Search

Supplementary Figure 5 from ARF Suppresses Tumor Angiogenesis through Translational Control of VEGFA mRNA

Supplementary Figure Legends 1-5 from ARF Suppresses Tumor Angiogenesis through Translational Control of VEGFA mRNA

Supplementary Figure 3 from ARF Suppresses Tumor Angiogenesis through Translational Control of VEGFA mRNA

Supplementary Figure 1 from ARF Suppresses Tumor Angiogenesis through Translational Control of VEGFA mRNA

Supplementary Figure 6 from ARF Suppresses Tumor Angiogenesis through Translational Control of VEGFA mRNA

Supplementary Figure 4 from ARF Suppresses Tumor Angiogenesis through Translational Control of VEGFA mRNA

Vascular endothelial growth factor A (VEGFA) is a specific mitogen for vascular endothelial cells that plays a critical role in cancer neoangiogenesis. Here, we report that the nucleolar tumor suppressor p19ARF suppresses VEGFA expression, acting at the level of mRNA translation without affecting the transcription of the VEGFA gene. Translational repression of VEGFA mRNA by p19ARF does not require p53, a major target of the ARF tumor suppressor pathway, but instead correlates with binding to nucleophosmin/B23. Maintaining VEGFA expression relies on nucleophosmin/B23, and downregulating this protein by RNAi or p19ARF leads to translational repression of VEGFA. p19ARF inhibits VEGFA-dependent tumor angiogenesis in nude mice. Additionally, p14ARF expression and microvessel density are inversely correlated in human colon carcinomas. Taken together, our results define a mechanism by which the ARF tumor suppressor targets the translational repression of specific oncogenes during neoplastic transformation. Cancer Res; 70(11); 4749–58. ©2010 AACR.

Supplementary Figure 2 from ARF Suppresses Tumor Angiogenesis through Translational Control of VEGFA mRNA

A 70-year-old man was diagnosed with hepatocellular carcinoma (HCC) with portal vein invasion and lung metastases, for which atezolizumab plus bevacizumab (ATZ/BEV) was initiated. After two months, computed tomography revealed tumor growth accompanied by ascites, right ventricular invasion, exacerbation of the lung metastases, and main portal vein invasion. However, continuation of ATZ/BEV caused remarkable size reductions in all lesions, finally resulting in the disappearance of the vascular invasion and lung metastases after nine cycles of treatment. The tumor growth was considered to reflect pseudoprogression, which is difficult to distinguish from hyperprogression. We herein report a remarkable HCC case of pseudoprogression on ATZ/BEV.

Plexin domain containing 2 (PLXDC2) is expressed in endothelial cells of tumor stroma, neural progenitor cells, and pluripotent stem cells, but their respective tissue expression pattern is not fully understood. In this study, we investigated the expression pattern of PLXDC2 in human hepatocellular carcinoma (HCC) tissues using a highly specific anti-PLXDC2 rabbit monoclonal antibody, which was recently developed. PLXDC2 was expressed in human HCC tissues including HCC cells, tumor vascular endothelial cells, and some infiltrating cells. The developed anti-PLXDC2 antibody allowed for highly specific and low background staining. Based on these current findings of PLXDC2 expression in human HCC tissues, the window may now be open to explore the role of PLXDC2 in human HCC.

Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary disease that causes a decline of kidney function that progresses in relation to the enlargement of kidney cysts.1 Tolvaptan, a selective vasopressin V2-receptor antagonist, suppresses the production of cAMP, which is expected to be beneficial for suppressing the growth of renal cysts in patients with ADPKD.2 The effectiveness of tolvaptan was demonstrated in a phase III international clinical trial (TEMPO 3:4 trial)3 and its use is recommended in the guidelines.4 Elevation of liver enzymes was reported in 4.9% of patients who received tolvaptan in the TEMPO 3:4 trial.3 Eight of 135 Japanese patients in the TEMPO 3:4 trial had a more than 3-fold increase above the upper limit of normal (ULN) in aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels; however, these levels recovered after the discontinuation of tolvaptan.5 Thus far, tolvaptan-associated liver enzyme elevation has recovered after cessation or dose reduction, and no cases of tolvaptan-associated severe liver failure have been reported. We herein report a patient with ADPKD who underwent liver transplantation for tolvaptan-associated acute liver failure.

Aim Clinical evaluations are generally used to verify the effectiveness of detoxification treatments for alcohol dependence, but new objective biomarkers are essential for accurate diagnosis. We aim to assess the accuracy of % carbohydrate-deficient transferrin (CDT) in a cohort of Japanese patients admitted to a psychiatric hospital specializing in alcohol dependence. In addition, we investigated the kinetics of %CDT during alcohol moderation or cessation. Methods The study cohort consisted of 126 alcohol-dependent patients. The levels of serum %CDT were assessed by the N Latex CDT direct immuno-nephelometric assay. Results Alcohol consumption was significantly correlated with %CDT. The only independent predictive factor of alcohol consumption was %CDT, with glutamyl transpeptidase (GGT) and albumin-bilirubin (ALBI) score proving insufficient. The cut-off value of %CDT was 1.9 % with high sensitivity and specificity in detecting alcohol abstinence beyond 30 days (68.6% sensitivity, 91.8% specificity) and excessive alcohol drinking (77.9% sensitivity, 77.1% specificity). %CDT levels were significantly decreased at 30-days of abstinence when compared with baseline. Notably, %CDT values were significantly changed even in the light alcohol drinking cohort (P = 0.0009), whereas GGT levels were not significantly changed. Conclusions Our results indicate that %CDT is an accurate and specific biomarker of alcohol consumption and is useful in detecting alcohol abstinence even in a low alcohol intake patient cohort. These results suggest that %CDT may be a useful objective biomarker of chronic alcohol abuse during clinical treatment for alcoholism. This article is protected by copyright. All rights reserved.

Despite the development of several therapeutic options, the prognosis of pancreatic cancer remains poor. One reason for this is the difficulty of diagnosing the disease at an early stage. For example, carbohydrate antigen (CA) 19-9, which is the most widely used biomarker for pancreatic cancer, cannot be used to detect the disease at early stages. Some studies have attempted to find novel biomarkers for pancreatic cancer. The aim of the present study was to find a novel diagnostic biomarker for pancreatic ductal adenocarcinoma (PDAC) in urine exosomes. Exosomes were isolated from urine and serum samples of patients with PDAC and control subjects, or culture media of cancer cell lines. MicroRNAs (miRNAs) were purified from exosomes. Novel biomarker candidates for PDCA were identisfied from urine exosome miRNA using expression profiling, and validated in a larger number of samples using 3D digital PCR. The results of a preliminary analysis of nine PDAC and seven control subjects revealed that the miR-3940-5p/miR-8069 ratio in urine exosomes was elevated in the patients with PDAC. Experiments using cultured cancer cell lines revealed that the elevation of the miR-3940-5p/miR-8069 ratio was specific for PDAC. Furthermore, the elevation of the miR-3940-5p/miR-8069 ratio in exosomes tended to be higher in the urine than in the serum of patients with PDAC. Validation experiments on 43 PDAC, 12 chronic pancreatitis and 25 control subjects demonstrated that the miR-3940-5p/miR-8069 ratio in urine exosomes was elevated in PDAC at a relatively early stage of the disease. When this ratio was used in combination with CA19-9 for the diagnosis of PDAC, the sensitivity and positive predictive value improved to 93.0 and 78.4%, respectively, when either of them was positive. Additionally, the positive predictive value reached 100% when both were positive. The negative predictive value also improved to 89.7% when both were negative. The miR-3940-5p/miR-8069 ratio in urine exosomes may be useful as a tool for the diagnosis of PDAC, particularly when used in combination with CA19-9.

AIM Hepatocellular carcinoma (HCC) patients with sarcopenia have a poor survival, but there are no predictive markers for survival relating to muscle mass and liver function. Therefore, we investigated whether the ratio between estimated glomerular filtration rates of serum creatinine (Scre) and serum cystatin C (Scys) (eGFRcre/eGFRcys) can be used as a predictive marker of survival in HCC patients. METHODS First, the correlation between Scre/Scys ratio and muscle mass was examined in 50 patients with chronic liver disease. Second, a change in Scre/Scys ratio relating to liver function was investigated in cirrhotic rats. Finally, the relationship between the eGFRcre/eGFRcys ratio and survival was assessed in 86 HCC patients. RESULTS The Scre/Scys ratio was correlated with skeletal muscle mass index (r = 0.331, P = 0.019) and psoas muscle area index (r = 0.397, P = 0.004) in chronic liver disease patients. In cirrhotic rats, Scre and Scre/Scys ratio were decreased corresponding with liver function. Thirty-five of 86 HCC patients died within the average follow-up period of 35 months. The patients with an eGFRcre/eGFRcys ratio

Hepatitis E virus subtype 3f (HEV-3f) strains are usually isolated in Europe and Thailand. Recently, HEV-3f strains were detected from six acute hepatitis E patients in Japan, none of whom had a history of travel to endemic areas. We inferred the origin and transmission route of the six HEV-3f strains. A time-scaled phylogenetic tree of the six strains with reference strains was constructed using a Bayesian statistical inference framework. The time-scaled tree indicated that the six strains independently derived from similar European strains between 2008 and 2014. The pattern suggested recent inflow of multiple HEV-3f strains from Europe to Japan. Japan imports a substantial amount of pork from European countries every year. The emergence of acute hepatitis cases caused by HEV-3f strains in Japan, in patients with no history of travel abroad, might be influenced by the increased opportunities to consume pork products imported from European countries.

Chronic liver disease patients often have complications, such as hepatocellular carcinoma (HCC) and acute bacterial infection. Model for end-stage liver disease and Child-Pugh scores are useful prognostic factors for chronic liver diseases but not for all chronic conditions, such as HCC. Our investigative aim targeted the prognostic abilities of neutrophil gelatinase-associated lipocalin (NGAL) in rat and human chronic liver diseases. Blood NGAL levels were measured by enzyme-linked immunosorbent assay in rats with cirrhosis and 96 patients with chronic liver disease and HCC. We examined the correlation between blood NGAL levels and liver functions as well as survival. In our rat model, liver NGAL expression was assessed by immunostaining, real-time quantitative polymerase chain reaction, and immunoblot. In rats with cirrhosis, blood NGAL levels were continuously and significantly elevated in the deceased group and were significantly correlated with liver functions. Liver NGAL, toll-like receptor 4, and interleukin-6 levels were increased in the deceased group compared to the survival group. Blood NGAL levels were significantly correlated with liver NGAL levels, indicating blood NGAL was derived from the liver. In patients with chronic liver disease, blood NGAL levels were associated with liver function and renal function. Blood NGAL levels were significantly increased in patients with chronic liver disease with HCC compared to without HCC. For the survival group, 38 out of 96 patients were dead in the average follow-up period of 9.9 months. The patients with blood NGAL ≤119 ng/mL had significantly longer rates of survival compared to patients with blood NGAL >119 ng/mL. Conclusion: Blood NGAL predicts the survival rate in rat and human chronic liver diseases. Our findings suggest blood NGAL may be prognostic of survival in chronic liver diseases complicated by HCC. (Hepatology Communications 2017;1:946-956).

Background Much evidence has demonstrated the influence of Hepatitis B virus (HBV) mutations on the clinical course of HBV infection. As large (L) protein plays a crucial role for viral entry, we hypothesized that mutations in the pre-S1 promoter region might affect the expression of L protein and subsequently change the biological characters of virus. Methods Patients infected with genotype C HBV were enrolled for analysis. HBV DNA sequences were inserted into a TA cloning vector and analyzed. To evaluate the effects of mutations in the pre-S1 promoter region, promoter activity and the expression of mRNA and L protein were analyzed using HepG2 cells. Results In total, 35 patients were enrolled and 13 patients (37.1%) had a single base substitution in the pre-S1 promoter region; the most frequent substitution was a G-to-A substitution at the 2765th base (G2765A) in the Sp1 region. The HBV viral load showed a negative correlation with the substitution ratio of the Sp1 region or G2765A (r = − 0.493 and − 0.473, respectively). Among those with a viral load ≤5.0 log IU/ml, patients with the G2765A substitution showed a significantly lower HBV viral load than those with the wild-type sequence. HepG2 cells transfected with the G2765A substitution vector showed reduced luciferase activity of the pre-S1 promoter, as well as reduced expression of pre-S1 mRNA and L protein. Furthermore, the G2765A substitution greatly reduced the L protein expression level of vector-produced virus particles. Conclusion G2765A substitution in the pre-S1 promoter reduced the expression of L protein and resulted in a low viral load and less severe disease in chronic HBV infections.

Aim To evaluate the clinical and molecular characteristics of hepatitis E virus (HEV) infection in Mie Prefecture, Japan, from 2004 through 2018. Methods The clinical information of hepatitis E cases was collected from 21 medical institutions in Mie Prefecture. The nucleotide sequences of infecting HEV strains were determined for cases with available serum samples. The origins or transmission routes were inferred from phylogenetic analyses of the nucleotide sequences. Results Fifty-three patients were diagnosed with HEV infection. The number of cases increased each year through 2012 and then decreased. Analyses of the clinical characteristics of the cases indicated that even mild cases were detected in the latter 10 years of the study. Nucleotide sequence analyses were undertaken on 38 of the 53 cases. The HEV subtype 3e (HEV-3e) strains identified for 13 cases were closely related to a swine HEV-3e strain that was isolated from the liver of a pig bred in Mie Prefecture. The number of cases infected with the indigenous Mie HEV-3e strains increased until 2012 but have not been reported since 2014. In the latter half of the study, cases involving various HEV strains of different genotypes and subtypes emerged. Conclusions The disappearance of indigenous Mie HEV-3e strains appeared to be the primary cause for the decrease in hepatitis E cases in Mie Prefecture. The disappearance might have been associated with improved hygienic conditions on pig farms or the closure of contaminated farms. The results suggest that indigenous HEV strains can be eradicated by appropriate management.

Objective We evaluated the modified diagnostic criteria for disseminated intravascular coagulation (DIC), which was published by the Japanese Society of Thrombosis and Hemostasis (JSTH), in 274 suspected DIC patients with hematopoietic injury. Materials and methods The diagnoses of the patients were as follows: DIC (n = 125); pre-DIC (n = 42) and non-DIC (n = 107). The efficacy of the diagnostic criteria for DIC was evaluated using a receiver operating characteristic (ROC) analysis. Results The area under the curve (ARC) and odd's ratio for the global coagulation test (GCT) scores in the diagnosis of “DIC” were high, while those for the diagnosis of “DIC and pre-DIC” were low, suggesting that the addition of antithrombin (AT) and soluble fibrin (SF)/thrombin antithrobin complex (TAT) was required to diagnose “DIC and pre-DIC”. Although the addition of the AT and SF/TAT values to the GCT did not increase its ability to predict a poor outcome, the JSTH's modified diagnostic criteria scores were correlated with the odds ratio for death. Discussion and conclusion The JSTH's modified diagnostic criteria for DIC, which included the GCT score, and the AT, and TAT/SF values, were useful for diagnosing DIC and pre-DIC, and predicting a poor outcome.

The aim of this study was to clarify the relationships among psychometric testing results, blood ammonia (NH3) levels, electrolyte abnormalities, and degree of inflammation, and their associations with the development of overt hepatic encephalopathy (HE) in liver cirrhosis (LC) patients. The relationships between covert HE and blood NH3, sodium (Na), and C-reactive protein (CRP) were examined in 40 LC patients. The effects of elevated NH3, hyponatremia, and elevated CRP on the development of overt HE were also investigated. The covert HE group had significantly lower serum Na levels and significantly higher serum CRP levels. During the median observation period of 11 months, 10 patients developed overt HE, and the results of multivariate analysis showed that covert HE and elevated blood NH3 were factors contributing to the development of overt HE. Electrolyte abnormalities and mild inflammation are involved in the pathogenesis of HE. Abnormal psychometric testing results and hyperammonemia are linked to subsequent development of overt HE.

Alcoholic liver disease (ALD) has become one of the most critical health problems in many countries, including Japan. Liver injury in ALD ranges from steatosis and steatohepatitis to fibrosis, cirrhosis, and hepatocellular carcinoma. Many factors are thought to contribute to the development and progression of ALD, particularly insulin resistance, generation of reactive oxygen species during alcohol metabolism, adipokines from visceral adipose tissue, and endotoxin derived from the gut. Although the pathogenesis of ALD has been widely investigated, the precise mechanisms are yet to be elucidated and many questions remain. This article reviews the possible mechanisms for the development of ALD identified to date.

BackgroundCopper (Cu) and zinc (Zn) are essential nutrients and cofactors of enzymatic reactions with their binding partner. Metallothionein (MT) plays an important role in protecting against heavy metals and oxidative injury, however it may also portend drug resistance and a worse prognosis for hepatocellular carcinoma (HCC) patients. The aim of this study was to determine the amount of Cu, Zn, Cu/Zn and MT in evaluating a group of patients with HCC, including those treated with lenvatinib.MethodsWe enrolled 175 patients with HCC (139 men, 36 women; mean age 71.1 years; hepatitis C virus n = 85, hepatitis B virus n = 19, hepatitis C virus and hepatitis B virus n = 2, non-alcoholic steatohepatitis n = 39, alcohol n = 25, others n = 5; Child-Pugh A n = 141, Child-Pugh B n = 30, Child-Pugh C n = 4; Barcelona clinic liver cancer (BCLC) stage 0 n = 38, stage A n = 56, stage B n = 39, stage C n = 38, stage D n = 4). We evaluated the associations between Cu, Zn and MT. The study outcome was liver cancer-specific survival. Moreover, we treated 12 HCC patients with lenvatinib and investigated the changes in MT during lenvatinib therapy.ResultsThe serum level of Cu was positively correlated with alanine aminotransferase and the BCLC stage. The serum level of Zn decreased concordant with liver disease progression. Patients with a Cu/Zn ratio≥0.999 had significantly improved rates of survival when compared to patients with a Cu/Zn ratioConclusionsThe Cu/Zn ratio could serve as a useful predictive marker for survival in cases of HCC. MT levels increased in HCC patients receiving lenvatinib therapy, and maybe a predictor of reduced survival.

Senescent cells accumulate in tissues during aging and are considered to underlie several aging‐associated phenotypes and diseases. We recently reported that the elimination of p19ARF‐expressing senescent cells from lung tissue restored tissue function and gene expression in middle‐aged (12‐month‐old) mice. The aging of lung tissue increases the risk of pulmonary diseases such as emphysema, and cellular senescence is accelerated in emphysema patients. However, there is currently no direct evidence to show that cellular senescence promotes the pathology of emphysema, and the involvement of senescence in the development of this disease has yet to be clarified. We herein demonstrated that p19ARF facilitated the development of pulmonary emphysema in mice. The elimination of p19ARF‐expressing cells prevented lung tissue from elastase‐induced lung dysfunction. These effects appeared to depend on reduced pulmonary inflammation, which is enhanced after elastase stimulation. Furthermore, the administration of a senolytic drug that selectively kills senescent cells attenuated emphysema‐associated pathologies. These results strongly suggest the potential of senescent cells as therapeutic/preventive targets for pulmonary emphysema.

Hepatocellular carcinoma (HCC) patients with sarcopenia have a poor survival, but there are no predictive markers for survival relating to muscle mass and liver function. Therefore, we investigated whether the ratio between estimated glomerular filtration rates of serum creatinine (Scre) and serum cystatin C (Scys) (eGFRcre/eGFRcys) can be used as a predictive marker of survival in HCC patients.First, the correlation between Scre/Scys ratio and muscle mass was examined in 50 patients with chronic liver disease. Second, a change in Scre/Scys ratio relating to liver function was investigated in cirrhotic rats. Finally, the relationship between the eGFRcre/eGFRcys ratio and survival was assessed in 86 HCC patients.The Scre/Scys ratio was correlated with skeletal muscle mass index (r = 0.331, P = 0.019) and psoas muscle area index (r = 0.397, P = 0.004) in chronic liver disease patients. In cirrhotic rats, Scre and Scre/Scys ratio were decreased corresponding with liver function. Thirty-five of 86 HCC patients died within the average follow-up period of 35 months. The patients with an eGFRcre/eGFRcys ratio 1.26 had significantly longer rates of survival compared to patients with an eGFRcre/eGFRcys ratio ≥1.26 (28.8 vs. 18.5 months, P = 0.001). Using multivariate Cox regression analyses, the patient-related eGFRcre/eGFRcys ratio (hazard ratio [HR], 4.178; P = 0.007), as well as the tumor-related factors α-fetoprotein (HR, 1.000; P 0.001) and Barcelona Clinic Liver Cancer stage (HR, 2.589; P 0.001), were independent predictors of survival.The Scre/Scys ratio is associated with muscle mass and liver function. Furthermore, the eGFRcre/eGFRcys ratio could serve as a useful predictive marker for survival of HCC.

Resveratrol is a natural polyphenol found in a wide variety of plants, including grapes, berries, and peanuts. Resveratrol can modulate a wide spectrum of molecular targets, including those involved in cancer signaling pathways. Here, we evaluated the role of resveratrol in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and examined the molecular mechanisms in the human hepatocellular carcinoma cell line HepG2. We used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay to assess cell viability, flow cytometry to analyze cell cycle and apoptosis, and immunoblotting to detect protein expression. Resveratrol decreased cell viability at a concentration of 100 μmol/l or higher. At a concentration of 50 μmol/l, resveratrol induced S phase arrest of the cell cycle without apoptosis. In addition, phospho-AMPK increased significantly in a dose-dependent manner. Resveratrol was found to synergistically augment TRAIL-induced apoptosis. The rates of early apoptosis were 3.4, 9.6, and 49.6% on treatment with 50 μmol/l resveratrol, 10 ng/ml TRAIL, and both reagents, respectively. Resveratrol significantly downregulated the expression of survivin in a dose-dependent manner. In conclusion, we found that that resveratrol could augment TRAIL sensitivity by downregulating survivin. These results suggest that combination resveratrol with TRAIL may be an effective new strategy for the treatment of hepatocellular carcinoma.

The innate immune system plays an important role as the first line of defense against many types of microbes. Accumulating reports suggest that human β-defensins (hBDs) are expressed by and have certain roles in some cancer cells. In this study, we investigated the roles of hBD-3 in colon cancer cells. The expression of hBD-3 was examined by reverse transcriptase-polymerase chain reaction analysis of colon cancer cell lines and immunohistochemical staining of colon cancer tissues. The effect of hBD-3 on proliferation of colon cancer was assessed using the MTT assay and a real-time cell analyzer, and the effect of hBD-3 on the migration of colon cancer cells was also examined. The results showed that hBD-3 is not expressed in colon cancer cells but is produced by tumor-infiltrating monocytes. Migration of colon cancer cells was significantly inhibited by hBD-3 in a dose-dependent manner, although proliferation of colon cancer cells was not affected by administration of hBD-3. Moreover, reduced expression of metastasis-associated 1 family, member 2 (MTA2) mRNA in colon cancer cells was associated with exposure to hBD-3. In conclusion, progression of colon cancer was inhibited by hBD-3 in a paracrine fashion. Therefore, hBD-3 may be a potent new agent for treating colon cancer.

Although several therapeutic options are available for hepatocellular carcinoma (HCC), the outcome is still very poor. One reason is the complexity of signal transduction in the pathogenesis of HCC. The aim of this study was to iden- tify new HCC-related genes and to investigate the functions of these genes in the pathogenesis and progression of HCC. Whole genomes of 15 surgically resected HCC specimens were examined for copy number alterations with compara- tive genomic hybridization. Gene expression was compared between HCC and normal liver tissues. The roles of the new genes in the progression of HCC were studied using cultured cell lines. Copy number gain in chromosome 8q was detected in 53% of HCC tissues examined. The gene that coded for collagen triple helix repeat containing 1 (CTHRC1), located at chromosome 8q22.3, was overexpressed in HCC compared with normal or liver cirrhosis tissues and identified as a new HCC-related gene. CTHRC1 deletion with short hairpin RNA significantly reduced proliferation, migration and invasion of HepG2 and Huh7 cells. In addition, mRNA of integrins β-2 and β-3 was downregulated, with deletion of CTHRC1 in these cells. Immunohistochemical staining on resected HCC tissues showing positive staining areas for CTHRC1 was significantly greater in poorly-differentiated HCC compared with well-differentiated HCC. Moreover, some cases showed strong staining for CTHRC1 in invasive areas of HCC. CTHRC1 has the potential to be a new biomarker for the aggressive HCC, and to be a new therapeutic target in treating HCC.

Aim The spleen is not believed to contribute to hematopoiesis in healthy adults. However, several reports have demonstrated that the spleen in adults contains a large number of hematopoietic stem/progenitor cells (HSC). Although splenectomy increases platelet and leukocyte counts, the effects of splenectomy on circulating HSC have not been elucidated. In this study, we evaluated the association between the number of circulating HSC and splenectomy in patients with hepatitis C virus (HCV)-associated liver cirrhosis (LC). Methods In 48 patients with various stages of HCV-associated chronic liver disease and seven patients with LC who underwent splenectomy, and 10 healthy volunteers, we determined the numbers of circulating CD34+ cells and colony-forming unit culture by flow cytometry and methylcellulose culture, respectively. Plasma stromal cell-derived factor-1α (SDF-1α) concentrations were measured using an enzyme-linked immunosorbent assay. Results The numbers of circulating CD34+ cells and colony-forming unit culture decreased but the plasma SDF-1α concentration increased with the progression of liver disease. There was an inverse correlation between the number of circulating HSC and the plasma SDF-1α concentration. The numbers of circulating HSC and platelets were determined before and after splenectomy in seven patients with LC. In these patients, the numbers of circulating HSC and platelets increased significantly after splenectomy and the enhancing effect persisted for a long time. Conclusion Our data suggest that the spleen plays an important role in modulating HSC dynamics in patients with HCV-associated chronic liver disease. Our results also imply that splenectomy may improve liver function in patients with LC.

Partial splenic embolization (PSE) or splenectomy is widely performed to increase platelet counts for interferon (IFN) therapy. The aim of the present study was to evaluate the long-term effects of splenectomy and subsequent IFN therapy in patients with hepatitis C virus (HCV)-related liver cirrhosis (LC). The present study included 19 patients with HCV-related LC who underwent splenectomy for thrombo-cytopenia caused by hypersplenism. IFN therapy was performed in all 19 patients. The effects of splenectomy and subsequent IFN therapy on peripheral blood counts, liver function, carcinogenesis and survival rates were evaluated. Splenectomy was safely performed in all patients without major complications with the exception of portal thrombosis, which, however, it did not affect liver function when treated appropriately. Thrombocytopenia improved and IFN therapy could be performed in all the patients. A sustained virological response (SVR) was not observed in patients with genotype 1 although it was observed in 75% of patients with genotype 2. Due to severe side effects, five patients did not undergo scheduled IFN therapy. Over 5 years, the mean platelet number increased from 5.2 x 10(4) to 16.8 x 10(4)/mm3 (P

Aim To characterize hepatitis E in Mie prefecture and to investigate whether raw pig liver sold as food in Mie is contaminated with hepatitis E virus (HEV) strains similar to those recovered from patients. Methods Seventeen patients with sporadic acute hepatitis E treated from 2004 to 2012 were studied. A total of 243 packages of raw pig liver from regional grocery stores were tested for the presence of HEV RNA. The partial genomic sequences of human and swine HEV isolates were determined and subjected to the phylogenetic analyses. Results The HEV isolates recovered from the 17 patients segregated into genotype 3 (n = 15) and genotype 4 (n = 2), and 15 genotype 3 isolates further segregated into 3e (n = 11) and 3b (n = 4). Pig liver specimens from 12 (4.9%) of the 243 packages had detectable HEV RNA. All 12 swine HEV isolates were grouped into genotype 3 (3a or 3b). Although no 3e strains were isolated from pig liver specimens, two 3b swine strains were 99.5–100% identical to two HEV strains recovered from hepatitis patients, within 412-nt partial sequences. Conclusion The 3e HEV was prevalent among hepatitis E patients. HEV RNA was detected in approximately 5% of pig liver sold as food. The presence of identical HEV strains between hepatitis patients and pig liver indicated that pigs play an important role as reservoirs for HEV in humans in Mie. Further studies are needed to clarify the source of 3e HEV in the animal and environmental reservoirs.

A 67-year-old male living in Tsu city, Mie prefecture, Japan was referred to our hospital for further examination of acute liver injury and was diagnosed as having clinical hepatitis E virus (HEV) infection in January 2010. The HEV strain (HE-JA11-1701) isolated from the patient belonged to genotype 3 and European-type subgenotype 3e. It was presumed that the patient had been infected from a wild boar (Sus scrofa leucomystax) because he consumed meat/viscera from a wild boar that he had captured himself as a hunter approximately 2 months before disease onset. A specimen of the boar meat/viscera that the patient had ingested was not available. However, the HE-JA11-1701 strain was 99.8% identical within the 412-nucleotide sequence of the open reading frame 2 region to a HEV strain (JBOAR012-Mie08) that had been recovered from a wild boar captured near the patient's hunting area in 2008. A phylogenetic analysis confirmed that the two HEV strains had a close genetic relationship and were segregated into subgenotype 3e, supported by a high bootstrap value of 99%. Of note, the HE-JA11-1701 and JBOAR012-Mie08 strains were remotely related to the 3e strains reported in Japan and European countries, with a nucleotide difference of 7.9-13.9%, reinforcing the uniqueness of the 3e strains obtained in the present study. These results strongly support our speculation that the patient developed acute hepatitis E via consumption of HEV-infected boar meat/viscera. Genetic analyses of HEV strains are useful for tracing infectious sources in sporadic cases of acute hepatitis E.

Senescent cells accumulate in many tissues as animals age and are considered to underlie several aging-associated pathologies. The tumor suppressors p19ARF and p16INK4a, both of which are encoded in the CDKN2A locus, play critical roles in inducing and maintaining permanent cell cycle arrest during cellular senescence. Although the elimination of p16INK4a-expressing cells extends the life span of the mouse, it is unclear whether tissue function is restored by the elimination of senescent cells in aged animals and whether and how p19ARF contributes to tissue aging. The aging-associated decline in lung function is characterized by an increase in compliance as well as pathogenic susceptibility to pulmonary diseases. We herein demonstrated that pulmonary function in 12-month-old mice was reversibly restored by the elimination of p19ARF-expressing cells. The ablation of p19ARF-expressing cells using a toxin receptor-mediated cell knockout system ameliorated aging-associated lung hypofunction. Furthermore, the aging-associated gene expression profile was reversed after the elimination of p19ARF. Our results indicate that the aging-associated decline in lung function was, at least partly, attributed to p19ARF and was recovered by eliminating p19ARF-expressing cells.

Background Helicobacter pylori (H. pylori) infection and eradication therapy have been known to influence gastric ghrelin and leptin secretion, which may lead to weight gain. However, the exact relationship between plasma ghrelin/leptin levels and H. pylori infection has remained controversial. The aim of this study was to investigate plasma ghrelin and leptin levels in H. pylori-positive and -negative patients, to compare the two levels of the hormones before and after H. pylori eradication, and to examine the correlation between body mass index (BMI) and active ghrelin or leptin levels, as well as that between atrophic pattern and active ghrelin or leptin levels. Methods Seventy-two H. pylori-positive patients who underwent upper gastrointestinal endoscopy, 46 diagnosed as having peptic ulcer and 26 as atrophic gastritis, were enrolled. Control samples were obtained from 15 healthy H. pylori-negative volunteers. The extent of atrophic change of the gastric mucosa was assessed endoscopically. Body weight was measured and blood was collected before and 12 weeks after H. pylori eradication therapy. Blood samples were taken between 8 and 10 AM after an overnight fast. Results Plasma ghrelin levels were significantly lower in H. pylori-positive patients than in H. pylori-negative patients. In particular, plasma active ghrelin levels were significantly lower in patients with gastritis compared with patients with peptic ulcer. Plasma ghrelin levels decreased after H. pylori eradication in both peptic ulcer and gastritis patients, while plasma leptin levels increased only in peptic ulcer patients. Plasma leptin levels and BMI were positively correlated, and active ghrelin levels and atrophic pattern were weakly negatively correlated in peptic ulcer patients. Conclusion H. pylori infection and eradication therapy may affect circulating ghrelin/leptin levels. This finding suggests a relationship between gastric mucosal injury induced by H. pylori infection and changes in plasma ghrelin and leptin levels.

Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease worldwide and its incidence is increasing concomitantly with the increase in the prevalence of metabolic syndrome. Fatty liver encompasses a broad pathological spectrum of disease, from relatively benign accumulation of fat (simple steatosis) to progressive non-alcoholic steatohepatitis (NASH), which is associated with necroinflammation and fibrosis. Approximately 20-30% of the Japanese population is estimated to have NAFLD, 10% of which is suggested to have NASH. The most worrisome feature of NASH is the potential progression to cirrhosis, hepatocellular carcinoma (HCC), and finally, mortality. Several factors, such as insulin resistance, adipokines, endotoxins and oxidative stress, are involved in the pathogenesis of NASH. However, the precise etiological mechanism of NAFLD/NASH has yet to be elucidated. This article reviews the clinical background, pathogenesis, new diagnostic approaches and future directions regarding NAFLD/NASH.

The RNA-binding protein Hzf (hematopoietic zinc finger) plays important roles in mRNA translation in cerebellar Purkinje cells and adipocytes. We along with others have reported that the expression of the Hzf gene is transcriptionally regulated by the p53 tumor suppressor protein. We show here that Hzf regulates p53 expression in cooperation with HuR. Hzf and HuR independently interact with the 3' untranslated region (UTR) of p53 mRNA, which facilitates the cytoplasmic localization of p53 mRNA in the presence of the ARF tumor suppressor protein. In the absence of Hzf and HuR, p53 induction by p19(ARF) is significantly attenuated, and the cells consequently acquire resistance to p19(ARF). Thus, these findings demonstrate that in addition to Mdm2 inhibition, p19(ARF) increases the concentration of p53 through posttranscriptional control of p53 mRNA and suggest critical roles for the RNA-binding proteins Hzf and HuR in p53 induction.

The risk factors for the development of hepatocellular carcinoma (HCC) in patients who have achieved a long-term sustained viral response (SVR) to interferon (IFN) are not fully understood. This study aimed to investigate the characteristics of patients who developed HCC after 10 years of achieving SVR. We retrospectively studied 5 patients with HCC which developed more than 10 years after the termination of IFN therapy. The clinical characteristics at the induction of IFN therapy were male gender, a mean age of 51.6±9.1 years, while 2 patients were moderate alcohol consumers. None of the 5 patients were positive for either HBs Ag or anti-HBc Ab. A histological examination at the initial IFN therapy showed the activity scores to be A2 in all cases, and the fibrosis scores at least F2. The clinical parameters at the diagnosis of HCC included fluctuating transaminase levels in all cases. These levels scarcely fell below the upper limits even after SVR was achieved. In 3 patients, liver tissues were obtained at the treatment of HCC. These tissues showed marked improvement in both activities and fibroses, but severe steatosis in 1 patient. To conclude, chronic hepatitis C patients who respond to IFN therapy should undergo long-term follow-up, even after the eradication of HCV, with special attention particularly to patients who had elevated transaminase levels and steatosis.

The outcome of hepatitis C virus (HCV) infection has been associated with antiviral CD4 T cell response, human leukocyte antigens (HLA) class II genotypes, and ethnicity. However, HLA class II molecules restrict the nature of CD4 T cell response, and HLA distributions differ between ethnic groups. In this study, we asked whether HLA class II genotypes associated with HCV clearance are shared between Caucasian and African Americans and whether they contribute to enhanced antiviral CD4 T cell response. In a cohort of 93 HCV-seropositive subjects from Northeast America with defined ethnicity, virological outcome, and HCV-specific CD4 T cell proliferation, we confirm the previously reported associations between HCV clearance and two HLA types (DQB1*03, DRB1*11) while identifying a new association with DRB3*02. Strikingly, these associations were identified only among Caucasian [DQB1*03: odds ratio (OR), 10.4; P = 0.031, DRB1*11: OR, 7.0, P = 0.019; DRB3*02: OR, 8.3, P = 0.005; DQB1*03–DRB3*02: OR, 13.5, P = 0.001) but not among African American patients. Furthermore, although HLA DQB1*03, DRB1*11, and DRB3*02 genotypes were associated with increased HCV-specific CD4 T cell response in univariate analyses, these associations were lost when controlling for virological outcomes. Conclusion: We conclude that the immunogenetic basis for HCV clearance differs between ethnic groups and that the association between HLA class II and HCV clearance is not directly explained by antiviral CD4 T cell response. (HEPATOLOGY 2008.)

Summary Background Radiofrequency ablation (RFA) therapy for hepatocellular carcinoma has enabled good local control to be possible. However, after successful local control, distant recurrences frequently occur in the remnant liver. Aim To identify the predictive factors for distant recurrence after RFA. Methods A total of 117 patients with initial non-advanced hepatocellular carcinoma with HCV who underwent RFA in our hospital were selected for this study. After transcatheter chemoembolization, RFA was performed under real-time computed tomography-fluoroscopic guidance. We studied survival rates, local (adjacent to treated tumour) and distant (intrahepatic site distant from the treated tumours) recurrence rates, as well as predictive factors for distant recurrence. Results After RFA, survival rates were 98.2% and 64.7% at 1 and 5 years, respectively. Child B patients had a significantly worse survival than Child A. Recurrence rates were 2.4% at 5 years for local, and 17.1% and 76.9% at 1 and 5 years, respectively, for distant. The Kaplan–Meier method revealed significantly high recurrence rates in cases with low albumin levels (Alb 60 IU/L), high alanine aminotransferase levels (ALT > 60 IU/L), low platelet counts (Plt 50 ng/mL). On multivariate analysis, low Alb levels and high AST levels were independent predictive factors for distant recurrence. Conclusions Although RFA enables good local control for initial hepatocellular carcinoma, distant recurrence is observed at high rates in HCV patients. Low albumin and high AST levels are predictive factors for distant recurrence.