed in JHH-1 cells and was significantly upregulated in 22 (61%) of 36 primary HCC tumors when compared with their non-tumorous counterparts. A copy number gain of EVI1 was observed in 24 (36%) of 66 primary HCC tumors. High EVI1 expression was significantly associated with larger tumor size and higher level of des-γ-carboxy prothrombin, a tumor marker for HCC. Knockdown of EVI1 resulted in increased induction of the cyclin-dependent kinase inhibitor p15(INK) (4B) by transforming growth factor (TGF)-ß and decreased expression of c-Myc, cyclin D1, and phosphorylated Rb in TGF-ß-treated cells. Consequently, knockdown of EVI1 led to reduced DNA synthesis and cell viability. Collectively, our results suggest that EVI1 is a probable target gene that acts as a driving force for the amplification at 3q26 in HCC and that the oncoprotein EVI1 antagonizes TGF-ß-mediated growth inhibition of HCC cells.

Influence of genes suppressing interferon effects in peripheral blood mononuclear cells during triple antiviral therapy for chronic hepatitis C.

The levels of expression of interferon-stimulated genes (ISGs) in liver are associated with response to treatment with pegylated interferon (PEG-IFN) plus ribavirin (RBV). However, associations between the responses of ISGs to IFN-based therapy and treatment efficacy or interleukin-28B (IL28B) genotype have not yet been determined. Therefore, we investigated the early responses of ISGs and interferon-lambdas (IFN-λs) in peripheral blood mononuclear cells (PBMCs) during PEG-IFN/RBV plus NS3/4 protease inhibitor (PI) therapy. We prospectively enrolled 50 chronic hepatitis C patients with HCV genotype 1, and collected PBMCs at baseline, 8 and 24 h after the initial administration of PEG-IFN/RBV/PI. Levels of mRNAs for selected ISGs and IFN-λs were evaluated by real-time PCR. All 31 patients with a favorable IL28B genotype and 13 of 19 with an unfavorable genotype achieved sustained virological responses (SVR). Levels of mRNA for A20, SOCS1, and SOCS3, known to suppress antiviral activity by interfering with the IFN signaling pathway, as well as IRF1 were significantly higher at 8 h in patients with an unfavorable IL28B genotype than in those with a favorable one (P = 0.007, 0.026, 0.0004, 0.0006, respectively), especially in the non-SVR group. Particularly, the fold-change of IRF1 at 8 h relative to baseline was significantly higher in non-SVR than in SVR cases with an unfavorable IL28B genotype (P = 0.035). In conclusion, levels of several mRNAs of genes suppressing antiviral activity in PBMCs during PEG-IFN/RBV/PI differed according to IL28B genotypes, paralleling treatment efficacy.

Evaluation of hepatocellular carcinoma development in patients with chronic hepatitis C by EOB-MRI.

AIM: To evaluate the efficacy of ethoxibenzyl-magnetic resonance imaging (EOB-MRI) as a predictor of hepatocellular carcinoma (HCC) development. METHODS: Between August 2008 and 2009, we studied 142 hepatitis C virus-infected patients (male 70, female 72), excluding those with HCC or a past history, who underwent EOB-MRI in our hospital. The EOB-MRI index [liver-intervertebral disc ratio (LI)] was calculated as: (post-liver intensity/post-intervertebral disc intensity)/(pre-liver intensity/pre-intervertebral disc intensity). RESULTS: The median follow-up period was 3.1 years and the patients were observed until the end of the study period (31 December, 2012). In the follow-up period, HCC occurred in 21 patients. The cumulative occurrence rates were 2.1%, 9.1%, and 14.1% at 1, 2, and 3 years, respectively. Using the optimal cut-off value of LI 1.46, on univariate analysis, age, aspartate amino transferase (AST), α-fetoprotein (AFP) ≥ 10, albumin, total cholesterol, prothrombin time, platelets, and LI < 1.46 were identified as independent factors, but on multivariate analysis, LI < 1.46: risk ratio 6.05 (1.34-27.3, P = 0.019) and AFP ≥ 10: risk ratio 3.1 (1.03-9.35, P = 0.045) were identified as independent risk factors. LI and Fib-4 index have higher area under the receiver operating characteristic curves than other representative fibrosis evaluation methods, such as Forn's index and AST-to-platelet ratio index. CONCLUSION: LI is associated with the risk of HCC occurrence in hepatitis C patients. LI may be a substitute for liver biopsy when evaluating this risk and its combined use with Fib-4 is a better predictive method of HCC progression.

Serum interferon-gamma-inducible protein-10 concentrations and IL28B genotype associated with responses to pegylated interferon plus ribavirin with and without telaprevir for chronic hepatitis C.

AIM: Several studies have shown that high pretreatment concentrations of serum interferon-γ-inducible protein-10 (IP-10) are correlated with non-response to pegylated interferon (PEG-IFN) plus ribavirin (RBV) for chronic hepatitis C (CHC). However, there are few reports on their effect on the Asian population. METHODS: We enrolled 104 Japanese genotype 1 CHC individuals treated with PEG-IFN/RBV and 45 with PEG-IFN/RBV/telaprevir, and evaluated the impact of pretreatment serum IP-10 concentrations on their virological responses. RESULTS: The pretreatment serum IP-10 concentrations were not correlated with IL28B genotype. The receiver-operator curve analysis determined the cut-off value of IP-10 for predicting a sustained virological response (SVR) as 300 pg/mL. In multivariate analysis, the IL28B favorable genotype and IP-10 concentration of less than 300 pg/mL were independent factors for predicting SVR. In a subgroup of patients with the IL28B favorable genotype, the SVR rate was higher in the patients with IP-10 of less than 300 than in those with 300 pg/mL or more, whereas no patient with the IL28B unfavorable genotype and IP-10 of 300 pg/mL or more achieved SVR. Among the patients treated with PEG-IFN/RBV/telaprevir, low pretreatment concentrations of serum IP-10 were associated with a very rapid virological response, defined as undetectable HCV RNA at week 2 after the start of therapy. CONCLUSION: Pretreatment serum IP-10 concentrations are associated with treatment efficacy in PEG-IFN/RBV and with early viral kinetics of hepatitis C virus in PEG-IFN/RBV/telaprevir therapy.

Alterations of the SWI/SNF chromatin remodelling subunit-BRG1 and BRM in hepatocellular carcinoma.

BACKGROUND: The SWI/SNF chromatin remodelling complex, which contains either brahma-related gene-1 (BRG1) or brahma (BRM) as the catalytic ATPase, functions as a master regulator of gene expression. AIMS: To examine alterations of BRG1 and BRM in hepatocellular carcinoma (HCC). METHODS: We investigated DNA copy number aberrations in human HCC cell lines using a high-density oligonucleotide microarray. We determined DNA copy numbers and expression levels of BRG1 and BRM genes in primary HCC tumours, and conducted further searches for mutations in BRG1 and BRM genes. RESULTS: Homozygous deletion of the BRG1 gene was found in HCC cell line SNU398. Copy number losses of BRG1 and BRM genes were observed in 14 (26%) and 7 (13%) of 54 primary HCC tumours respectively. We found four somatic missense mutations in the BRG1 gene in two of 36 primary HCC tumours, but no mutations in BRM gene. Expression of BRM mRNA, but not BRG1 mRNA, was significantly reduced in primary HCC tumours, compared to non-tumour tissue counterparts. Immunohistochemical analyses of non-tumour liver tissues showed that BRM protein was expressed in hepatocytes and bile-duct epithelial cells, whereas BRG1 protein was expressed in bile-duct epithelial cells, but not in hepatocytes. BRM protein expression was lost in nine (22.5%) of 40 HCC tumours. Loss of BRM protein expression was significantly associated with poor overall survival. CONCLUSION: Reduced expression of BRM may contribute to the carcinogenesis of HCC. Although deletions and mutations in BRG1 gene were identified, the role of BRG1 in HCC tumourigenesis remains unclear.

Epigenetic silencing of miR-335 and its host gene MEST in hepatocellular carcinoma.

MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous silencers of target genes. Some tumor-suppressive miRNAs are known to be epigenetically silenced by promoter DNA methylation in cancer. In the present study, we aimed to identify miRNA genes that are silenced by DNA hypermethylation in hepatocellular carcinoma (HCC). We screened for miRNA genes with promoter DNA hypermethylation using a genome-wide methylation microarray analysis in HCC cells. It was found that miR-335, which is harbored within an intron of its protein-coding host gene, MEST, was downregulated by aberrant promoter hypermethylation via further methylation assays, including methylation-specific PCR, combined bisulfite and restriction analysis, bisulfite sequencing analysis and 5-aza-2'-deoxycytidine treatment. The expression levels of miR-335 significantly correlated with those of MEST, supporting the notion that the intronic miR-335 is co-expressed with its host gene. The levels of miR-335/MEST methylation were significantly higher in 18 (90%) out of 20 primary HCC tumors, compared to their non-tumor tissue counterparts (P<0.001). The expression levels of miR-335 were significantly lower in 25 (78%) out of 32 primary HCC tumors, compared to their non-tumor tissue counterparts (P=0.001). Furthermore, the expression levels of miR-335 were significantly lower in HCC tumors with distant metastasis compared to those without distant metastasis (P=0.02). In conclusion, our results indicate that expression of miR-335 is reduced by aberrant DNA methylation in HCC.

SOX2 identified as a target gene for the amplification at 3q26 that is frequently detected in esophageal squamous cell carcinoma.

SOX2 is a transcription factor with a high-mobility group DNA-binding domain that functions as a master regulator during embryogenesis and organogenesis. We investigated DNA copy number aberrations in esophageal squamous cell carcinoma (ESCC) cell lines using a high-density oligonucleotide microarray and found frequent amplification at the chromosomal region 3q26. The estimated extent of the minimal overlapping region of amplification was 1.3 Mb. This chromosomal region includes a single gene, SOX2. The SOX2 protein was overexpressed in cell lines in which the gene was amplified. Knockdown experiments showed that SOX2 promotes proliferation of ESCC cells. Genes potentially modulated by SOX2 were determined by expression array analyses combined with small interfering RNA cell-transfection studies. A copy number gain of SOX2 (>2-fold) was observed in 6 of the 40 primary ESCCs (15%). Immunohistochemical study revealed that expression of the SOX2 protein was significantly elevated in 62 of the 89 ESCC tumors (70%), compared with their nontumorous counterparts, and that upregulated expression of SOX2 was associated with poor differentiation of ESCC. Our results suggest that SOX2 is likely to be a target of the 3q26 amplification and may therefore be involved in the development or progression of ESCC.

PEG10 is a probable target for the amplification at 7q21 detected in hepatocellular carcinoma.

DNA copy number aberrations in human hepatocellular carcinoma (HCC) cell lines were investigated using a high-density oligonucleotide microarray, and a novel amplification at the chromosomal region 7q21 was detected. Molecular definition of the amplicon indicated that PEG10 (paternally expressed gene 10), a paternally expressed imprinted gene, was amplified together with CDK14 (cyclin-dependent kinase 14; previously PFTAIRE protein kinase 1, PFTK1) and CDK6 (cyclin-dependent kinase 6). An increase in PEG10 copy number was detected in 14 of 34 primary HCC tumors (41%). PEG10, but not CDK14 or CDK6, was significantly overexpressed in 30 of 41 tumors (73%) from HCC patients, compared with their nontumorous counterparts. These results suggest that PEG10 is a probable target, acting as a driving force for amplification of the 7q21 region, and may therefore be involved in the development or progression of HCCs.

Steatosis and hepatic expression of genes regulating lipid metabolism in Japanese patients infected with hepatitis C virus.

PURPOSE: Steatosis is a histological finding associated with the progression of chronic hepatitis C. The aims of this study were to elucidate risk factors associated with steatosis and to evaluate the association between steatosis and hepatic expression of genes regulating lipid metabolism. METHODS: We analyzed 297 Japanese patients infected with hepatitis C virus and a subgroup of 100 patients who lack metabolic factors for steatosis. We determined intrahepatic mRNA levels of 18 genes regulating lipid metabolism in these 100 patients using real-time reverse transcription-polymerase chain reaction. Levels of peroxisome proliferator-activated receptor alpha and sterol regulatory element-binding protein 1 proteins were assessed by immunohistochemistry. RESULTS: Steatosis was present in 171 (57%) of 297 patients. The presence of steatosis was independently associated with a higher body mass index, higher levels of gamma-glutamyl transpeptidase and triglyceride, and a higher fibrosis stage. Steatosis was present in 43 (43%) of 100 patients lacking metabolic factors. Levels of mRNA and protein of peroxisome proliferator-activated receptor alpha, which regulates beta-oxidation of fatty acid, were lower in patients with steatosis than in patients without steatosis. CONCLUSIONS: These findings indicate that impaired degradation of lipid may contribute to the development of hepatitis C virus-related steatosis.