Establishment and characterization of a highly metastatic hepatocellular carcinoma cell line.

The prevalence of alcohol-related hepatocellular carcinoma (HCC) has been increasing during the last decade. Cancer research requires cell lines suitable for both in vitro and in vivo assays. However, there is a lack of cell lines with a high in vivo metastatic capacity for this HCC subtype. Herein, a new HCC cell line was established, named HCC-ZJ, using cells from a patient diagnosed with alcohol-related HCC. The karyotype of HCC-ZJ was 46, XY, del (p11.2). Whole-exome sequencing identified several genetic variations in HCC-Z that occur frequently in alcohol-associated HCC, such as mutations in TERT, CTNNB1, ARID1A, CDKN2A, SMARCA2, and HGF. Cell counting kit-8 assays, colony formation assays, and Transwell assays were performed to evaluate the proliferation, migration, and sensitivity to sorafenib and lenvatinib of HCC-Z in vitro. HCC-ZJ showed a robust proliferation rate, a weak foci-forming ability, a strong migration capacity, and a moderate invasion tendency in vitro. Finally, the tumorigenicity and metastatic capacity of HCC-Z were evaluated using a subcutaneous xenograft model, an orthotopic xenograft model, and a tail-veil injection model. HCCZJ exhibited strong tumorigenicity in the subcutaneous xenograft and orthotopic tumor models. Moreover, HCC-ZJ spontaneously formed pulmonary metastases in the orthotopic tumor model. In summary, a new HCC cell line derived from a patient with alcohol-related HCC was established, which showed a high metastatic capacity and could be applied for in vitro and in vivo experiments during pre-clinical research.Highlights• An alcohol-related HCC cell line, HCC-ZJ, was established• HCC-ZJ was applicable for in vitro functional experiment and gene editing• HCC-ZJ was applicable for in vivo tumor growth and spontaneous metastasis models.

Kinesin family member 18B activates mTORC1 signaling via actin gamma 1 to promote the recurrence of human hepatocellular carcinoma.

The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is frequently reported to be hyperactivated in hepatocellular carcinoma (HCC) and contributes to HCC recurrence. However, the underlying regulatory mechanisms of mTORC1 signaling in HCC are not fully understood. In the present study, we found that the expression of kinesin family member 18B (KIF18B) was positively correlated with mTORC1 signaling in HCC, and the upregulation of KIF18B and p-mTOR was associated with a poor prognosis and HCC recurrence. Utilizing in vitro and in vivo assays, we showed that KIF18B promoted HCC cell proliferation and migration through activating mTORC1 signaling. Mechanistically, we identified Actin gamma 1 (γ-Actin) as a binding partner of KIF18B. KIF18B and γ-Actin synergistically modulated lysosome positioning, promoted mTORC1 translocation to lysosome membrane, and prohibited p70 S6K from entering lysosomes for degradation, which finally led to the enhancement of mTORC1 signaling transduction. Moreover, we found that KIF18B was a direct target of Forkhead box M1, which explains the potential mechanism of KIF18B overexpression in HCC. Our study highlights the potential of KIF18B as a therapeutic target for the treatment of HCC.

The high level of IL-1ß in the serum of ACLF patients induces increased IL-8 expression in hUC-MSCs and reduces the efficacy of hUC-MSCs in liver failure.

BACKGROUND: Stem cells play a therapeutic role mainly through immunoregulation. However, the immunomodulatory function of stem cells may be affected by inflammation-related factors in patients' serum. Therefore, this study aims to investigate the possible mechanism by which acute-on-chronic liver failure (ACLF) patient serum influences the efficacy of hUC-MSCs. METHODS: The serum of surviving and dead ACLF patients was collected to culture hUC-MSCs in vitro, and the hUC-MSCs cultured in the serum of ACLF patients were used to treat acute liver failure (ALF) rats. The therapeutic effect on the rats was evaluated by a survival curve, the transaminase level and liver histopathology. The expression of cytokines in hUC-MSCs was detected by Q-PCR and ELISA. RESULTS: Serum pretreatment reduced the therapeutic effect of hUC-MSCs on ALF, especially pretreatment in the serum from dead ACLF patients. After hUC-MSCs were cultured in the serum of surviving or dead ACLF patients, the most differentially expressed factor was IL-8. Interfering with the expression of IL-8 in hUC-MSCs can improve the therapeutic effect of hUC-MSCs on ALF. The high level of IL-1ß in the serum of dead ACLF patients causes the increased expression of IL-8 in hUC-MSCs through the activation of the NF-κB signaling pathway. Meanwhile, we found that the neutralizing IL-1ß in serum from dead ACLF patients can improve the therapeutic effect of hUC-MSCs on ALF. CONCLUSION: The high level of IL-1ß in ACLF serum can promote the expression of IL-8 in hUC-MSCs through the NF-κB signaling pathway, thus reducing the effect of hUC-MSCs on ALF.

Improved bone and renal safety in younger tenofovir disoproxil fumarate experienced chronic hepatitis B patients after switching to tenofovir alafenamide or entecavir.

INTRODUCTION AND OBJECTIVES: Renal and bone impairment has been reported in chronic hepatitis B (CHB) patients receiving long-term tenofovir disoproxil fumarate (TDF) therapy. This study aimed to assess the incidence of renal and bone impairment in CHB patients with long-term TDF therapy and to identify the changes in bone mineral density (BMD) and renal function in these patients after switching to entecavir (ETV) or tenofovir alafenamide (TAF). MATERIALS AND METHODS: This retrospective study collected clinical data from CHB patients who received TDF monotherapy over 96 weeks. The changes in BMD and renal function were analyzed after 96 weeks of switching antiviral regimens (ETV or TAF) or maintenance TDF. RESULTS: At baseline, 154 patients receiving TDF monotherapy over 96 weeks were enrolled, with a younger median age of 36.75 years, 35.1% (54/154) of patients experienced elevated urinary ß2 microglobulin and 20.1% (31/154) of patients had reduced hip BMD (T<-1). At week 96, among the 123 patients with baseline normal BMD, patients who maintained TDF (n=85) had experienced a decrease in hip BMD, while patients who switched antiviral regimens (n=38) experienced an increase (-13.97% vs 2.34%, p<0.05). Among patients with a baseline reduced BMD (n=31), the alterations in BMD were similar in patients who maintained TDF (n=5) and those who switched antiviral regimens (n=26) (-15.81% vs 7.35%, p<0.05). Irrespective of baseline BMD status, renal function decreased significantly in patients who maintained TDF and improved in patients who switched antiviral regimens. CONCLUSIONS: Younger CHB patients on long-term TDF therapy are at high risk for bone and renal impairment, with the risk being reduced when switched to ETV or TAF.

Effectiveness and safety of tenofovir amibufenamide and its comparison with tenofovir alafenamide in patients with chronic hepatitis B: results from a retrospective real-world study.

Background/aim: Tenofovir amibufenamide (TMF) has shown potent antiviral efficacy in randomized clinical studies. This study aimed to reveal the effectiveness and safety of tenofovir amibufenamide in the real world and compared tenofovir amibufenamide to tenofovir alafenamide (TAF) in patients with chronic hepatitis B (CHB). Methods: In this retrospective study, tenofovir amibufenamide-treated chronic hepatitis B patients were divided into treatment-naive (TN) and treatment-experienced (TE) groups. Furthermore, tenofovir alafenamide-treated patients were enrolled using the propensity score matching method (PSM). We assessed the virological response (VR, HBV DNA < 100 IU/mL) rate, renal function, and blood lipid changes during 24 weeks of treatment. Results: Virologic response rates at week 24 were 93% (50/54) in the treatment-naive group and 95% (61/64) in the treatment-experienced group. The ratios of alanine transaminase (ALT) normalization were 89% (25/28) in the treatment-naive group and 71% (10/14) in the treatment-experienced group (p = 0.306). Additionally, serum creatinine decreased in both the treatment-naive and treatment-experienced groups, (-4.44 ± 13.55 µmol/L vs. -4.14 ± 9.33 µmol/L, p = 0.886), estimated glomerular filtration rate (eGFR) increased (7.01 ± 12.49 ml/min/1.73 m2 vs. 5.50 ± 8.16 ml/min/1.73 m2, p = 0.430), and low-density lipoprotein cholesterol (LDL-C) levels increased (0.09 ± 0.71 mmol/L vs. 0.27 ± 0.68 mmol/L, p = 0.152), whereas total cholesterol/high-density lipoprotein cholesterol (TC/HDL-C) levels decreased continuously from 3.26 ± 1.05 to 2.49 ± 0.72 in the treatment-naive group and from 3.31 ± 0.99 to 2.88 ± 0.77 in the treatment-experienced group. Using propensity score matching, we further compared virologic response rates between the tenofovir amibufenamide and tenofovir alafenamide cohorts. Virologic response rates in treatment-naive patients were higher in the tenofovir amibufenamide cohort [92% (35/38) vs. 74% (28/38), p = 0.033]. Virologic response rates in treatment-experienced patients showed no statistical difference between the tenofovir amibufenamide and tenofovir alafenamide cohorts. Conclusion: Tenofovir amibufenamide had profound antiviral effectiveness and no adverse effects on renal function or blood lipids. Additionally, tenofovir amibufenamide was more efficient than tenofovir alafenamide in inhibiting viral replication, which needs to be demonstrated in future studies.

Dengzhan Shengmai capsule attenuates cardiac fibrosis in post-myocardial infarction rats by regulating LTBP2 and TGF-ß1/Smad3 pathway.

BACKGROUND: Cardiac fibrosis contributes to myocardial remodeling after myocardial infarction (MI), which may facilitate the progression to end-stage heart failure. Dengzhan Shengmai capsule (DZSMC), a traditional Chinese formula derived from Shen-mai powder, has shown remarkable therapeutic effects against cardiovascular diseases. However, the effect of DZSMC on cardiac fibrosis and its potential mechanism are ill-defined. PURPOSE: To evaluate the effects of DZSMC on cardiac fibrosis after myocardial infarction (MI) and investigate its underlying mechanism. METHOD: In vivo, MI rat models were established by permanently ligation of left anterior descending coronary arteries (LAD) and then were intragastrically treated with DZSMC or captopril for 5 weeks. Ex vivo, an everted intestinal sac model was used to study the intestinal absorption components of DZSMC, which were further identified through an ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS) method. In vitro, a myocardium fibrotic model was constructed by stimulating primary cardiac fibroblasts (CFs) with 1 µM Ang II. Subsequently, the absorbent solution of DZSMC from the intestinal sac was performed on the cell models to further elucidate its anti-fibrotic effects and underling mechanism. RESULTS: In vivo results showed that DZSMC significantly improved cardiac function and inhibited pathological myocardial fibrosis in post-MI rats in a dose dependent manner. Histological analysis and western blot results demonstrated that DZSMC treatment significantly reduced the expression of extracellular matrix (ECM)-related proteins, including LTBP2, TGF-ßR1, Smad3 and pSmad3, in myocardial tissue of MI rats. Ex vivo results showed that 18 absorbed components were identified, mainly consisting of phenolic acids, flavonoids and lignans, which may be responsible for the anti-fibrotic effects. Further in vitro results validated that DZSMC attenuated myocardial fibrosis by suppressing the expression of LTBP2, TGF-ß1 and pSmad3. CONCLUSION: DZSMC ameliorates cardiac function and alleviates cardiac fibrosis, which may be mediated by inhibition of CFs activation and reduction of excessive ECM deposition via LTBP2 and TGF-ß1/Smad3 pathways.

Tumor Microenvironment Responsive Nanomicelle with Folic Acid Modification Co-Delivery of Doxorubicin/Shikonin for Triple Negative Breast Cancer Treatment.

Triple negative breast cancer (TNBC), which has poor prognosis, easily develops drug resistance and metastasizes. In general, those TNBC characteristics are related to a high activation of the epithelial-mesenchymal transition (EMT) pathway, which is inhibited by shikonin (SKN). Therefore, the synergistic therapy of SKN and doxorubicin (DOX) will increase anti-tumor efficacy and reduce metastasis. In this study, we prepared the folic acid-linked PEG nanomicelle (NM) grafted with the DOX (denoted as FPD) to load the SKN. We prepared the SKN@FPD NM according to the effective ratio of dual drugs, where the drug loadings of DOX and SKN were 8.86 ± 0.21% and 9.43 ± 0.13%, with 121.8 ± 1.1 nm of its hydrodynamic dimension and 6.33 ± 0.16 mV of zeta potential, respectively. The nanomaterials significantly slowed down the release of DOX and SKN over 48 h, leading to the release of pH-responsive drugs. Meanwhile, the prepared NM inhibited the activity of MBA-MD-231 cells in vitro. Further in vitro study revealed that the SKN@FPD NM increased the DOX uptake and significantly reduced the metastasis of MBA-MD-231 cells. Overall, these active-targeting NMs improved the tumor-targeting of small molecular drugs and effectively treated TNBC.

Upregulation of microRNA-125b-5p alleviates acute liver failure by regulating the Keap1/Nrf2/HO-1 pathway.

Background: Acute liver failure (ALF) and acute-on-chronic liver failure (ACLF) are the two most common subtypes of liver failure. They are both life-threatening clinical problems with high short-term mortality. Although liver transplantation is an effective therapeutic, its application is limited due to the shortage of donor organs. Given that both ACLF and ALF are driven by excessive inflammation in the initial stage, molecules targeting inflammation may benefit the two conditions. MicroRNAs (miRNAs) are a group of small endogenous noncoding interfering RNA molecules. Regulation of miRNAs related to inflammation may serve as promising interventions for the treatment of liver failure. Aims: To explore the role and mechanism of miR-125b-5p in the development of liver failure. Methods: Six human liver tissues were categorized into HBV-non-ACLF and HBV-ACLF groups. Differentially expressed miRNAs (DE-miRNAs) were screened and identified through high-throughput sequencing analysis. Among these DE-miRNAs, miR-125b-5p was selected for further study of its role and mechanism in lipopolysaccharide (LPS)/D-galactosamine (D-GalN) -challenged Huh7 cells and mice in vitro and in vivo. Results: A total of 75 DE-miRNAs were obtained. Of these DE-miRNAs, miR-125b-5p was the focus of further investigation based on our previous findings and preliminary results. We preliminarily observed that the levels of miR-125b-5p were lower in the HBV-ACLF group than in the HBV-non-ACLF group. Meanwhile, LPS/D-GalN-challenged mice and Huh7 cells both showed decreased miR-125b-5p levels when compared to their untreated control group, suggesting that miR-125b-5p may have a protective role against liver injury, regardless of ACLF or ALF. Subsequent results revealed that miR-125b-5p not only inhibited Huh7 cell apoptosis in vitro but also relieved mouse ALF in vivo with evidence of improved liver histology, decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and reduced tumor necrosis factor-α (TNF-α) and IL-1ß levels. Based on the results of a biological prediction website, microRNA.org, Kelch-like ECH-associated protein 1 (Keap1) was predicted to be one of the target genes of miR-125b-5p, which was verified by a dual-luciferase reporter gene assay. Western blot results in vitro and in vivo showed that miR-125b-5p could decrease the expression of Keap1 and cleaved caspase-3 while upregulating the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1(HO-1). Conclusion: Upregulation of miR-125b-5p can alleviate acute liver failure by regulating the Keap1/Nrf2/HO-1 pathway, and regulation of miR-125b-5p may serve as an alternative intervention for liver failure.

Serum Pregenomic RNA Combined With Hepatitis B Core-Related Antigen Helps Predict the Risk of Virological Relapse After Discontinuation of Nucleos(t)ide Analogs in Patients With Chronic Hepatitis B.

Background and Aim: Cessation of nucleos(t)ide analogs (NAs) therapy in patients with chronic hepatitis B (CHB) is uncommon. Although criteria for discontinuation appear in some guidelines, the indicators for assessing discontinuation of NAs are limited, whether NAs can be safely ceased remains a difficult clinical issue. Our study aimed to investigate the role of serum pregenomic RNA (pgRNA) and hepatitis B core-related antigen (HBcrAg) at the end of treatment (EOT) in guiding the safe discontinuation of NAs in CHB patients. Methods: This is a retrospective study, clinical data of all CHB patients who discontinued NAs treatment at West China Hospital between June 2020 and January 2021 were collected, including EOT pgRNA, HBcrAg, hepatitis B surface antigen (HBsAg), etc. All patients should meet the Asian-Pacific guideline for discontinuation. Observing virological relapse (VR) rates during 1 year of NAs discontinuation and analyzing the relationship between EOT pgRNA, HBcrAg, and VR. Results: A total of 64 patients were enrolled in this study and 33 (51.5%) patients experienced VR in 1 year. EOT pgRNA positivity (OR = 14.59, p = 0.026) and EOT higher HBcrAg levels (OR = 14.14, p = 0.001) were independent risk factors for VR. The area under the receiver-operating characteristic (AUROC) value of EOT HBcrAg for VR was 0.817 (p < 0.001), optimal cut-off value was 3.3 log10 U/mL. Patients with EOT pgRNA positivity and EOT HBcrAg >3.3 log10 U/mL were more likely to experience VR after discontinuation of NAs (88.9 vs. 45.5%, p = 0.027). Conclusion: According to current guidelines, a higher VR rate occurs after cessation of NAs. EOT pgRNA positivity and higher HBcrAg level carries a higher risk of VR. Combining these novel markers can better help us assess whether patients can safely cease NAs treatment.

Dynamics of Serum Pregenome RNA in Chronic Hepatitis B Patients Receiving 96-Month Nucleos(t)ide Analog Therapy.

Background: Tissue covalently closed circular DNA (cccDNA) can reflect the activity of HBV replication. However, it is impractical to assess intrahepatic cccDNA in every outpatient. Serum pregenome RNA (pgRNA) is transcribed from intrahepatic cccDNA and may reflect the activity of intrahepatic cccDNA. We explored the dynamics and the potential role of serum pgRNA in patients receiving long-term NAs treatment. Methods: Serum pgRNA, HBV DNA, HBsAg, HBeAg, and ALT levels were quantified, and the relationships between serum pgRNA and these common clinical indicators before and after the treatment were investigated. Results: Serum pgRNA showed dynamic change during the 96-month NAs therapy, and serum pgRNA levels were positive and detectable in 19 patients with undetectable serum HBV DNA. Serum pgRNA showed strong and positive correlation with serum HBV DNA (r = 0.693, p < 0.001) and serum HBsAg levels (r = 0.621, p < 0.001) at baseline. Patients with HBeAg seroconversion had lower baseline serum pgRNA levels (p = 0.002). The area under the curve (AUC) of baseline serum pgRNA for predicting HBeAg seroconversion was 0.742 (95% CI: 0.606-0.850) with 63.16% sensitivity and 80.56% specificity. The cumulative HBeAg seroconversion rate was higher in patients with low serum pgRNA (p = 0.001). Conclusion: Serum pgRNA of low level at baseline or great decline at month 6 may independently predict the high incidence of undetectable serum pgRNA at year 4 following NAs therapy, and the baseline serum pgRNA may serve as a novel predictor for HBeAg seroconversion during NAs therapy.

The aberrant upregulation of exon 10-inclusive SREK1 through SRSF10 acts as an oncogenic driver in human hepatocellular carcinoma.

Deregulation of alternative splicing is implicated as a relevant source of molecular heterogeneity in cancer. However, the targets and intrinsic mechanisms of splicing in hepatocarcinogenesis are largely unknown. Here, we report a functional impact of a Splicing Regulatory Glutamine/Lysine-Rich Protein 1 (SREK1) variant and its regulator, Serine/arginine-rich splicing factor 10 (SRSF10). HCC patients with poor prognosis express higher levels of exon 10-inclusive SREK1 (SREK1L). SREK1L can sustain BLOC1S5-TXNDC5 (B-T) expression, a targeted gene of nonsense-mediated mRNA decay through inhibiting exon-exon junction complex binding with B-T to exert its oncogenic role. B-T plays its competing endogenous RNA role by inhibiting miR-30c-5p and miR-30e-5p, and further promoting the expression of downstream oncogenic targets SRSF10 and TXNDC5. Interestingly, SRSF10 can act as a splicing regulator for SREK1L to promote hepatocarcinogenesis via the formation of a SRSF10-associated complex. In summary, we demonstrate a SRSF10/SREK1L/B-T signalling loop to accelerate the hepatocarcinogenesis.

A COVID-19 Patient Discharged According to Strict Discharge Standards: Viral Negativity in Both Nasopharynx and Feces.

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) currently has spread all over the world. However, the dynamic characteristics of SARS-CoV-2 infections have not previously been described in detail. Here, we report a cured patient in West China Hospital, and describe the dynamic detection of SARS-CoV-2-RNA in different specimens and viral specific IgM and IgG antibodies in blood. The findings suggest that the fecal SARS-CoV-2-RNA negativity may be considered as a new standard for de isolation. Serum IgM and IgG antibodies detection were helpful for early diagnosis of SARS-CoV-2 infection and judgment of patients in recovery stage, respectively.

Pain pathways and potential new targets for pain relief.

Pain is an unpleasant sensory and emotional experience that affects a sizable percentage of people on a daily basis. Sensory neurons known as nociceptors built specifically to detect damaging stimuli can be found throughout the body. They transmit information about noxious stimuli from mechanical, thermal, and chemical sources to the central nervous system and higher brain centers via electrical signals. Nociceptors express various channels and receptors such as voltage-gated sodium and calcium channels, transient receptor potential channels, and opioid receptors that allow them to respond in a highly specific manner to noxious stimuli. Attenuating the pain response can be achieved by inhibiting or altering the expression of these pain targets. Achieving a deeper understanding of how these receptors can be affected at the molecular level can lead to the development of novel pain therapies. This review will discuss the mechanisms of pain, introduce the various receptors that are responsible for detecting pain, and future directions in pharmacological therapies.

Signature network-based survey of the effects of a traditional Chinese medicine on heart failure.

ETHNOPHARMACOLOGICAL RELEVANCE: Heart failure (HF) after myocardial infarction (MI) is one of the most common disabling and painful diseases. A traditional Chinese medicine (TCM) formula, Shengmaisan, is known as a multitarget medicine that is widely used clinically to treat heart failure (HF) in Asian countries. However, its mechanism has not been comprehensively demonstrated. AIM OF THE STUDY: To use a prediction network to figure out which disease link SMZ mainly alleviates in HF and find biomarkers related to myocardial fibrosis in the serum for clinical reference. MATERIALS AND METHODS: In this article, we collected a large amount of actual measurement data and our own proteomics data, along with the biomarkers of heart failure staging under study to establish a precise network. Then, we tested and verified the medicinal effect of SMZ in treatment of HF after MI by Measurement of left ventricular wall thickness and ejection fraction by echocardiography. Then we tested the serum level of the potential targets of SMZ predicting by the network we developed using ELISA. RESULTS: the cardiac ejection fraction and retarding the thinning of the anterior wall of the left ventricle increased after treating with SMZ. The serum level of EGFR and MAPK1 decreased in the groups treated with SMZ. CONCLUSION: SMZ can improve the cardiac function of rats with MI by increasing the cardiac ejection fraction and retarding the thinning of the anterior wall of the left ventricle. In addition, SMZ could delay heart failure mainly by inhibiting the progression of myocardial fibrosis through decreasing the EGFR and MAPK1 levels.

USP5 facilitates non-small cell lung cancer progression through stabilization of PD-L1.

PD-L1(CD274) is a well-known immunosuppressive molecule, which confers immunoescape features to cancer cells and has become one of the major targets in cancer immunotherapies. Understanding the regulatory mechanisms that control PD-L1 protein expression is important for guiding immune checkpoint blockade therapy. Here, we showed that ubiquitin specific peptidase 5 (USP5) was a novel PD-L1 deubiquitinase in non-small cell lung cancer (NSCLC) cells. USP5 directly interacted with PD-L1 and deubiquitinated PD-L1, therefore enhances PD-L1 protein stability. Meanwhile, USP5 protein levels were highly elevated and positively correlated to PD-L1 levels in NSCLC tissues, and were closely correlated with poor prognosis of these patients. In addition, knockdown of USP5 retarded tumor growth in the Lewis lung carcinoma mouse model. Thus, we identified that USP5 was a new regulator of PD-L1 and targeting USP5 is a promising strategy for cancer therapy.

Antiviral Therapy for Chronic HBV Infection With Persistently Normal Alanine Aminotransferase: Controversy and Consensus.

ALT is one of the most sensitive biochemical indexes to reflect liver injury. It is generally believed that hepatitis B virus (HBV) infected patients with normal ALT levels are in either immune tolerance or low replication stage of the natural history of hepatitis B, and there is no or only mild inflammation in liver tissue, so antiviral therapy is not recommended. However, chronic HBV-infected patients with normal ALT levels are not always in a stable state. A considerable number of patients will develop active hepatitis or occult progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Therefore, whether antiviral therapy should be recommended for chronic HBV infection with normal ALT level has been a hot topic in clinical practice. In this paper, the definition of immune tolerance, the relationship between ALT and liver inflammation, and the benefits of antiviral therapy were reviewed, and we hope it will be helpful for clinicians to have a deeper understanding of whether antiviral therapy should be considered for chronic HBV infection with normal ALT.

Retrospective Analysis of the Clinical Efficacy of N-Acetylcysteine in the Treatment of Hepatitis B Virus Related Acute-on-Chronic Liver Failure.

Objective: HBV-related acute-on-chronic liver failure (HBV-ACLF) has a high mortality due to severe intrahepatic cholestasis and coagulation dysfunction, thus new treatment measures are urgently needed to improve the therapeutic effect. This study aimed to observe the efficacy of N-acetylcysteine (NAC) in the treatment of HBV-ACLF. Methods: The data of patients with HBV-ACLF admitted to West China Hospital from October 2019 to August 2020 were collected retrospectively, and they were divided into treatment group and control group according to whether they had received additional NAC treatment. The improvement of biochemistry, coagulation function and disease severity score after 14 days of hospitalization were analyzed between two groups. Results: A total of 90 HBV-ACLF patients were included, including 42 patients in treatment group and 48 patients in control group. Compared with baseline, serum TBil, DBil, TBA, GGT and ALP in two groups both decreased significantly, while PTA increased significantly. Interesting, the decrease of serum TBil, DBil and TBA and the increase of PTA in treatment group were all significantly than these in control group. Additionally, more patients in treatment group than control group changed from CTP grade C to grade B. Subgroup analysis of CTP grade C patients showed that the decrease of serum TBil, DBil and TBA and the increase of PTA in treatment group were significantly than these in control group. Conclusion: The NAC treatment may help to improve intrahepatic cholestasis and coagulation dysfunction of HBV-ACLF.

Cell heterogeneity, rather than the cell storage solution, affects the behavior of mesenchymal stem cells in vitro and in vivo.

BACKGROUND: Mesenchymal stem cells (MSCs) have to be expanded in vitro to reach a sufficient cell dose for the treatment of various diseases. During the process of expansion, some obstacles remain to be overcome. The purpose of this study was to investigate the effects of storage solutions and heterogeneity on the behavior of MSCs in vitro and in vivo. METHODS: Umbilical cord MSCs (UC-MSCs) of similar sizes within normal ranges were suspended in three different storage solutions, phosphate buffer solution, normal saline, and Dulbecco's modified Eagle medium. Then, the ultrastructure, viability, and safety of these cells were compared. Other two UC-MSC populations of different sizes were categorized based on their mean diameters. The ultrastructure, proliferation, immunosuppression, hepatic differentiation potential, and number of senescent cells were investigated and compared. The survival rates of mice after the infusion of UC-MSCs of different sizes were compared. RESULTS: For UC-MSCs suspended in different storage solutions, the cell apoptosis rates, ultrastructure, and survival rates of mice were similar, and no differences were observed. Cells with a diameter of 19.14 ± 4.89 µm were categorized as the larger UC-MSC population, and cells with a diameter of 15.58 ± 3.81 µm were categorized as the smaller population. The mean diameter of the larger UC-MSC population was significantly larger than that of the smaller UC-MSC population (p < 0.01). Smaller UC-MSCs had more powerful proliferation and immunosuppressive potential and a higher nucleus-cytoplasm ratio than those of large UC-MSCs. The number of cells positive for ß-galactosidase staining was higher in the larger UC-MSC population than in the smaller UC-MSC population. The survival rates of mice receiving 1 × 106 or 2 × 106 smaller UC-MSCs were 100%, both of which were higher than those of mice receiving the same amounts of larger UC-MSCs (p < 0.01). The cause of mouse death was explored and it was found that some larger UC-MSCs accumulated in the pulmonary capillary in dead mice. CONCLUSION: Different storage solutions showed no significant effects on cell behavior, whereas heterogeneity was quite prevalent in MSC populations and might limit cells application. Hence, it is necessary to establish a more precise standardization for culture-expanded MSCs.