Varied immune responses of HBV-specific B cells in patients undergoing pegylated interferon-alpha treatment for chronic hepatitis B.

BACKGROUND & AIMS: The changes of HBV-specific B-cells in chronic hepatitis B (CHB) patients underwent pegylated interferon-alfa (PEG-IFNα) treatment and achieved functional cure remain unclear. We aimed to evaluate the alterations in HBV-specific B-cells during treatment and therefore explored the mechanism of functional recovery of HBsAg-specific B-cells. METHODS: We included 39 nucleos(t)ide analogues-treated CHB patients who received sequential combination therapy with PEG-IFNα and 8 treatment-naive CHB patients. HBV-specific B-cells were characterized ex vivo using fluorescent labeled HBsAg and HBcAg. The frequency, phenotype, and subsets of HBV-specific B-cells and follicular helper T cells (Tfh-cells) were detected using flow cytometry. The functionality of HBV-specific B-cells was quantified through ELISpot assays. RESULTS: During treatment, the fraction of activated memory B-cells (MBCs) among HBsAg-specific B-cells and the expression of IgG, CXCR3, and CD38 increased. Antibody-secretion capacity of HBsAg-specific B-cell was restored after treatment only in patients with a functional cure and it showed a positive correlation with serum hepatitis B surface antibody levels. The phenotype and function of HBsAg-specific B-cells differed between patients with and without functional cure. Patients with functional cure exhibited IgG+ classical MBCs and plasmablasts in HBsAg-specific B-cells. HBcAg-specific B-cells displayed both attenuated antibody secretion with reduced IgG expression and an IgM+ atypical type of MBCs after treatment, irrespective of with and without functional cure. The number of CD40L+ Tfh-cells increased after PEG-IFNα treatment and positively correlated with HBsAg-specific B-cell activation. CONCLUSIONS: After PEG-IFNα treatment, HBsAg- and HBcAg-specific B-cells exhibit various changes in antibody secretion. Their functional differences are reflected in the alterations in phenotypes and subtypes. The presence of CD40L+ Tfh-cells is associated with the active recovery of HBsAg-specific B-cells. IMPACT AND IMPLICATIONS: HBV-related complications and hepatocellular carcinoma remain the leading causes of mortality from chronic liver disease worldwide, and a cure is rarely achieved with antiviral therapies. Elucidating the immunological mechanisms underlying the functional cure of CHB patients offers a promising therapeutic strategy for viral clearance, such as therapeutic vaccine. We analyzed the alterations in HBV-specific B-cells in patients treated with PEG-IFNα and identified novel pathways for immunotherapeutic boosting of B cell immunity.

Monocyte distribution width as a promising biomarker for differential diagnosis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma.

Objective: We aimed to investigate the association and diagnostic value of monocyte distribution width (MDW) for chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Methods: MDW levels were measured in 483 individuals (103 CHB, 77 LC, 153 HCC, and 150 controls). MDW was detected using UniCel Dx900 for specific cell volume parameters and the distribution of cell volumes. Results: Our findings revealed a dynamic upward change in MDW levels across different stages of chronic liver disease, from CHB to LC and HCC. In CHB, MDW levels were highest among HBeAg-positive CHB patients and exhibited a negative correlation with HBV markers while positively correlating with ALT levels. In LC, MDW showed a positive association with the pathological progression of LC, demonstrating consistency with CP scores. MDW proved to be equally effective as traditional detection for diagnosing LC. In HCC, MDW was positively correlated with HCC occurrence and development, with higher levels observed in the high MDW group, which also exhibited elevated AFP levels, MELD scores, and 90-day mortality rates. MDW surpassed predictive models in its effectiveness for diagnosing HCC, as well as CHB and LC, with respective areas under the curve of 0.882, 0.978, and 0.973. Furthermore, MDW emerged as an independent predictor of HCC. Conclusion: MDW holds significant diagnostic efficacy in identifying CHB, LC, and HCC. These findings suggest that MDW could serve as a promising biomarker for predicting the severity of liver diseases and aid in rational clinical treatment strategies.

A novel microfluidic chip-based digital PCR method for enhanced sensitivity in the early diagnosis of colorectal cancer via mSEPT9.

BACKGROUND: To enhance the sensitivity of plasma methylated Septin9 gene (mSEPT9) detection in colorectal cancer (CRC) screening, we developed a microfluidic chip-based digital PCR (dPCR) method suitable for low-concentration samples, aiming to apply it for mSEPT9 detection in CRC diagnosis. METHODS: Our microfluidic chip-based dPCR method utilized specific primers and probes with locked nucleic acids (LNAs) modifications for mSEPT9 detection. We evaluated its performance, including detection limit, specificity, and linear range, comparing it with a commercial qPCR reagent kit using the same samples (95 CRC, 23 non-CRC). RESULTS: The LNAs-modified dPCR method showed a linear range of 100-104 copies/µL and a detection limit of 100 copies/µL. Clinical testing revealed that our dPCR method exhibited a sensitivity of 82.11 % and specificity of 95.65 % for CRC diagnosis, outperforming the commercial qPCR kit (sensitivity: 58.95 %, specificity: 91.30 %), particularly in Stage I with a diagnostic sensitivity of 90.91 %. Combining mSEPT9 and carcinoembryonic antigen (CEA) improved diagnostic sensitivity to 91.49 %. CONCLUSIONS: Our accurate microfluidic chip-based dPCR method, especially in combination with CEA, holds promise for effective CRC screening and timely interventions, offering enhanced mSEPT9 quantification over conventional qPCR.

Oncostatin M Induces IFITM1 Expression to Inhibit Hepatitis B Virus Replication Via JAK-STAT Signaling.

BACKGROUND & AIMS: Functional cure is achieved by a limited number of patients with chronic hepatitis B (CHB) after nucleotide analogue(s) and interferon treatment. It is urgent to develop therapies that can help a larger proportion of patients achieve functional cure. The present study was designed to explore the anti-hepatitis B virus (HBV) potency of interleukin-6 family cytokines and to characterize the underlying mechanisms of the cytokine displaying the highest anti-HBV potency. METHODS: HBV-infected cells were used to screened the anti-HBV potency of interleukin-6 family cytokines. The concentration of oncostatin M (OSM) in patients with chronic HBV infection was examined by enzyme-linked immunosorbent assay. The underlying mechanism of OSM anti-HBV was explored through RNA-seq. C57BL/6 mice injected with rAAV8-1.3HBV were used to explore the suppression effect of OSM on HBV in vivo. RESULTS: OSM is the most effective of the interleukin-6 family cytokines for suppression of HBV replication (percentage of average inhibition: hepatitis B surface antigen, 34.44%; hepatitis B e antigen, 32.52%; HBV DNA, 61.57%). Hepatitis B e antigen-positive CHB patients with high OSM levels had lower hepatitis B surface antigen and hepatitis B e antigen than those with low levels. OSM activated JAK-STAT signaling pathway promoting the formation of STAT1-IRF9 transcription factor complex. Following this, OSM increased the expression of various genes with known functions in innate and adaptive immunity, which was higher expression in patients with CHB in immune clearance phase than in immune tolerance phase (data from GEO: GSE65359). Interferon-induced transmembrane protein 1, one of the most differentially expressed genes, was identified as an HBV restriction factor involved in OSM-mediated anti-HBV effect. In vivo, we also found OSM significantly inhibited HBV replication and induced expression of antiviral effector interferon-induced transmembrane protein 1. CONCLUSIONS: Our study shows that OSM remodels the immune response against HBV and exerts potent anti-HBV activity, supporting its further development as a potential therapy for treating CHB.

SIRT1 and miR-34a-5p Expression in PBMCs as Potential Biomarkers for Patients With Type 2 Diabetes With Cognitive Impairments.

CONTEXT: Patients with type 2 diabetes mellitus (T2DM) are at significantly increased risk of Alzheimer disease (AD). However, no biomarkers are available for early identification of patients with T2DM with cognitive impairment (T2DM-CI). Mitochondrial dysfunction is linked to AD. Silent Information Regulator 1 (SIRT1), which is responsible for regulating mitochondrial biogenesis, and its related miRNAs were also altered in AD. OBJECTIVE: This study aimed to determine whether mitochondrial function in peripheral blood mononuclear cells (PBMCs) of patients with T2DM-CI was altered and if these alterations could be used as biomarkers. METHODS: A total of 374 subjects were enrolled, including AD, T2DM-CI, T2DM-nCI (T2DM without cognitive impairment), and healthy controls. The mitochondrial function was determined using a commercial assay kit. The mitochondrial DNA (mtDNA) content, the expression of SIRT1, and selected miRNAs in PBMCs were measured by quantitative polymerase chain reaction. The correlations and diagnostic accuracy were assessed using the Spearman correlation coefficient or receiver operating characteristics analysis, respectively. RESULTS: We found significant changes in mitochondrial function in PBMCs of patients with AD compared with controls (all P < .05), which were not found in T2DM-CI. However, mtDNA content and SIRT1 mRNA expression were lower in PBMCs of patients with T2DM-CI, while miR-34a-5p expression was higher than in patients with T2DM-nCI (all P < .05). A combination of SIRT1 and miR-34a-5p demonstrated excellent discrimination between T2DM-CI and T2DM-nCI (area under the curve = 0.793; sensitivity: 80.01%; specificity: 78.46%). Furthermore, correlation analysis revealed a link between miR-34a-5p expression and hyperglycemia in T2DM-CI. CONCLUSION: Our findings revealed that there was an alteration of mitochondria at the peripheral level in patients with T2DM-CI. SIRT1 combined with miR-34a-5p in PBMCs performed well in identifying patients with T2DM-CI and may be a promising biomarker.

Detection of serum large and middle hepatitis B virus surface proteins: A novel potential diagnostic and prognostic biomarker for chronic hepatitis B.

BACKGROUND: The significance of large (LHB) and middle (MHB) HBV surface proteins in chronic hepatitis B (CHB) remains uncertain. This study investigates the role of LHB and MHB in different infection phases and liver diseases. METHODS: Serum samples from 217 patients with HBV chronic infection, CHB, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) were subjected to quantification of LHB and MHB using ELISA. RESULTS: Positive correlations were observed among LHB, MHB, and LHB/HBsAg, with HBV serum markers including HBsAg, HBeAg, and HBV DNA. (P < 0.0001). In HBeAg-positive chronic infection, LHB and MHB were higher than in HBeAg-positive CHB (P < 0.01). In HBeAg-negative chronic infection, LHB and MHB were lower than in HBeAg-negative CHB (P < 0.01). ROC analysis identified LHB and MHB as potential discriminators of CHB and chronic infection. LC and HCC exhibited lower LHB, MHB, and MHB/HBsAg than CHB (P < 0.05). Multivariate analysis found that age and the MHB/HBsAg serve as independent factors for the progression of CHB to end stage of liver disease. CONCLUSIONS: LHB and MHB emerge as novel biomarkers distinguishing chronic infection and CHB. MHB/HBsAg shows promise as a predictor for CHB progression.

A genetic variant in the CD40 gene is related to HBV infection in the Chinese Han population.

BACKGROUND: CD40 is an important immune costimulatory molecule that has recently been found to be associated with chronic hepatitis B. This study aims to explore the association between CD40 polymorphisms and HBV infection, as well as to investigate the impact of different rs1883832 genotypes on CD40 expression and its effect on the progression of chronic HBV infection. METHODS: We genotyped rs1883832 in 3433 individuals using MassARRAY, and quantified the CD40 expression, including CD40 mRNA, sCD40, and mCD40. The CD40 and HBV infection indicators were assessed to investigate the potential function of rs1883832 in suppressing HBV replication in HepG2.2.15 and HepAD38, CD40L in cytotoxic t lymphocytes (CTLs) and interferon-γ, TNF-α, granzyme B, and perforin were measured to elucidate the mechanism by which CD40 inhibits HBV replication. RESULTS: Our study revealed that the frequencies of CC genotype and C allele of rs1883832 were significantly higher in immune recovery compared to chronic hepatitis B. Individuals with CC genotype exhibited significantly elevated CD40 in serum and B cells compared to TT genotypes in chronic hepatitis B. Additionally, CD40 is capable of inhibiting HBV replication and transcription in hepatocytes by means of interaction with CD40L. A significant negative correlation was found between HBV DNA, HBeAg, and mCD40. Conversely, the expressions of ALT and mCD40 showed a positive correlation, which aligns with the trend of CD40L. CONCLUSIONS: rs1883832 C allele may have a protective role in HBV immune recovery. This protective effect could potentially be attributed to the regulation of CD40 expression. The activation of the anti-HBV immune response, which occurs through binding CD40L on CTL, can suppress HBV DNA replication and potentially facilitate immune recovery in HBV infection.

Diagnostic value of cerebrospinal fluid Neutrophil Gelatinase-Associated Lipocalin for differentiation of bacterial meningitis from tuberculous meningitis or cryptococcal meningitis: a prospective cohort study.

BACKGROUND: The early differential diagnosis between bacterial meningitis (BM) and tuberculous meningitis (TBM) or cryptococcal meningitis (CM) remains a significant clinical challenge. Neutrophil Gelatinase-Associated Lipocalin (NGAL) has been reported as a novel inflammatory biomarker in the early stages of infection. This study aimed to investigate whether cerebrospinal fluid (CSF) NGAL can serve as a potential biomarker for distinguishing between BM and TBM or CM. METHODS: We prospectively enrolled the patients with suspected CNS infections at admission and divided them into three case groups: BM (n = 67), TBM (n = 55), CM (n = 51), and an age- and sex-matched hospitalized control (HC, n = 58). Detected the CSF NGAL and assessed its diagnostic accuracy in distinguishing between BM and TBM or CM. Additionally, longitudinally measured the CSF NGAL levels in patients with BM to evaluate its potential as a monitoring tool for antibacterial treatment. RESULTS: The concentration of CSF NGAL in BM was significantly higher than in TBM, CM, and HC (all P < 0.05), while the serum NGAL did not show significant differences among the three case groups. The ROC analysis demonstrated that CSF NGAL presented a good diagnostic performance with an AUC of 0.834 (0.770-0.886) and at the optimal cutoff value of 74.27 ng/mL with 70.15% sensitivity and 77.36% specificity for discriminating BM with TBM and CM. Additionally, the CSF NGAL in the convalescent period of BM was significantly lower than in the acute period (P < 0.05). CONCLUSIONS: CSF NGAL may serve as a potential biomarker for distinguishing between acute BM and TBM or CM. Additionally, it holds clinical significance in monitoring the effectiveness of antibiotic therapy for BM.

Development and validation of a diagnostic model for AFP-negative hepatocellular carcinoma.

PURPOSE: AFP appears to be negative in about 30% of overall hepatocellular carcinoma (HCC). Our study aimed to develop a nomogram model to diagnose AFP-negative HCC (AFPN-HCC). PATIENTS AND METHODS: The training set included 294 AFPN-HCC patients, 159 healthy objects, 63 patients with chronic hepatitis B(CHB), and 64 patients with liver cirrhosis (LC). And the validation set enrolled 137 healthy controls objects, 47 CHB patients and 45 patients with LC. LASSO, univariate, and multivariable logistic regression analysis were performed to construct the model and then transformed into a visualized nomogram. The receiver operating characteristic (ROC) curves, the calibration curve, decision curve analysis (DCA), and clinical impact curve (CIC) were further used for validation. RESULTS: Four variables including age, PIVKA-II, platelet (PLT) counts, and prothrombin time (PT) were selected to establish the nomogram. The area under the curve (AUC) of the ROC to distinguish AFPN-HCC patients was 0.937(95% CI 0.892-0.938) in training set and 0.942(95% CI 0.921-0.963) in validation set. We also found that the model had high diagnostic value for small-size HCC (tumor size < 5 cm) (AUC = 0.886) and HBV surface antigen-positive AFPN-HCC (AUC = 0.883). CONCLUSIONS: Our model was effective for discrimination of AFPN-HCC from patients with benign liver diseases and healthy controls, and might be helpful for the diagnosis for AFPN-HCC.

Identification reproducible microbiota biomarkers for the diagnosis of cirrhosis and hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a malignant tumor with high incidence in China, which is mainly related to chronic hepatitis B (CHB) and liver cirrhosis (LC) caused by hepatitis B virus (HBV) infection. This study aimed to identify reproducible gut microbial biomarkers across Chinese population for LC and HCC diagnosis. In this study, a group of 21 CHB, 25 LC, 21 HCC and 15 healthy control (HC) were examined, and used as the training data. Four published faecal datasets from different regions of China were collected, totally including 121 CHB, 33 LC, 70 HCC and 96 HC. Beta diversity showed that the distribution of community structure in CHB, LC, HCC was significantly different from HC. Correspondingly, 14 and 10 reproducible differential genera across datasets were identified in LC and HCC, respectively, defined as LC-associated and HCC-associated genera. Two random forest (RF) models based on these reproducible genera distinguished LC or HCC from HC with an area under the curve (AUC) of 0.824 and 0.902 in the training dataset, respectively, and achieved cross-region validations. Moreover, AUCs were greatly improved when clinical factors were added. A reconstructed random forest model on eight genera with significant changes between HCC and non-HCC can accurately distinguished HCC from LC. Conclusively, two RF models based on 14 reproducible LC-associated and 10 reproducible HCC-associated genera were constructed for LC and HCC diagnosis, which is of great significance to assist clinical early diagnosis.

ATP13A2 is a Prognostic Biomarker and Correlates with Immune Infiltrates in Hepatocellular Carcinoma.

PURPOSE: To explore the expression and role of ATPase cation transporting 13A2 (ATP13A2) on hepatocellular carcinoma (HCC) progression and prognosis. METHODS: The level of ATP13A2 in 63 HCC tissues was evaluated by quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry. Then, the prognostic value of ATP13A2 for HCC was explored. GO and KEGG pathway enrichments were performed to predict ATP13A2-mediated biological functions. Besides, the correlations between ATP13A2 and key regulators involved in cell cycle and metastasis, the status of different tumor-infiltrating immune cells was investigated. RESULTS: ATP13A2 was frequently upregulated in 63 HCC tissues relatively to matched non-tumor tissues. The level of ATP13A2 significantly correlated with tumor stage and tumor grade. HCC patients with higher levels of ATP13A2 had a worse prognosis. Moreover, multivariate survival analysis supported ATP13A2 to be an independent prognostic factor for HCC. GO and KEGG analysis indicated a potential role of ATP13A2 on regulating cell cycle, metastasis, and immune infiltrates. Especially, the level of ATP13A2 was positively correlated with CCNB1, CCND3, CDC25B, CDK4, Vimentin, MMP9, MMP14, and LMNB2. A positive correlation was noticed between ATP13A2 and infiltration levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, dendritic cells, monocytes, M2 macrophages, and exhausted T cells in HCC. CONCLUSION: Upregulation of ATP13A2 is a common feature as well as an independent prognostic biomarker for HCC. ATP13A2 are associated with key regulators involved in cell cycle, metastasis, and immune infiltrates in HCC, and may act as a potential immunotherapy target for HCC.

IFIT3 Is Increased in Serum from Patients with Chronic Hepatitis B Virus (HBV) Infection and Promotes the Anti-HBV Effect of Interferon Alpha via JAK-STAT2 In Vitro.

Increasing evidence indicates that interferon alpha (IFN-α) therapy is an effective treatment option for a subgroup of patients with chronic hepatitis B virus (HBV) infection. It has been confirmed that interferon-induced protein with tetratricopeptide repeats 3 (IFIT3), a member of the interferon-stimulated genes (ISGs), could inhibit the replication of various viruses. However, its effect on HBV replication is unclear. The present study sought to explore the role and mechanism of IFIT3 in IFN-α antiviral activities against HBV. IFIT3 mRNA levels in the peripheral blood of 108 treatment-naive patients and 70 healthy controls were analyzed first. The effect of IFIT3 on the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway under the dual intervention of IFN-α and HBV was also explored in vitro. Treatment-naive individuals exhibited elevated levels of IFIT3 mRNA compared to the controls (P < 0.0001). Mechanistically, the knockdown of IFIT3 inhibited the phosphorylation of signal transducer and activator of transcription 2 (STAT2), whereas the overexpression of IFIT3 produced the opposite effect in vitro. Meanwhile, the overexpression of IFIT3 enhanced the expression of IFN-α-triggered ISGs, including myxovirus resistance A (MxA), 2'-5'-oligoadenylate synthetase 1 (OAS1), and double-stranded RNA-activated protein kinase (PKR), while a weaker induction of IFN-α-triggered ISGs was observed in ruxolitinib-treated cells. After decreasing IFIT3 expression by validated small hairpin RNAs (shRNAs), the levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and HBV DNA secreted by HepG2 cells transiently transfected with the pHBV1.2 plasmid were increased. Our findings suggest that IFIT3 works in a STAT2-dependent manner to promote the antiviral effect of IFN-α through the JAK-STAT pathway in HBV infection in both human hepatocytes and hepatocarcinoma cells. IMPORTANCE Our study contributes new insights into the understanding of the functions and roles of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3), which is one of the interferon-stimulated genes induced by hepatitis B virus infection in human hepatocytes and hepatocarcinoma cells, and may help to identify targeted genes promoting the efficacy of interferon alpha.

Emerging role of mitochondria in response to HBV infection.

Hepatitis B is a major global health problem that potentially life-threatening liver infection caused by the hepatitis B virus (HBV), which can lead to death due to liver cirrhosis and hepatocellular carcinoma (HCC). A considerable of research has demonstrated that mitochondrial dysfunction exists in patients with HBV infection, indicating that there is clinical relation between HBV infection and mitochondrial alterations. To explore the complex interplay between the functions of mitochondria and HBV infection in greater depth, we systematically summarized these mitochondrial alterations due to HBV infection in recent years. The liver is the central organ of metabolism that is a mitochondria-rich tissue and represents strong defense and regeneration capabilities in the body. Infested cells and their microenvironment must upregulate energy production for proliferation, growth, and effector functions to restrain the damage imposed by HBV. The changes in metabolic pathways caused by HBV infection are nothing more than those in the cytoplasm and mitochondria. Thus, this article brings into focus the effects of novel reprogramming of inner and outer mitochondria on HBV infection and then derives novel insights and new approaches for HBV diagnosis and therapy.

Compartmentalisation of Hepatitis B virus X gene evolution in hepatocellular carcinoma microenvironment and the genotype-phenotype correlation of tumorigenicity in HBV-related patients with hepatocellular carcinoma.

Hepatitis B virus (HBV) exists as quasispecies (QS). However, the evolutionary characteristics of haplotypes of HBV X gene in the hepatocellular carcinoma (HCC) microenvironment remain unclear. Mutations across X gene are essential for the tumorigenicity of HBV X protein (HBx). However, the functional phenotypes of many mutant HBx remain unknown. This study aims to compare the characteristics of X gene evolution between tumour and non-tumour tissues in HCC patients and investigate the tumorigenic phenotype of HBx harbouring mutation T81P/S101P/L123S. This study included 24 HCC patients. Molecular cloning of X gene was performed to analyse characteristics of haplotypes in liver tissues. HCC cell lines stably expressing wild-type or mutant HBx and subcutaneous tumour xenograft mouse model were used to assess HBx-T81P/S101P/L123S tumorigenicity. The mean heterogeneity of HBV QS across X gene in tumour tissues was lower than that in non-tumour tissues. A location bias was observed in X gene clones with genotype C or D in tumour tissues compared to those with genotype B. Mutations in genotype-C or - D clones were mainly clustered in the dimerization region and aa110-aa140 within the transactivation region. A novel mutation combination at residues 81, 101 and 123 was identified in tumour tissues. Further, HBx-T81P/S101P/L123S promotes cell proliferation and increases genomic instability, which was mediated by MYC. This study elucidates the compartmentalized evolution patterns of HBV X gene between intra tumour and non-tumour tissues in HCC patients and provides a new mechanism underlying HBV-driven hepatocarcinogenesis, suggesting a potential viral marker for monitoring HCC.

Adenosine Triphosphate in Serum as a Promising Biomarker for Differential Diagnosis of Hepatitis B Disease Progression.

The need to be diagnosed with liver biopsy makes the clinical progression of chronic HBV infection diagnosis a challenge. Existing HBV serum biochemical assays are used throughout clinical but have limited effects. Studies have shown that mitochondrial function is tightly coupled to HBV infection. Here, we verified the diagnostic value of serum Adenosine Triphosphate (ATP) as a potential marker for differential HBV infection progress by detecting the level of ATP in the serum from a wide spectrum of HBV-infected populations, and confirmed the role of ATP in the deterioration of HBV infection-related diseases through HBV-infected cells and mouse models. The results showed that there were significantly lower serum ATP levels in HBeAg-positive CHB patients compared with healthy controls. And during the progression of CHB to liver cirrhosis and hepatocellular carcinoma, the ATP level was increased but not higher than healthy controls. The area under the curve (AUC) of serum ATP was 0.9063 to distinguish HBeAg-positive CHB from healthy, and another AUC was 0.8328 in the CHB against the HCC group. Preliminary exploration of the mechanism indicated that the decline of serum ATP was due to impaired mitochondria in CHB patients. Our data provide evidence that serum ATP distinguishes the various progress of HBV infection-related diseases and expands diagnostic biomarkers for HBeAg-positive CHB patients with healthy controls.

CCN1 Promotes Inflammation by Inducing IL-6 Production via α6ß1/PI3K/Akt/NF-κB Pathway in Autoimmune Hepatitis.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease with unknown etiology. CCN1, an extracellular matrix-associated protein, is associated with carcinoma, inflammation, liver fibrosis, and even autoimmune diseases. However, the role that CCN1 plays in AIH has remained undetermined. In this study, expression of CCN1 in liver was detected by real-time PCR, western blot and immunohistochemistry (IHC). CCN1 level in serum was detected by ELISA. Diagnostic value of CCN1 was determined by receiver operating characteristic (ROC) curve analysis. CCN1 conditional knockout (CCN1 fl/fl Cre+) mice were generated by mating CCN1 fl/fl C57BL/6J and CAG-Cre-ERT C57BL/6J mice. Autoimmune hepatitis mice model was induced by concanavalin A (ConA). IKKα/ß, IκBα, NF-κB p65 and Akt phosphorylation were determined by western blot. NF-κB p65 nuclear translocation was examined by immunofluorescence. Here, we found that CCN1 was over-expressed in hepatocytes of AIH patients. CCN1 level also increased in serum of AIH patients compared to healthy controls (HC). ROC curve analysis results showed that serum CCN1 was able to distinguish AIH patients from HD. In ConA induced hepatitis mice model, CCN1 conditional knockout (CCN1 fl/fl Cre+) attenuated inflammation by reducing ALT/AST level and IL-6 expression. In vitro, CCN1 treatment dramatically induced IL-6 production in LO2 cells. Moreover, the production of IL-6 was attenuated by CCN1 knockdown. Furthermore, we showed that CCN1 could activate IL-6 production via the PI3K/Akt/NF-κB signaling pathway by binding to α6ß1 receptor. In summary, our results reveal a novel role of CCN1 in promoting inflammation by upregulation of IL-6 production in AIH. Our study also suggests that targeting of CCN1 may represent a novel strategy in AIH treatment.

Serum-Derived Exosomal miR-140-5p as a Promising Biomarker for Differential Diagnosis of Anti-NMDAR Encephalitis With Viral Encephalitis.

Background: Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is the most common type of autoimmune encephalitis. Early recognition and treatment, especially distinguishing from viral encephalitis (VE) in the early stages, are crucial for the outcomes of patients with anti-NMDAR encephalitis. Compared with plasma microRNAs (miRNAs), exosomal miRNAs are more abundant and not easy to degrade. Moreover, exosomes can pass through the blood-brain barrier. This study aimed to explore the clinical value of serum exosomal miRNAs in the differential diagnosis of anti-NMDAR encephalitis with VE. Method: Serum samples from a total of 30 patients with anti-NMDAR encephalitis, 30 patients with VE, and 30 cases of control patients hospitalized in the same period were collected. Firstly, the serum exosomes were isolated and identified by transmission electron microscope (TEM), nanoparticle-tracking analyzer (NTA), and Western blot (WB). The expression levels of let-7b and miR-140-5p from serum exosomes were detected by real-time quantitative PCR (qPCR). At the same time, we also detected complement 3 (C3), complement 4 (C4), and high sensitivity CRP (hs-CRP) expression levels in three groups. Finally, we analyzed the difference and diagnostic value of the test results. Results: Isolated particles showed identical characteristics to the exosomes through TEM, NTA, and WB analyses. Compared with the VE group and control group, the expression of miR-140-5p was significantly upregulated in serum exosomes of the NMDAR group. In contrast, the serum C3 in the NMDAR group was significantly lower than the other two groups. ROC curve analysis showed the area under the curve (AUC) of serum exosomal miR-140-5p and serum C3 was 0.748 (76.67% sensitivity and 73.33% specificity) and 0.724 (76.67% sensitivity and 60% specificity) to distinguish anti-NMDAR encephalitis from VE, respectively. The AUC of serum exosomal miR-140-5p combined with serum C3 was 0.811, the sensitivity was 70.00%, and the specificity was 86.67%. Conclusion: Serum exosomal miR-140-5p combined with serum C3 would be a promising marker in the differential diagnosis of anti-NMDAR encephalitis with VE.

Circulating Cysteine Rich Protein 61 (Cyr61, CCN1) in Chinese Adults: Distribution and Reference Intervals.

BACKGROUND: The circulating levels of Cyr61 (also known as CCN1) may prove to have great clinical value in the diagnosis, monitoring and prognosis of many disorders in humans. However, the reference intervals (RIs) for this analyte in human subjects have not previously been well established. Therefore, establishing RIs and determining the distribution of circulating Cyr61 levels are very important for future clinical studies and could provide an orientation value for exploring its clinical usefulness. METHODS: The Cyr61 levels in 2,514 healthy Chinese Han subjects (1,250 males and 1,264 females, aged 18 - 88 years, recruited from 4 hospitals in Shanghai and Fujian) were measured with a sandwich ELISA (R&D Systems, USA). The RIs were determined in a manner consistent with the Clinical and Laboratory Standards Institute guidelines. RESULTS: The levels of serum Cyr61 showed a non-Gaussian distribution. A statistically significant difference was observed between the males and females such that the median level of Cyr61 in the males was significantly higher than that in the females. Furthermore, the Cyr61 levels significantly increased with age in the female group whereas no difference was observed among the different age groups among the males. The RIs for serum Cyr61 were 3.3 - 184 pg/mL and 5.0 - 182 pg/mL in females aged 18 - 45 and 46 - 88 years, respectively. The RI for serum Cyr61 was 4.0 - 198 pg/mL in the males. CONCLUSIONS: The RIs for serum Cyr61 were established among Chinese Han individuals. The effects of age and gender on the distribution characteristics of serum Cyr61 were studied, revealing that the RIs were gender and, in females, age-specific, which may suggest that a female hormone, estrogen plays a role in the regulation of Cyr61 expression in vivo.

IL-17 and IL-21 polymorphisms in relation to HBV related hepatocellular carcinoma in Chinese Han population.

OBJECTIVE: Inflammatory cytokine gene polymorphisms may influence the hepatic and extrahepatic HBV-related disease. In this study, we aimed to investigate the relationship between polymorphisms of IL-17, IL-21 gene and HBV related hepatocellular carcinoma in Chinese Han population. METHODS: We performed a multi-center study comprised 866 HBV-related hepatocellular carcinoma (HCC) patients and 1086 unrelated patients with a diagnosis of chronic hepatitis B (CHB) as control to evaluate the effects of IL-17 (rs4711998), IL-21 SNPs (rs12508721, rs13143866 and rs2221903) and the susceptibility of HCC. MassARRAY technology was utilized to genotype. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum IL-17 and IL-21 level. Quantitative real time polymerase chain reaction (qRT-PCR) was used to analyze the serum viral loads. RESULTS: In logistic regression analysis, our results showed the frequency of rs4711998 allele G in CHB group was significantly higher than that in HCC group (P = 0.042, 0.859(0.743-0.994)), and it is present only among females. Compared to HCC group, rs13143866 A allele was more likely to appear in HCC group (P = 0.015, 1.268 (1.049-1.532)). The frequency of AA also showed different between HCC group and CHB groups (P = 0.011, 3.135 (1.292-7.603)), which showed strong sex-specific relationships. ELISA showed a higher serum IL-17 and IL-21 expression in HCC patients compared to CHB patients (P all <0.05). Haplotype rs12508721C/rs13143866A/rs2221903T in male HCC group was statistically higher than in male CHB group(P = 0.013) but not in females (P > 0.05). CONCLUSION: We suggested rs4711998 allele A as risk factors for women to develop HBV related-HCC in Chinese Han population. rs13143866 allele A as risk factors to develop HBV related-HCC in Chinese male population. Male patients with haplotype rs12508721C/rs13143866A/rs2221903T may with 1.3-fold risk for HBV-related HCC.