Hepatitis B Virus (HBV) Upregulates TRAIL-R3 Expression in Hepatocytes Resulting in Escape From Both Cell Apoptosis and Suppression of HBV Replication by TRAIL.

BACKGROUND: Hepatitis B virus (HBV) evades host immunity by regulating intracellular signals. To clarify this immune tolerance mechanism, we performed gene expression analysis using HBV-infected humanized mouse livers. METHODS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 3 (TRAIL-R3) was significantly upregulated in livers of HBV-infected human hepatocyte transplanted mice by cDNA microarray and next-generation sequencing. We analyzed the significance of TRAIL-R3 upregulation in HBV infection using human hepatocyte transplanted mice and HepG2 cell lines. RESULTS: TRAIL-R3 induction by HBV infection was verified by in vitro and in vivo HBV replication models, and induction was inhibited by antiviral nucleot(s)ide analogue treatment. TRAIL-R3 transcription was regulated by the TRAIL-R3 promoter at -969 to -479 nucleotides upstream from the transcription start site, and by hepatitis B x (HBx) via activation of nuclear factor-κB (NF-κB) signal. TRAIL not only induced cell apoptosis but also inhibited HBV replication. TRAIL-R3 upregulation could inhibit both TRAIL-dependent apoptosis in HBV-infected hepatocytes and TRAIL-mediated suppression of HBV replication. CONCLUSIONS: These results suggest a mechanism by which HBV persists by escaping host immunity through upregulation of TRAIL-R3. Development of novel drugs to inhibit this escape system might lead to complete HBV elimination from human hepatocytes.

Usefulness of linked color imaging in the early detection of superficial esophageal squamous cell carcinomas.

BACKGROUND AND AIMS: Linked color imaging (LCI) improved the visibility of gastric cancer and colorectal flat lesions. This study aimed to investigate the usefulness of LCI in detecting superficial esophageal squamous cell carcinomas (SESCC). METHODS: We enrolled 37 consecutive SESCC patients (46 SESCCs) diagnosed using LCI and blue laser imaging bright mode (BLI-BRT) and treated in Hiroshima University Hospital between April 2018 and November 2018. Eight professional endoscopists compared images obtained on non-magnifying BLI-BRT and LCI versus conventional white light imaging (WLI). Identification and boundary diagnosis of SESCC with LCI and BLI-BRT were compared with WLI. Changes in lesion visibility were clarified. Interobserver agreement was assessed. Clinicopathological features of lesion that influence visibility with LCI were assessed. RESULTS: In LCI, 37% (17/46) of cases had improved visibility and 63% (29/46) had unchanged visibility (interobserver agreement = 0.74). Among cases with multiple lugol voiding lesions (LVLs), ΔE between the lesion and background mucosa was significantly higher in LCI than in WLI (20.8 ± 7.9 vs 9.2 ± 6.1, P < 0.05). No significant differences were found in tumor size, morphological type, color, depth, and smoking or drinking history. However, multiple LVLs were significantly higher among cases with improved versus unchanged visibility. On BLI-BRT, 39% (18/46) of cases had improved visibility and 61% (28/46) had unchanged visibility (interobserver agreement = 0.60). CONCLUSION: Almost the same as BLI-BRT, LCI improves SESCC visibility compared with WLI. This is useful for cases with multiple LVLs. In cases without background coloration (BGC), LCI may make SESCC more visible than BLI-BRT.

Gastric Cancer with Submucosal Invasion after Successful Helicobacter pylori Eradication: A Propensity Score-Matched Analysis of Patients with Annual Patient Endoscopic Survey.

BACKGROUND AND AIM: The incidence of gastric cancer occurring after successful Helicobacter pylori eradication has been increasing. We aimed to clarify the influence of eradication therapy on the ability to diagnose early gastric cancer after successful H. pylori eradication in patients who underwent annual endoscopic screening. METHODS: A total of 220 patients (179 men; mean age 71.0 years) had differentiated-type early gastric cancer that was discovered through annual endoscopic screening. Patients were categorized into 2 groups: the H. pylori-eradicated group (n = 81) and the non-eradicated control group (n = 139). After matching patients by propensity scores, we retrospectively analyzed the clinicopathological characteristics of 162 patients (81 patients in each group). Furthermore, we compared the characteristics of gastric cancer with submucosal invasion between the 2 groups. RESULTS: The prevalence of early gastric cancer with submucosal invasion was significantly higher in the eradicated group than in the control group, both before propensity score matching (16.0 vs. 7.2%, respectively; p = 0.038) and after propensity score matching of 81 pairs (16.0 vs. 4.9%, respectively; p = 0.021). In the comparative analysis of gastric cancer with submucosal invasion, there was no difference between the 2 groups with respect to factors influencing the ability to diagnose its presence endoscopically. CONCLUSION: H. pylori eradication therapy increased the prevalence of differentiated-type gastric cancer with submucosal invasion despite patients' completion of annual endoscopic screening after eradication.

CTL-associated and NK cell-associated immune responses induce different HBV DNA reduction patterns in chronic hepatitis B patients.

The activation of hepatitis B virus (HBV)-related hepatitis is associated with both natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). We analyzed the association between the immune response and changes in the proportion of Pre-S deletion variants. We quantified Pre-S deleted HBV (HBV-del) and wild-type HBV (HBV-wt) DNA levels in sera obtained from HBV-infected mice and chronic hepatitis B patients. In chronic hepatitis B patients, the HBV-del proportion usually increased during or after ALT elevation but did not occur during all ALT elevations. To clarify this difference in the immunological responses, we performed in vivo analyses using HBV-infected human hepatocyte chimeric mice. Although HBV-del proportions did not change in mice with NK cell-associated hepatitis or in mice treated with entecavir, the proportions sharply increased in mice with CTL-associated hepatitis. Furthermore, the number of patients in which HBV-del proportions were greater than 5% was significantly higher in chronic hepatitis B patients than in asymptomatic carriers (P = 0.023). We identified associations between virological response in chronic hepatitis B patients and two different immune responses. The proportion of HBV-del variants could be a useful biomarker for distinguishing between chronic hepatitis and asymptomatic carriers.

Taking Warfarin with Heparin Replacement and Direct Oral Anticoagulant Is a Risk Factor for Bleeding after Endoscopic Submucosal Dissection for Early Gastric Cancer.

BACKGROUND: Although bleeding after endoscopic submucosal dissection (ESD) for early gastric cancer (EGC) remains problematic, especially in patients taking anticoagulants, there are differing views on the ideal and optimal management for these patients. This study investigated the risk of bleeding after ESD in patients taking anticoagulants. METHODS: We enrolled 61 consecutive patients taking anticoagulants (anticoagulant group) and 968 patients taking no antithrombotic agents (non-antithrombotic group) treated with ESD for EGC between December 2010 and October 2016. We analyzed the risk factors for bleeding after ESD in relation to the various clinical factors. RESULTS: Incidences of bleeding after ESD were significantly higher (14%; 11/76) in the anticoagulant group compared to the non-antithrombotic group (3%; 40/1,167). Moreover, bleeding after ESD was significantly more common in patients in the warfarin monotherapy group (14%; 5/37) and in the direct oral anticoagulant (DOAC) monotherapy group (22%; 4/18), compared to the non-antithrombotic group. Multivariate analysis revealed that dialysis, the use of anticoagulants, and an operation time ≥75 min were independent risk factors for bleeding after ESD. CONCLUSIONS: Our data suggest that patients who take warfarin and receive heparin bridging, and those who take DOAC medication, are prone to bleeding after ESD for EGC.

Evaluation of the visibility of early gastric cancer using linked color imaging and blue laser imaging.

BACKGROUND: Blue laser imaging (BLI) and linked color imaging (LCI) are the color enhancement features of the LASEREO endoscopic system, which provide a narrow band light observation function and expansion and reduction of the color information, respectively. METHODS: We examined 82 patients with early gastric cancer (EGC) diagnosed between April 2014 and August 2015. Five expert and 5 non-expert endoscopists retrospectively compared images obtained on non-magnifying BLI bright mode (BLI-BRT) and LCI with those obtained via conventional white light imaging (WLI). Interobserver agreement was also assessed. RESULTS: In experts' evaluation of the images, an improvement in visibility was observed in 73% (60/82) and 20% (16/82) of cases under LCI and BLI-BRT, respectively. In non-experts' evaluation of the images, an improvement in visibility was observed in 76.8% (63/82) and 24.3% (20/82) of cases under LCI and BLI-BRT, respectively. There were no significant differences between experts and non-experts in the evaluation of the images. The improvement in visibility was significantly higher with LCI than with BLI-BRT in experts and non-experts (p < 0.01). With regard to tumor color on WLI, the improvement in the visibility of reddish and whitish tumors was significantly higher than that of isochromatic tumors when LCI was used. The improvement in visibility with LCI was observed in 71% (12/17) and 74% (48/65) of patients with and without Helicobacter pylori (Hp) eradication, respectively; no significant difference in improvement was observed between these groups. The interobserver agreement was good to satisfactory at ≥ 0.62. CONCLUSIONS: In conclusion, our study showed that LCI improved the visibility of EGC, regardless of the level of endoscopists' experience or Hp eradication in patients, particularly for EGCs with a reddish or whitish color. The improvement in visibility was significantly higher with LCI than that with BLI.

Clinical outcomes of endoscopic resection for superficial non-ampullary duodenal tumors.

Background and study aims Because superficial non-ampullary duodenal epithelial tumors (SNADETs) are relatively rare, studies evaluating the outcomes of endoscopic resection (ER) for SNADETs are limited. Therefore, this study aimed to evaluate the clinical validity of ER for SNADETs. Patients and methods The study participants included 163 consecutive patients (108 men; mean age, 61.5 ±â€Š11.3 years) with 171 SNADETs, excluding patients with familial adenomatous polyposis resected by ER, at Hiroshima University Hospital between May 2005 and September 2016. Clinicopathological features and the outcomes of ER for 171 cases were retrospectively analyzed. Additionally, the prognosis of 135 patients with more than 12 months' follow-up was analyzed. Results Mean diameter of SNADETs was 10.7 ±â€Š7.2 mm. Most of the SNADET cases were classified as category 3 (71 %, 121/171), but some were category 5 (2 %, 3/171). En bloc resection rates were 93 % (146/157), 100 % (7/7), and 86 % (6/7) in endoscopic mucosal resection (EMR), polypectomy, and in endoscopic submucosal dissection (ESD) cases, respectively. Complete resection rates were 90 % (141/157), 100 % (7/7), and 71 % (5/7) in EMR, polypectomy, and ESD cases, respectively. Emergency surgery was performed in two patients with intraoperative perforation and in two with delayed perforation without artificial ulcer bed closure after ER. Since endoscopic closure of ulcer by clipping was performed, delayed perforation has not occurred. Local recurrence occurred in 1.2 %, but no metastasis to lymph nodes or other organs occurred after ER. No patient died of primary SNADETs. Conclusion Our data supported the clinical validity of ER for SNADETs. However, delayed perforation should be given much attention.

Clinical Usefulness of Dual Red Imaging in Gastric Endoscopic Submucosal Dissection: A Pilot Study.

BACKGROUND/AIMS: Dual red imaging (DRI) is a new, image-enhanced endoscopy technique. There are few reports about the usefulness of DRI during gastric endoscopic submucosal dissection (ESD). We aimed to examine the usefulness of DRI in endoscopic hemostasis during gastric ESD. METHODS: We enrolled a total of 20 consecutive patients who underwent gastric ESD. Five endoscopists compared DRI with white light imaging (WLI) for the visibility of blood vessels and bleeding points while performing endoscopic hemostasis. RESULTS: The visibility of blood vessels was increased in 56% (19/34) of the cases, and the visibility of bleeding points was improved in 55% (11/20) of the cases with the use of DRI compared with the use of WLI. CONCLUSION: DRI improved the visibility of blood vessels and bleeding points in cases with oozing bleeding, blood pooling around the bleeding points, and multiple bleeding points.

Comparison of intracellular responses between HBV genotype A and C infection in human hepatocyte chimeric mice.

BACKGROUND AND AIMS: The clinical course and responsiveness to antiviral treatments differs among hepatitis B virus (HBV) genotypes. However, the cause of these differences is unclear. In the present study, we compared mRNA expression profiles in human hepatocyte chimeric mice infected with HBV genotypes A and C. METHODS: Fifteen chimeric mice were prepared and divided into the following three groups: uninfected control mice, HBV genotype A-infected mice, and HBV genotype C-infected mice. Human hepatocytes were collected from these mouse livers and gene expression analyses were performed using next-generation RNA sequencing. RESULTS: Although similar pathways were influenced by HBV infection, including inflammation mediated by chemokine and cytokine signaling, p53, and integrin signaling pathways, expression levels of up-regulated genes by HBV genotype A or C infection were quite different. In HBV genotype A-infected hepatocytes, 172 genes, including KRT23 and C10orf54, were significantly more highly expressed than in HBV genotype C-infected cells, whereas 10 genes, including SPX and IER3, were expressed at significantly lower levels. Genes associated with the p53 pathway and the inflammation mediated by chemokine and cytokine signaling pathway were more highly expressed in cells with HBV genotype A infection, whereas genes associated with CCKR signaling map and oxidative stress response were more highly expressed in cells with HBV genotype C infection. CONCLUSION: Several differences in gene expression with respect to HBV genotype A and C infection were detected in human hepatocytes. These differences might be associated with genotypic difference in the clinical course or responsiveness to treatment.

The association between serum cytokine and chemokine levels and antiviral response by entecavir treatment in chronic hepatitis B patients.

BACKGROUND: Although nucleoside/nucleotide analogue therapy is thought to suppress chronic hepatitis B (CHB) via regulation of inflammatory cytokines/chemokines, the mechanism is still unclear. In this study, serum cytokine/chemokine levels were measured in CHB patients treated with entecavir, and the association with antiviral response was analysed. METHODS: A total of 78 Japanese patients with CHB were enrolled, and serum cytokine/chemokine levels were measured at baseline and at 12, 24 and 48 weeks of entecavir treatment using the MULTIPLEX kit. RESULTS: Antiviral response to entecavir treatment was significantly associated with hepatitis B surface antigen (HBsAg) titre and serum interferon-gamma-inducible protein 10 (IP-10) level (12w; P=0.0002; OR=0.020 [95% CI 0.002, 0.156], P=0.003; OR=0.042 [95% CI 0.005, 0.336], respectively). HBe-positive patients whose serum macrophage-derived chemokine (MDC) level was lower (<582.83 pg/ml) and IP-10 level was higher (≥1,323.13 pg/ml) achieved hepatitis B e antigen (HBeAg) loss earlier than those who remained HBeAg-positive (P=0.044). HBsAg reduction by entecavir treatment was significantly associated with higher initial tumour necrosis factor-alpha (TNF-α) level (≥15.20 pg/ml) and higher alanine aminotransferase level (≥73 IU/l; P=0.009; OR=18.460 [95% CI 2.044, 166.709], P=0.022; OR=7.709 [95% CI 1.341, 44.327], respectively). CONCLUSIONS: Results of the present study indicate that changes in cytokine/chemokine levels following entecavir therapy are associated with response to antiviral therapy in CHB patients. Monitoring of serum cytokine/chemokine levels could be useful for predicting reduction of HBV DNA and HBsAg and HBe seroconversion.

Endoplasmic reticulum-mediated induction of interleukin-8 occurs by hepatitis B virus infection and contributes to suppression of interferon responsiveness in human hepatocytes.

The events in the immune response to hepatitis B virus (HBV) remain unclear. We analyzed the direct influence of HBV on gene expression in human hepatocytes under immunodeficient conditions using a human hepatocyte chimeric mouse model. HBV-infected or non-infected chimeric mouse livers were collected, and gene expression profiles were compared. Since IL-8 was the most significantly up-regulated gene at 8 weeks after HBV infection, we focused on IL-8 and found that HBx and the large HBs (L-HBs) protein induce transcription of IL-8 via endoplasmic reticulum stress. This stress induces IL-8 transcription via NFAT activation and contributes to suppression of interferon responsiveness in HBV-infected human hepatocytes. In the present study, we identified a novel regulatory mechanism in which the L-HBs protein activates IL-8 via endoplasmic reticulum stress, suggesting a key role for IL-8 in the immune response to HBV and a potential new target for antiviral treatments of HBV infection.

Reduction of hepatitis B surface antigen in sequential versus add-on pegylated interferon to nucleoside/nucleotide analogue therapy in HBe-antigen-negative chronic hepatitis B patients: a pilot study.

BACKGROUND: Although pegylated interferon (PEG-IFN) and nucleotide/nucleoside analogue (NA) combination therapy is considered to be optimal for accelerating serum hepatitis B surface antigen (HBsAg) reduction, the effect is limited, and the best approach to PEG-IFN treatment for chronic hepatitis B patients during long-term NA therapy has yet to be determined. METHODS: A total of 21 hepatitis B e antigen-negative chronic hepatitis B patients whose HBV DNA levels were suppressed to undetectable levels by NA therapy were administrated PEG-IFN-α2a for 48 weeks (sequential therapy: 10, add-on therapy: 11). Factors associated with HBsAg reduction by PEG-IFN therapy were analysed. RESULTS: During PEG-IFN treatment, HBsAg levels were reduced by 0.48 log IU/ml. More than 1 log IU/ml of HBsAg reduction was observed in eight patients (sequential therapy: six, add-on therapy: two), and one patient with sequential therapy achieved HBsAg loss. By univariate analysis, sequential therapy was marginally associated with more than 1 log IU/ml HBsAg reduction during PEG-IFN treatment (P=0.060). After PEG-IFN treatment, only five patients, including the patient with HBsAg loss, achieved more than 0.5 log IU/ml of HBsAg reduction by 1 year after PEG-IFN treatment. By univariate analysis, sequential therapy was significantly associated with HBsAg reduction after PEG-IFN treatment (P=0.012). In addition, alanine aminotransferase elevation during PEG-IFN therapy and lower serum interleukin-8 level at the end of PEG-IFN treatment were also significantly associated with HBsAg reduction by 1 year after PEG-IFN treatment (P=0.038, P=0.044, respectively). CONCLUSIONS: Sequential therapy may be superior to add-on therapy in reducing HBsAg levels during long-term NA therapy in chronic hepatitis B patients.

Clinical Usefulness of the VS Classification System Using Magnifying Endoscopy with Blue Laser Imaging for Early Gastric Cancer.

BACKGROUND: Blue laser imaging (BLI) enables the acquisition of more information from tumors' surfaces compared with white light imaging. Few reports confirm the validity of magnifying endoscopy (ME) with BLI (ME-BLI) for early gastric cancer (EGC). We aimed to assess the detailed endoscopic findings from EGCs using ME-BLI. METHODS: We enrolled 386 consecutive patients with 417 EGCs that were diagnosed using ME-BLI and resected by endoscopic submucosal dissection. Using the VS classification system, three highly experienced endoscopists (HEEs) and three less experienced endoscopists (LEEs) evaluated the demarcation line (DL), microsurface pattern (MSP), and microvascular pattern (MVP) within the endoscopic images of EGCs obtained using ME-BLI, assigning high-confidence (HC) or low-confidence (LC) levels. We investigated the clinicopathological features associated with each confidence level. RESULTS: The HEEs' evaluations determined the presence of DL in 99%, irregular MSP in 96%, and irregular MVP in 96%, and the LEEs' evaluations determined the presence of DL in 98%, irregular MSP in 95%, and irregular MVP in 95% of the EGCs. When DL was present, HC levels in the Helicobacter pylori- (H. pylori-) eradicated group and noneradicated group were evident in 65% and 89%, a difference that was significant (p < 0.001). CONCLUSIONS: In the diagnosis of EGC with ME-BLI, the VS classification system with ME-NBI can be applied, but identifying the DL after H. pylori was difficult.

Clinical usefulness of magnifying endoscopy for non-ampullary duodenal tumors.

Study aims This study aimed to investigate the clinical usefulness of magnifying endoscopy (ME) for non-ampullary duodenal tumors. Patients and methods We enrolled 103 consecutive patients with non-ampullary duodenal tumors that were observed by ME with narrow-band imaging (ME-NBI) and had pit pattern analysis before endoscopic resection at Hiroshima University Hospital before December 2014. ME-NBI images were classified as Type B or Type C according to the Hiroshima classification, and pit patterns were classified as regular or irregular. We studied the clinicopathological features and diagnoses with ME-NBI and pit pattern analyses according to the Vienna classification (category 3: 73 patients; category 4: 30 patients). Results Category 4 lesions were significantly larger than category 3 lesions. According to ME-NBI images, category 4 Type C lesions (83 %) were significantly more common than category 4 Type B lesions (17 %). According to pit pattern analyses, category 4 irregular lesions 4 (77 %) were significantly more common than category 4 regular lesions (23 %). The accuracies of using Type C ME-NBI images and irregular pit patterns to diagnose category 4 lesions were 87 % and 84 %, the sensitivities were 83 % and 77 %, and the specificities were 89 % and 88 %, respectively. There was no significant difference between ME-NBI and pit pattern analyses for diagnosing the histologic grade of non-ampullary duodenal tumors. Conclusion Our study showed that ME-NBI and pit pattern analysis had equivalent abilities to determine the histologic grade of non-ampullary duodenal tumors. ME-NBI may be more useful because it is a simple, less time-consuming procedure.