Fragmentomics of urinary cell-free DNA in nuclease knockout mouse models.

Urinary cell-free DNA (ucfDNA) is a potential biomarker for bladder cancer detection. However, the biological characteristics of ucfDNA are not well understood. We explored the roles of deoxyribonuclease 1 (DNASE1) and deoxyribonuclease 1-like 3 (DNASE1L3) in the fragmentation of ucfDNA using mouse models. The deletion of Dnase1 in mice (Dnase1-/-) caused aberrations in ucfDNA fragmentation, including a 24-fold increase in DNA concentration, and a 3-fold enrichment of long DNA molecules, with a relative decrease of fragments with thymine ends and reduction of jaggedness (i.e., the presence of single-stranded protruding ends). In contrast, such changes were not observed in mice with Dnase1l3 deletion (Dnase1l3-/-). These results suggested that DNASE1 was an important nuclease contributing to the ucfDNA fragmentation. Western blot analysis revealed that the concentration of DNASE1 protein was higher in urine than DNASE1L3. The native-polyacrylamide gel electrophoresis zymogram showed that DNASE1 activity in urine was higher than that in plasma. Furthermore, the proportion of ucfDNA fragment ends within DNase I hypersensitive sites (DHSs) was significantly increased in Dnase1-deficient mice. In humans, patients with bladder cancer had lower proportions of ucfDNA fragment ends within the DHSs when compared with participants without bladder cancer. The area under the curve (AUC) for differentiating patients with and without bladder cancer was 0.83, suggesting the analysis of ucfDNA fragmentation in the DHSs may have potential for bladder cancer detection. This work revealed the intrinsic links between the nucleases in urine and ucfDNA fragmentomics.

ALT to qHBsAg ratio predicts long-term HBsAg seroclearance after entecavir cessation in Chinese patients with chronic hepatitis B.

BACKGROUND & AIMS: Factors predicting HBsAg seroclearance after treatment cessation, irrespective of nucleos(t)ide analogue (NA) resumption, have important clinical implications. We evaluated predictors of long-term HBsAg seroclearance after entecavir cessation. METHODS: This study followed-up Chinese patients with chronic hepatitis B from two previous studies of entecavir cessation. All patients were non-cirrhotic, HBeAg-negative, with undetectable HBV DNA (<20 IU/ml) at end-of-treatment (EOT). They were monitored closely for 48 weeks with regular HBV DNA, quantitative HBsAg (qHBsAg) and alanine aminotransferase (ALT) measurements. Entecavir was resumed at HBV DNA >2,000 IU/ml, irrespective of ALT levels. After the initial 48 weeks, patients were assessed every 6 months, regardless of entecavir resumption, to monitor for HBsAg seroclearance. RESULTS: A total of 194 patients (63.4% male, mean age 49.9 years, on entecavir for a median of 47.2 months) were recruited; 94 (48.5%) and 158 (81.4%) patients had EOT qHBsAg <100 IU/ml and <1,000 IU/ml, respectively; 151 (77.8%) patients were eventually resumed on entecavir. After follow-up for a median of 70.7 (51.0-118.2) months, 28 (14.4%) patients had HBsAg seroclearance. qHBsAg levels at weeks 36 and 48 after EOT independently predicted HBsAg seroclearance (both p <0.01), whereas qHBsAg from EOT to week 24 only trended towards statistical significance. The ratio of ALT/qHBsAg at all time points from EOT to week 48 independently predicted HBsAg seroclearance (hazard ratios ranging from 1.003-1.028, all p <0.01) with excellent diagnostic performance (area under the receiver-operating characteristic curve 0.799-0.933, negative predictive value >90% at different time points), regardless of whether entecavir was resumed. CONCLUSIONS: The ALT/qHBsAg ratio after entecavir cessation predicts HBsAg seroclearance, even in patients who were resumed on treatment. Its use may mitigate the risk of severe hepatitis flares in patients managed by observation without treatment resumption. IMPACT AND IMPLICATIONS: Current predictors of HBsAg seroclearance after finite nucleos(t)ide analogue (NA) therapy have suboptimal predictive value. We demonstrated that the ALT/qHBsAg ratio may be able to reflect the balance between host control and virological activity. The ALT/qHBsAg ratio at different time points from end-of-treatment till week 48 independently and accurately predicted HBsAg seroclearance in patients who have stopped entecavir. The ALT/qHBsAg ratio may be utilized by clinicians for patient selection and retreatment decisions in finite NA therapy.

Outcomes of High-Grade Immune Checkpoint Inhibitor Hepatitis in Hospitalized and Nonhospitalized Patients.

BACKGROUND & AIMS: Guidelines recommend hospitalization for severe immune checkpoint inhibitor (ICI) hepatitis. We compared patient outcomes in the inpatient versus outpatient settings. METHODS: We conducted a multicenter, retrospective cohort study of 294 ICI-treated patients who developed grade 3-4 ICI hepatitis. The primary outcome was time to alanine aminotransferase (ALT) normalization (≤40); secondary outcomes included time to ALT ≤100 U/L and time to death. To account for confounding by indication, inverse probability of treatment weighting was applied to perform Cox regression. A sensitivity analysis was performed excluding patients with grade 4 hepatitis. RESULTS: One hundred and sixty-six patients (56.5%) were hospitalized for a median of 6 (interquartile range, 3-11) days. On inverse probability of treatment weighting Cox regression, hospitalization was not associated with time to ALT normalization (hazard ratio [HR], 1.11; 95% confidence interval [CI], 0.86-1.43; P = .436) or time to ALT ≤100 U/L (HR, 1.11; 95% CI, 0.86-1.43; P = .420). In the sensitivity analysis limited to patients with grade 3 hepatitis, hospitalization was also not associated with time to ALT normalization (HR, 1.11; 95% CI, 0.83-1.50; P = .474) or time to ALT ≤100 U/L (HR, 1.19; 95% CI, 0.90-1.58; P = .225). In a subgroup analysis of 152 patients with melanoma, hospitalization was not associated with reduced risk of all-cause death (HR, 0.93; 95% CI, 0.53-1.64; P = .798). Notably, despite their Common Terminology Criteria for Adverse Events classification of high-grade hepatitis, 94% of patients had "mild" liver injury based on International Drug-Induced Liver Injury Criteria. CONCLUSIONS: Hospitalization of patients with high-grade ICI hepatitis was not associated with faster hepatitis resolution and did not affect mortality. Routine hospitalization may not be necessary in all patients with high-grade ICI hepatitis and Common Terminology Criteria for Adverse Events criteria may overestimate severity of liver injury.

Nuclease deficiencies alter plasma cell-free DNA methylation profiles.

The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation were explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in DNASE1L3-deficient mice was significantly hypomethylated, while cfDNA in DNASE1-deficient mice was hypermethylated. The cfDNA hypomethylation in DNASE1L3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs). These findings were absent in DNASE1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affects cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology, and cfDNA fragmentation.

Cigarette Smoking Is Associated With Lower Chance of Hepatitis B Surface Antigen Seroclearance and Altered Host Immunity.

Cigarette smoking is associated with worse clinical outcomes in patients with chronic hepatitis B (CHB) infection, but the effects on hepatitis B surface antigen (HBsAg) seroclearance are unclear. This study aimed to investigate the effect of active smoking on HBsAg seroclearance (SC) and its impact on peripheral blood lymphocytes in patients with CHB infection. Longitudinal follow-up data was retrieved in 7833 antiviral-treated CHB subjects identified from a centralised electronic patient record database (Part 1). Phenotypic analysis of peripheral blood mononuclear cells (PBMCs) from 27 CHB-infected patients (6 active smokers; 13 with SC) was performed by flow cytometry to assess programmed death-1 (PD-1) expression and proportion of regulatory T cells (CD4+CD25+CD127lo). Effector function of HBV-specific T cells was examined by comparing granzyme B (GZMB) and transforming growth factor beta (TGFß) production in undepleted PBMCs and Treg-depleted PBMCs after 7 days in vitro stimulation with HBV envelope protein overlapping peptides (Part 2). Over a median follow-up of 5 years, smoking was associated with lower probability of SC (aHR 0.70, 95% CI 0.57-0.87). PD-1 expression was increased in CD4+T cells, CD8+T cells and CD20+B cells among smokers compared to non-smokers and positively correlated with pack years (all p < 0.05). Treg depletion led to partial functional recovery of HBV-specific T cells, with significantly bigger magnitude in smokers (p = 0.0451, mean difference = 4.68%) than non-smokers (p = 0.012, mean difference = 4.2%). Cigarette smoking is associated with lower chance of HBsAg seroclearance, higher PD-1 expression on lymphocytes, and impairment of effector functions of HBV-specific T cells in CHB.

Glycemic burden and the risk of adverse hepatic outcomes in patients with chronic hepatitis B with type 2 diabetes.

BACKGROUND AND AIMS: Type 2 diabetes (T2D) is common among patients with chronic hepatitis B infection (CHB) and has been associated with increased risk of carcinogenesis, including HCC. We investigated factors associated with HCC and fibrosis progression among patients with CHB with T2D (CHB+T2D). APPROACH AND RESULTS: Chinese patients with CHB were prospectively recruited for the incidence of HCC and fibrosis progression defined by transient elastography. Among patients with CHB+T2D, glycemic control was assessed by mean glycated hemoglobin (HbA1c) and HbA1c variability determined using HbA1c measurements in the 5 years preceding recruitment. A total of 2330 patients with CHB were recruited (mean age 54.6 ±11.8 years old, 55.5% male, 57.9% antiviral-treated), with 671 (28.8%) having CHB+T2D (mean T2D duration 7.2 ± 4.6 years, mean HbA1c 7.2 ± 0.9%). T2D was independently associated with HCC (HR 2.080, 95% CI 1.343-3.222) and fibrosis progression (OR 4.305, 95% CI 3.416-5.424) in the overall cohort. In patients with CHB+T2D, factors reflecting glycemic burden (T2D duration [HR 1.107, 95% CI 1.023-1.198]), mean HbA1c (HR 1.851, 95% CI 1.026-3.339), time reaching target HbA1c (HbA1c-TRT; HR 0.978, 95% CI 0.957-0.999), liver stiffness (HR 1.041-1.043), and smoking (HR 2.726-3.344) were independently associated with HCC (all p < 0.05), but not HbA1c variability or controlled attenuation parameter. The same glycemic burden-related factors (T2D duration, mean HbA1c, and HbA1c-TRT), in addition to baseline fasting glucose, baseline HbA1c, AST and antiviral therapy, were independently associated with fibrosis progression at 3 years. CONCLUSIONS: High glycemic burden was associated with HCC development and fibrosis progression among patients with CHB+T2D, highlighting the importance of glycemic control in reducing liver-related complications.

High end-of-treatment hepatitis B core-related antigen levels predict hepatitis flare after stopping nucleot(s)ide analogue therapy.

BACKGROUND AND AIMS: Accurate biomarkers to predict outcomes following discontinuation of nucleos(t)ide analogue (NA) therapy are needed. We evaluated serum hepatitis B core-related antigen (HBcrAg) level as a biomarker for predicting outcomes after NA discontinuation. METHODS: Patients with HBeAg-negative chronic hepatitis B (CHB) without cirrhosis were enrolled in a prospective trial evaluating clinical outcomes until 96 weeks after NA discontinuation. End of treatment (EOT) and off-treatment levels of serum HBcrAg, HBsAg, HBV RNA and HBV DNA were used to predict key clinical outcomes including hepatitis flare (ALT ≥5 × ULN and HBV DNA > 2000 IU/mL). The SCALE-B score was calculated for the purposes of model validation. RESULTS: HBcrAg was tested amongst 65 participants. The median age was 54 years, 54% were male and 83% were Asian. HBcrAg was detectable in 86% patients. HBcrAg level ≥4 log U/mL at EOT was predictive of hepatitis flare [8/10 (80%) vs. 17/55 (31%), p = .001]. The presence of either HBcrAg ≥4 log U/mL or detectable HBV RNA at EOT predicted for both biochemical relapse and hepatitis flare. The SCALE-B model at EOT predicted for virological relapse, biochemical relapse, hepatitis flare and HBsAg loss in this cohort. An increase in the serum HBcrAg level off-treatment was also associated with hepatitis flare. No participant with EOT HBcrAg level ≥4 log U/mL achieved HBsAg loss. CONCLUSIONS: High levels of serum HBcrAg predict for hepatitis flare after stopping NA therapy and low likelihood of HBsAg loss at week 96. People with high levels of serum HBcrAg are not suitable candidates for NA discontinuation.

Plasma interferon-gamma-inducible-protein 10 level as a predictive factor of spontaneous hepatitis B surface antigen seroclearance in chronic hepatitis B patients.

BACKGROUND AND AIM: Spontaneous seroclearance of hepatitis B surface antigen (HBsAg) is a rare event that occurs in patients that are chronically infected with the hepatitis B virus. As the functional cure and ultimate treatment endpoint of chronic hepatitis B (CHB), HBsAg seroclearance is an important milestone in the natural history of CHB and serves great clinical value. This study aims to identify host and viral factors associated with HBsAg seroclearance. METHODS: This is a retrospective study carried out in the Queen Mary Hospital, Hong Kong. By analyzing the plasma retrieved from the serum archive (collected during 2011-2021) of 100 CHB patients attending the hospital's liver clinic, the longitudinal cytokine profiles between the HBsAg-losers and the control groups were compared. RESULTS: Data revealed that plasma levels of IP-10 were significantly lower at 3-5 years prior to HBsAg seroclearance compared with patients who remained HBsAg positive (P < 0.05). Receiver operating characteristic curve analysis reveals that plasma IP-10 levels at multiple time points before HBsAg seroclearance return area under receivor-operating characteristic curve (AUC) greater than 0.7. Plasma IP-10 levels at 42.39 pg/mL produced an AUC = 0.723 with 74.0% sensitivity and 75.5% specificity to predict subsequent HBsAg seroclearance in the next 3-5 years. Low plasma IP-10 identified 91.4% patients with quantitative HBsAg < 100 IU/mL who would subsequently develop HBsAg seroclearance, compared with 37% with higher plasma IP-10 levels (P < 0.001). CONCLUSIONS: Low plasma levels of IP-10 are associated with subsequent HBsAg seroclearance, suggesting potential clinical utilities of measurement of IP-10 in predicting HBsAg seroclearance, especially among patients with low HBsAg.

Regression of liver fibrosis after HBsAg loss: A prospective matched case-control evaluation using transient elastography and serum enhanced liver fibrosis test.

BACKGROUND AND AIM: We assessed the effect of hepatitis B surface antigen (HBsAg) seroclearance (HBsAg-loss) on liver fibrosis regression in patients with chronic hepatitis B (CHB) infection. METHOD: CHB patients with recent documented HBsAg-loss were age- and gender-matched with treatment-naïve HBeAg-negative CHB infection. Paired assessment with transient elastography and enhanced liver fibrosis (ELF) measurements were performed and repeated at 3 years. Fibrosis regression was arbitrarily defined as decrease in ≥ 1 fibrosis stage by ELF, or combining with reduction > 30% in liver stiffness. RESULTS: A total of 142 HBsAg-loss and 142 CHB subjects were recruited (median age 58.1 years, 51.4% male). A total of 1.8% (1.4% HBsAg-loss vs 2.1% CHB) achieved combined endpoint of fibrosis regression at 3 years. When ELF-only definition of fibrosis regression was used, 14.5% HBsAg-loss and 16.9% CHB subjects achieved this endpoint, which was significantly associated with baseline ELF (hazard ratio (HR) 1.827, 95% confidence interval (CI) 1.085-3.075) and time since HBsAg-loss (HR 2.688, 95% CI 1.257-5.748). While increasing time since HBsAg-loss increased the proportion of ELF-defined fibrosis regression, increasing age was also associated with significant fibrosis. Age of achieving HBsAg-loss (ageSC) was independently associated with high baseline ELF values. Up to 52.3% and 63.8% subjects with ageSC > 50 had advanced fibrosis/cirrhosis at baseline and 3 years, respectively, compared with 5.9% and 20.6% in subjects with ageSC < 50. CONCLUSION: Fibrosis regression occurred in a minority of subjects achieving HBsAg-loss, which was not significantly different compared with subjects with persistent overt CHB. Subjects after achieving HBsAg-loss, especially among those with ageSC > 50, should receive ongoing surveillance for liver-related complications.

Critical care unit bed availability and postoperative outcomes: a multinational cohort study.

BACKGROUND: Critical care beds are a limited resource, yet research indicates that recommendations for postoperative critical care admission based on patient-level risk stratification are not followed. It is unclear how prioritisation decisions are made in real-world settings and the effect of this prioritisation on outcomes. METHODS: This was a prespecified analysis of an observational cohort study of adult patients undergoing inpatient surgery, conducted in 274 hospitals across the UK and Australasia during 2017. The primary outcome was postoperative morbidity at day 7. Logistic regression models were used to evaluate the relationship between critical care admission and patient and health system factors. The causal effect of critical care admission on outcome was estimated using variation in critical care occupancy as a natural experiment in an instrumental variable analysis. RESULTS: A total of 19,491 patients from 248 hospitals were eligible for analysis, of whom 2107 were directly admitted to critical care postoperatively. Postoperative morbidity occurred in 2829/19,491 (15%) patients. Increasing surgical risk was associated with critical care admission, as was increased availability of critical care beds (odds ratio (95%CI) 1.04 (1.01-1.06), p = 0.002) per available bed; however, the probability of admission varied significantly between hospitals (median odds ratio 3.05). There was no evidence of a difference in postoperative morbidity with critical care admission (odds ratio (95%CI) 0.91 (0.57-1.45), p = 0.710). DISCUSSION: Postoperative critical care admission is variable and related to bed availability. Statistical methods that adjust for unobserved confounding lowered the estimates of harm previously reported to have been associated with postoperative critical care admission. Our findings provide a rationale for a clinical trial which would evaluate any potential benefits for postoperative critical care admission for patients in whom there is no absolute indication for admission.

Effect of Antiviral Treatment on Hepatitis B Virus Integration and Hepatocyte Clonal Expansion.

BACKGROUND: This study investigated the effect of nucleos(t)ide analogue (NUC) treatment on hepatitis B virus (HBV) DNA integration and hepatocyte clonal expansion, both of which are implicated in hepatocellular carcinoma (HCC) in chronic hepatitis B. METHODS: Twenty-eight patients receiving NUCs (11 lamivudine, 7 telbivudine, 10 entecavir) were included. All had liver biopsies at baseline and year 1, and 7 had a third biopsy at year 10. HBV DNA integration and hepatocyte clone size were assessed by inverse polymerase chain reaction. RESULTS: All patients had detectable HBV integration at baseline, with a median integration frequency of 1.01 × 109 per liver and hepatocyte clone size of 2.41 × 105. Neither integration frequency nor hepatocyte clone size correlated with age and HBV virologic parameters. After 1 year of treatment, HBV integration was still detectable in all patients, with a median of 5.74 × 108 integration per liver (0.22 log reduction; P = .008) and hepatocyte clone size of 1.22 × 105 (0.40 log reduction; P = .002). HBV integration remained detectable at year 10 of treatment, with a median integration frequency of 4.84 × 107 integration per liver (0.93 log reduction from baseline) and hepatocyte clone size of 2.55 × 104 (1.02 log reduction from baseline). From baseline through year 1 to year 10, there was a decreasing trend in both integration frequency and hepatocyte clone size (P = .066 and.018, respectively). CONCLUSIONS: NUCs reduced both HBV DNA integration and hepatocyte clonal expansion, suggesting another alternative pathway besides direct viral suppression to reduce HCC risk. Our findings supported the notion for a long-term NUC treatment to prevent HCC.

A High-Performance Hybrid Biofuel Cell with a Honeycomb-Like Ti3 C2 Tx /MWCNT/AuNP Bioanode and a ZnCo2 @NCNT Cathode for Self-Powered Biosensing.

This work focusses on developing a hybrid enzyme biofuel cell-based self-powered biosensor with appreciable stability and durability using murine leukemia fusion gene fragments (tDNA) as a model analyte. The cell consists of a Ti3 C2 Tx /multiwalled carbon nanotube/gold nanoparticle/glucose oxidase bioanode and a Zn/Co-modified carbon nanotube cathode. The bioanode uniquely exhibits strong electron transfer ability and a high surface area for the loading of 1.14 × 10-9  mol cm-2 glucose oxidase to catalyze glucose oxidation. Meanwhile, the abiotic cathode with a high oxygen reduction reaction activity negates the use of conventional bioenzymes as catalysts, which aids in extending the stability and durability of the sensing system. The biosensor offers a 0.1 fm-1 nm linear range and a detection limit of 0.022 fm tDNA. Additionally, the biosensor demonstrates a reproducibility of ≈4.85% and retains ≈87.42% of the initial maximal power density after a 4-week storage at 4 °C, verifying a significantly improved long-term stability.

Effect of corticosteroid dosing on outcomes in high-grade immune checkpoint inhibitor hepatitis.

BACKGROUND AND AIMS: Consensus guidelines recommend high-dose corticosteroids (1-2 mg/kg/day methylprednisolone equivalents) for treating grade ≥3 immune checkpoint inhibitor (ICI) hepatitis. We examined the effect of corticosteroid dosing on time to alanine aminotransferase (ALT) normalization, need for additional immunosuppression, and steroid-related complications. APPROACH AND RESULTS: We conducted a retrospective cohort study of 215 ICI-treated patients from 2010 to 2020 who developed grade ≥3 (ALT > 200 U/L) ICI hepatitis. Patients were grouped by initial corticosteroid dose (≥1.5 mg/kg or <1.5 mg/kg methylprednisolone equivalents). Propensity scores were calculated predicting the risk of receiving the higher steroid dose and used in inverse probability of treatment weighted (IPTW) logistic or Cox regression. The 87 patients in the ≥1.5 mg/kg group received higher initial (2.0 vs. 0.8 mg/kg/day, p < 0.001) and maximum (2.0 vs. 1.0 mg/kg/day, p < 0.001) steroid doses than the 128 patients in the <1.5 mg/kg group. There was no difference between the higher versus lower-dose groups in development of steroid-refractory hepatitis (OR 1.22, 95% CI 0.79-1.89, p = 0.365) on IPTW-logistic regression. In patients with steroid-responsive disease, there was no difference between the two groups in time to ALT normalization using either standard Cox regression (HR 1.02, 95% CI 0.72-1.45, p = 0.903) or IPTW-Cox regression (HR 1.09, 95% CI 0.78-1.51, p = 0.610). The ≥1.5 mg/kg group had longer exposure to corticosteroids (median 60 vs. 44 days, p = 0.005) and higher incidences of infection (18.4% vs. 7.0%, relative risk [RR] 2.6, 95% CI 1.2-5.6, p = 0.011) and hyperglycemia requiring treatment (23.3% vs. 7.8%, RR 3.0, 95% CI 1.5-6.0, p = 0.001). CONCLUSIONS: In patients with high-grade ICI hepatitis, initial treatment with 1 mg/kg/day methylprednisolone equivalents provides similar hepatitis outcomes with reduced risk of steroid-related complications when compared with higher-dose regimens.

miR-181b regulates vascular endothelial aging by modulating an MAP3K3 signaling pathway.

Endothelial cell (EC) aging plays a vital role in the pathogenesis of cardiovascular disease (CVD). MicroRNAs have emerged as crucial regulators of target gene expression by inhibiting mRNA translation and/or promoting mRNA degradation. We identify an aging-related and oxidative stress-responsive microRNA, miR-181b, that inhibits endothelial cell apoptosis and senescence. In gain- or loss-of-function studies, miR-181b regulated the expression of key apoptosis markers (Bcl2, Bax, cleaved-Caspase3) and senescence markers (p16, p21, γH2AX) and the ratio of apoptotic cells (TUNEL-positive) and senescent cells (SA-ßgal-positive) in H2 O2 -induced ECs. Mechanistically, miR-181b targets MAP3K3 and modulates a MAP3K3/MKK/MAPK signaling pathway. MAP3K3 knockdown recapitulated the phenotype of miR-181b overexpression and miR-181b was dependent on MAP3K3 for regulating EC apoptosis and senescence. In vivo, miR-181b expression showed a negative correlation with increasing age in the mouse aorta. Endothelial-specific deficiency of miR-181a2b2 increased the target MAP3K3, markers of vascular senescence (p16, p21), and DNA double-strand breaks (γH2AX) in the aorta of aged mice. Collectively, this study unveils an important role of miR-181b in regulating vascular endothelial aging via an MAP3K3-MAPK signaling pathway, providing new potential therapeutic targets for antiaging therapy in CVD.

Early Liver Specialist Consultation is Associated With Faster Biochemical Resolution of Severe Immune Checkpoint Inhibitor-Induced Hepatitis.

BACKGROUND: We evaluated the impact of gastroenterology/hepatology consultation, as recommended by guidelines, on the management of severe immune checkpoint inhibitor (ICI)-induced hepatitis. METHODS: We conducted a multicenter, retrospective cohort study of 294 patients who developed grade ≥3 (alanine aminotransferase [ALT] >200 U/L) ICI-induced hepatitis, with early gastroenterology/hepatology consultation defined as occurring within 7 days of diagnosis. The primary outcome was time to ALT normalization (≤40 U/L), and the secondary outcome was time to ALT improvement to ≤100 U/L. RESULTS: A total of 117 patients received early consultation. In the 213 patients with steroid-responsive hepatitis, early consultation was not associated with faster ALT normalization (hazard ratio [HR], 1.12; 95% CI, 0.83-1.51; P=.453). A total of 81 patients developed steroid-refractory hepatitis, with 44 (54.3%) receiving early consultation. In contrast to the patients whose hepatitis responded to steroid treatment, early consultation in those with steroid-refractory disease was associated with faster ALT normalization (HR, 1.89; 95% CI, 1.12-3.19; P=.017) and ALT improvement to ≤100 U/L (HR, 1.72; 95% CI, 1.04-2.84; P=.034). Notably, additional immunosuppressive therapy for steroid-refractory disease was initiated sooner after diagnosis in the early consult group (median 7.5 vs 13.0 days; log-rank P=.001). When time to additional immunosuppression was added as a covariate to the Cox model in mediation analysis, early consultation was no longer associated with time to ALT normalization (HR, 1.39; 95% CI, 0.82-2.38; P=.226) or with time to ALT improvement to ≤100 U/L (HR, 1.25; 95% CI, 0.74-2.11; P=.404). Time to additional immunosuppression remained associated with faster ALT normalization and faster ALT improvement to ≤100 U/L in the model, suggesting that the faster hepatitis resolution in the early consultation group was primarily attributable to earlier initiation of additional immunosuppression. CONCLUSIONS: Early gastroenterology/hepatology consultation is associated with faster resolution of biochemical abnormalities in patients with steroid-refractory hepatitis. This beneficial effect appears to be mediated by earlier initiation of additional immunosuppressive therapy in those receiving early consultation.

Anti-HBc: a significant host predictor of spontaneous HBsAg seroclearance in chronic hepatitis B patients - a retrospective longitudinal study.

BACKGROUND AND AIM: In chronic hepatitis B infection (CHB), seroclearance of hepatitis B surface antigen (HBsAg) is associated with favourable clinical outcomes compared to those with persistent HBsAg seropositivity, and thus considered as a desired treatment endpoint. This current study explores the possibility of serum antibody to hepatitis B core antigen (anti-HBc) as a potential predictive factor of HBsAg seroclearance. METHODS: This is a retrospective study that analyzed the plasma samples of CHB patients using the LUMIPULSE® G1200 analyzer. The longitudinal anti-HBc level between patients who subsequently achieved HBsAg seroclearance (S-losers) and those with persistent HBsAg-positivity (controls) were compared at multiple time points before the event. RESULTS: A total of 240 subjects (120 S-losers and 120 controls; age- and gender-matched) were included (mean age 56.42 ± 10.81, 65% male). Compared to controls, S-losers had significantly lower plasma anti-HBc levels prior to HBsAg seroclearance, with a significant trend of declining plasma anti-HBc 8-5 years prior to HBsAg seroclearance (p < 0.01), while such trend was not observed in controls. ROC curve analysis revealed that plasma anti-HBc at multiple time points before HBsAg seroclearance return AUC greater than 0.7. Plasma anti-HBc level at the cut-off value of 82.50 COI was 68.3% sensitive and 90% specific for HBsAg seroclearance within 1 year. Combining with quantitative HBsAg < 100 IU/mL, anti-HBc < 82.5 COI identified 88.2% patients who would develop HBsAg seroclearance within 1 year. CONCLUSION: Plasma anti-HBc level began to decline 10 years prior to HBsAg seroclearance and can serve as a potential predictor for subsequent HBsAg seroclearance.

Stanniocalcin 1 is a serum biomarker and potential therapeutic target for HBV-associated liver fibrosis.

Stanniocalcin 1 (STC1), a secreted protein, is upregulated in human cancers including hepatocellular carcinoma (HCC). While most HCCs develop from chronic liver disease, which involves progressive parenchymal injury and fibrosis, the role of STC1 in this preneoplastic stage remains poorly understood. In this study we investigated the clinical relevance and functional significance of secreted STC1 in liver fibrosis. To this end, the STC1 level was determined in the serum samples of chronic hepatitis B patients and correlated with the degree of liver fibrosis. Diagnostic performance of STC1 was analysed by area under the receiver operating characteristic curve (AUROC), sensitivity, specificity, positive predictive value, and negative predictive value. The results were compared with other well-characterised serum biomarkers for liver fibrosis: Aspartate transaminase to Platelet Ratio Index (APRI) and Fibrosis-4 (FIB-4). The functional role of STC1 was interrogated by in vitro experiments using cell line models. Expression of fibrogenic markers was quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Our results showed that the serum STC1 level in chronic hepatitis B patients was positively correlated with the degree of liver fibrosis and showed a stepwise increase in accordance with the severity of fibrosis. The AUROCs for detecting significant fibrosis (>9.0 kPa) and cirrhosis (>12.0 kPa) was 0.911 and 0.880, respectively. STC1 demonstrated a superior specificity and positive predictive value when compared to APRI and FIB-4. Consistent with this, STC1 was elevated in the liver tissues and sera of CCl4 -treated mice showing marked liver fibrosis. In vitro, STC1 was secreted by the human hepatic stellate cell line LX2. Human recombinant STC1 (rhSTC1) induced expression of fibrogenic markers in LX2 cells. The profibrogenic phenotype conferred by rhSTC1 or TGF-ß1 in LX2 cells could be attenuated using anti-STC1 antibody. Taken together, STC1 is a specific serum biomarker for HBV-associated liver fibrosis. STC1 functionally promotes liver fibrogenesis and is a potential actionable target. © 2022 The Pathological Society of Great Britain and Ireland.

Long-term serological, virological and histological responses to RNA inhibition by ARC-520 in Chinese chronic hepatitis B patients on entecavir treatment.

OBJECTIVE: We examined the serological, virological (in serum and liver) and histological profiles in chronic hepatitis B virus (HBV) patients during and after completion of multiple dose (MD) ARC-520. DESIGN: The present phase 1b study was a multidose, open-label extension cohort of patients that had received single dose ARC-520 in our previous study. Eight patients received 4-9 4 weekly doses of MD ARC-520 and entecavir. Liver biopsies were performed in six patients. Intrahepatic and serum HBV DNA, HBV RNA and viral antigens were measured. RESULTS: All patients had 28.9-30.4 months of follow-up after the last MD. All three hepatitis B e antigen (HBeAg)-positive patients had profound reductions in hepatitis B surface antigen (HBsAg), HBeAg, hepatitis B core-related antigen and HBV RNA with two undergoing HBeAg seroconversion. One further achieved HBsAg seroconversion (anti-HBs level of 25.1 IU/L) and the remaining two had HBsAg reductions of -1.7 and -3.5 log IU/mL >30 months after MD. Among the five HBeAg-negative patients, four had modest HBsAg reduction >29 months after completion of MD and one achieved HBsAg seroconversion (anti-HBs level of 152.5 IU/L) and was negative for liver HBsAg staining. Entecavir was successfully stopped in this patient 12 months after HBsAg seroconversion. Temporally related alanine aminotransferase elevations preceded by HBsAg reductions were observed in three patients suggesting immune activation. HBcAg staining was negative in all six biopsied patients. Two patients with <10% HBsAg positive staining of hepatocytes had correspondingly low serum HBsAg levels of 1.5 and 11.5 IU/mL. CONCLUSIONS: MD ARC-520 therapy achieved sustained and profound reductions of viral antigens and HBV RNA. HBsAg seroclearance was achievable. TRIAL REGISTRATION NUMBER: NCT02065336.

Hepatitis B virus X protein promotes hepatocarcinogenesis via the activation of HMGA2/STC2 signaling to counteract oxidative stress-induced cell death.

Chronic hepatitis B virus (HBV) infection can cause oxidative stress and induce cell death. The mechanisms by which cells overcome oxidative stress to survive remain largely unknown. Here, we used human sera, liver tissues and cell lines to study how HBV modulates cellular pathways to counteract oxidative stress-induced cell death. We found high-mobility group AT-hook 2 (HMGA2), an architectural transcription factor is upregulated in hepatocellular carcinoma (HCC) tissues and cell lines. Elevated serum HMGA2 is significantly associated with viral load in HBV carriers, and HBV-related HCC. We showed that HBV X protein (HBx) encoded by HBV-induced cell growth via HMGA2 activation. The growth-promoting effect is abolished when HMGA2 is suppressed. Ectopic HBx expression induced DNA damage and oxidative stress. HMGA2 silencing reduced oxidative stress in HBx-expressing cells. Cytoprotective stanniocalcin 2 (STC2) protein is a downstream target of HMGA2. Consistent with the findings in HMGA2, STC2 mRNA and protein expression are upregulated in HCC tissues. Elevated serum STC2 is also associated with viral load in HBV carriers, and HCC. STC2 is transcriptionally upregulated by HBx and HMGA2 to elicit cytoprotection against apoptosis. STC2 knockdown disrupted Bax/Bcl-2 balance that increased cytochrome c release, caspase 3/7 activity and apoptosis, and thus abolished the growth-promoting effect of HMGA2. Clinical relevance of HBx/HMGA2/STC2 signaling is evidenced by the significant correlation of serum HMGA2/STC2 in active HBV infection and HCC. These findings reveal a novel HBx regulatory HMGA2/STC2 pathway in counteracting reactive oxygen species-induced cell death. HMGA2 and STC2 may be therapeutic targets for prevention of hepatocarcinogenesis in chronic HBV infection.

Enhanced Liver Fibrosis Score Stratifies Hepatocellular Carcinoma Risk in Patients With Hepatitis B Surface Antigen Seroclearance.

In this prospective study involving 337 chronic hepatitis B patients who achieved spontaneous hepatitis B surface antigen (HBsAg) seroclearance (SC), serum enhanced liver fibrosis (ELF) before SC was associated with hepatocellular carcinoma (HCC) (hazard ratio 2.588), and ELF <10.8 was associated with >97% reduction in risk of HCC development in patients with age SC ≥ 50 (n = 190).