Endoplasmic reticulum stress promotes HBV production by enhancing use of the autophagosome/multivesicular body axis.

BACKGROUND AND AIMS: HBV infection has been reported to trigger endoplasmic reticulum (ER) stress and initiate autophagy. However, how ER stress and autophagy influence HBV production remains elusive. Here, we studied the effect of tunicamycin (TM), an N-glycosylation inhibitor and ER stress inducer, on HBV replication and secretion and examined the underlying mechanisms. APPROACH AND RESULTS: Protein disulfide isomerase (an ER marker), microtubule-associated protein 1 light chain 3 beta (an autophagosome [AP] marker), and sequestosome-1 (a typical cargo for autophagic degradation) expression were tested in liver tissues of patients with chronic HBV infection and hepatoma cell lines. The role of TM treatment in HBV production and trafficking was examined in hepatoma cell lines. TM treatment that mimics HBV infection triggered ER stress and increased AP formation, resulting in enhanced HBV replication and secretion of subviral particles (SVPs) and naked capsids. Additionally, TM reduced the number of early endosomes and HBsAg localization in this compartment, causing HBsAg/SVPs to accumulate in the ER. Thus, TM-induced AP formation serves as an alternative pathway for HBsAg/SVP trafficking. Importantly, TM inhibited AP-lysosome fusion, accompanied by enhanced AP/late endosome (LE)/multivesicular body fusion, to release HBsAg/SVPs through, or along with, exosome release. Notably, TM treatment inhibited HBsAg glycosylation, resulting in impairment of HBV virions' envelopment and secretion, but it was not critical for HBsAg/SVP trafficking in our cell systems. CONCLUSIONS: TM-induced ER stress and autophagic flux promoted HBV replication and the release of SVPs and naked capsids through the AP-LE/MVB axis.

CCDC88A/GIV promotes HBV replication and progeny secretion via enhancing endosomal trafficking and blocking autophagic degradation.

Hepatitis B virus (HBV) particles are thought to be secreted from hepatocytes through multivesicular bodies (MVBs); however, the cellular trafficking mechanisms prior to this process remain elusive. It has been reported that CCDC88A/GIV expression, which is involved in multiple aspects of vesicular trafficking, changes dynamically at different phases of chronic HBV infection. In this study, we focused on the role of CCDC88A/GIV in HBV replication. In the liver tissues of chronically HBV-infected patients, HBV infection significantly enhanced CCDC88A/GIV expression, and increased endoplasmic reticulum (ER) stress and autophagosome formation without changing endosome formation. Additionally, colocalization of SHBsAg with early endosomes (~30.2%) far exceeded that with autophagosomes (~3.2%). In hepatoma cells, CCDC88A/GIV and its downstream proteins, DNM2 (dynamin 2; a CCDC88A/GIV effector), CLTC and RAB5A significantly enhanced HBV replication and endosome formation but inhibited autophagosome formation. Blocking endocytosis disrupted HBsAg trafficking to endosomes and caused its accumulation in the ER lumen, which triggered ER stress to initiate the unfolded protein response (UPR). Therefore, HBsAg trafficking into autophagosomes was increased, and the lysosomal activity and maturation, which was inhibited by HBV infection, were restored. Meanwhile, core particles were prevented from entering MVBs. CCDC88A/GIV and its other effector, GNAI3, decreased autophagic flux by enhancing the insulin-induced AKT-MTOR pathway, thereby inhibiting HBV antigens autophagic degradation. In conclusion, CCDC88A/GIV enhanced HBV replication by increasing endosomal trafficking and reducing autophagic degradation of HBV antigens, suggesting that CCDC88A/GIV-mediated endosomal trafficking plays an important role in HBV replication and progeny secretion.Abbreviations: ACTB: actin beta; AO: acridine orange; ATF6: activating transcription factor 6; CCDC88A/GIV: coiled-coil domain containing 88A; CLTC: clathrin heavy chain; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DNM2: dynamin 2; ER: endoplasmic reticulum; ERN1: endoplasmic reticulum to nucleus signaling 1; EIF2A: eukaryotic translation initiation factor 2A; FBS: fetal bovine serum; GNAI3: G protein subunit alpha i3; HBV: hepatitis B virus; HBV RIs: HBV replication intermediates; HBcAg: HBV core protein; HBsAg: HBV surface antigen; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MVBs: multivesicular bodies; MTOR: mechanistic target of rapamycin kinase; PDI: protein disulfide isomerase; PHH: primary human hepatocyte; pSM2: a HBV replication-competent plasmid; HSPA5/BIP: heat shock protein family A (Hsp70) member 5; SQSTM1/p62: sequestosome 1; siRNA: small interfering RNA; SEM: standard error of the mean; UPR: unfolded protein response.

Human umbilical cord-derived mesenchymal stem cells improve the function of liver in rats with acute-on-chronic liver failure via downregulating Notch and Stat1/Stat3 signaling.

BACKGROUND: Effective treatments for acute-on-chronic liver failure (ACLF) are lacking. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have been applied in tissue regeneration and repair, acting through paracrine effects, cell fusion, and actual transdifferentiation. The present study was designed to investigate the therapeutic potential of hUC-MSCs in acute-on-chronic liver injury (ACLI) and ACLF rat models. METHODS: Wistar rats aged 6 weeks were intraperitoneally administered porcine serum (PS) at a dose of 0.5 mL twice per week for 11 weeks to generate an immune liver fibrosis model. After 11 weeks, rats with immune liver fibrosis were injected intravenously with lipopolysaccharide (LPS) to induce an ACLI model or combined LPS and D-galactosamine (D-GalN) to induce an ACLF model. The rats with ACLI or ACLF were injected intravenously with 2×106 hUC-MSCs, 4×106 hUC-MSCs, or 0.9% sodium chloride as a control. The rats were sacrificed at 1, 2, 4, and 6 weeks (ACLI rats) or 4, 12, and 24 h (ACLF rats). The blood and liver tissues were collected for biochemical and histological investigation. RESULTS: The application of hUC-MSCs in rats with ACLI and ACLF led to a significant decrease in the serum levels of ALT, AST, TBil, DBil, ALP, ammonia, and PT, with ALB gradually returned to normal levels. Inflammatory cell infiltration and collagen fiber deposition in liver tissues were significantly attenuated in ACLI rats that received hUC-MSCs. Inflammatory cell infiltration and apoptosis in liver tissues of ACLF rats that received hUC-MSCs were significantly attenuated. Compared with those in the rats that received 0.9% sodium chloride, a significant reduction in proinflammatory cytokine levels and elevated serum levels of hepatocyte growth factor (HGF) were found in ACLF rats that received hUC-MSCs. Furthermore, Notch, IFN-γ/Stat1, and IL-6/Stat3 signaling were inhibited in ACLI/ACLF rats that received hUC-MSCs. CONCLUSIONS: hUC-MSC transplantation can improve liver function, the degree of fibrosis, and liver damage and promote liver repair in rats with ACLI or ACLF, mediated most likely by inhibiting Notch signaling and reversing the imbalance of the Stat1/Stat3 pathway.

Immunostimulatory siRNA with a uridine bulge leads to potent inhibition of HBV and activation of innate immunity.

BACKGROUND: Hepatitis B virus (HBV) infection is difficult to cure. HBV-specific immune tolerance plays a key role in HBV persistence, and enhancing cellular and humoral immunity will improve the control of HBV infection. The purpose of the study was to explore the anti-HBV and immunostimulatory effects of msiRNAs that introduce unpaired uridine bulges in the passenger strand. METHODS: msiRNAs targeting the HBV S and X genes were designed and named msiHBs and msiHBx, respectively. HepG2 cells were cotransfected with siRNA or msiRNA and the HBV replication-competent plasmid pHY106-wta or pHY106-X15. HepG2.215 cells were transfected with siRNA or msiRNA. The levels of HBsAg, HBeAg, and the cytokines TNF-α, IFN-α, IFN-ß, IL-1α, and IL-6 in the culture supernatant was detected by ELISA. The levels of intracellular HBV RNA, nuclear HBV replication intermediates, and HBV DNA in the supernatant were measured by quantitative RT-PCR and PCR. The levels of HBV replication intermediates were detected by Southern blotting. Peripheral blood mononuclear cells were transfected with siRNA or msiRNA, and the levels of secreted cytokines IFN-α and IFN-ß were detected by ELISA. The bioactivity of type I interferons in the supernatants was detected by the virus protection assay. RESULTS: msiHBx treatment led to a significant decrease in HBsAg (to a negative level) and HBV DNA (95.5%) in the supernatant and intrahepatocellular HBV replication intermediates (89.8%) in HepG2 cells with transient HBV replication and in HepG2.2.15 cells. There was no significant difference between msiHBx and siHBx in terms of the reduction in HBV proteins and HBV replication (P > 0.05). Compared with siHBx, msiHBx treatment of HepG2 cells transfected with the HBV replication-competent plasmid led to a significant increase in the levels of the antiviral cytokines TNF-α (3.3-fold), IFN-α (1.4-fold), and IFN-ß (2.5-fold) (P < 0.01), without upregulation of the proinflammatory cytokines IL-1α and IL-6. The virus protection assay results showed msiHBx-mediated type I interferons effectively protected L929 cells against ECMV infection. CONCLUSIONS: msiHBx could effectively inhibit HBV expression and replication and induce an antiviral innate immune response without proinflammatory activation. The dual RNAi and immunostimulatory activity of msiRNAs may play an important role in the control of HBV infection.

The Efficacy of Contrast Transthoracic Echocardiography and Contrast Transcranial Doppler for the Detection of Patent Foramen Ovale Related to Cryptogenic Stroke.

BACKGROUND: Patent foramen ovale (PFO) has been linked to the pathophysiology of cryptogenic stroke. Contrast transesophageal echocardiography (cTEE) is the current gold standard for PFO diagnosis, but it has the disadvantage of being semi-invasive and does not exempt from risks. As a diagnostic test, the efficacy of contrast transthoracic echocardiography (cTTE) and contrast transcranial Doppler (cTCD) is controversial. This study is aimed at investigating the efficacy of cTTE and cTCD versus cTEE in PFO detection, exploring a more cost-effective and reliable method for the diagnosis of PFO related to cryptogenic stroke. METHODS: From August 2019 to January 2020, a total of 213 patients with suspected PFO were included in our study. All patients underwent cTEE, cTCD, and cTTE examinations. cTTE3 was named for using a cutoff of 3 beats to detect PFO during cTTE, and cTTE5 represented a cutoff of 5 beats. A cutoff of cTCD grade III was named cTCD III. A cutoff of grade IV was named cTCD IV. cTTE3+cTCD IV was used for the combination of a cutoff of 3 beats during cTTE with grade IV of cTCD. cTTE5+cTCD III combined a cutoff of 5 beats during cTTE with cTCD grade III. Taking cTEE as the gold standard, we compared the sensitivity, specificity, negative likelihood ratio (-LR), and misdiagnosis rate for PFO detection among the above methods. RESULTS: A total of 161 of 213 (76%) patients had PFO confirmed by cTEE. With the spontaneous Valsalva maneuver, the sensitivity, specificity, negative likelihood ratio (-LR), and misdiagnosis rate of cTTE3 in PFO diagnosis were 60%, 90%, 44%, and 10%, respectively, and those for cTTE5 were 76%, 78%, 31% and 22%, respectively. The sensitivity, specificity, negative likelihood ratio (-LR), and misdiagnosis rate of cTCD III were 80%, 71%, 29%, and 29%, respectively, while those for cTCD IV were 55%, 90%, 49%, and 10%, respectively. When cTTE and cTCD were combined to diagnose PFO, the specificity and misdiagnosis rate were significantly improved, especially cTTE3+cTCD IV, with 100% specificity and a misdiagnosis rate of 0. CONCLUSION: cTTE or cTCD can be used for preliminary PFO related to cryptogenic stroke findings. The combination of the two methods can improve the specificity of PFO diagnosis, especially using the cutoff of cTTE3+cTCD IV.

The Value of Contrast-Enhanced Transesophageal Echocardiography in the Detection of Cardiac Right-to-Left Shunt Related with Cryptogenic Stroke and Migraine.

PURPOSE: To analyze the characteristics of right-to-left shunt (RLS) in patients with cryptogenic stroke and migraine by contrast-enhanced transesophageal echocardiography (c-TEE). METHODS: The study population consisted of 330 patients with cryptogenic stroke and 330 patients with migraine who suspected PFO. All of them received c-TEE examination successfully. In terms of c-TEE analyses, RLS could be diagnosed when microbubbles were visualized in the transition from the right atrium to the left atrium. For semiquantitative analysis, a small amount of RLS was grade 1, indicating 1-10 microvesicles per frame could be seen in the left atrium, a moderate amount of RLS was grade 2, indicating 11-30 microvesicles per frame could be seen in the left atrium, and a large amount of RLS was grade 3, indicating more than 30 microvesicles per frame, or the left atrium is filled with microvesicles. RESULTS: A total of 660 patients were analyzed in the study. PFO-RLS was detected in 348 (348/660, 52.7%) cases by TEE, while in 392 (392/660, 59.3%) cases by c-TEE. Simultaneously, P-RLS was detected in 239 (239/660, 36.2%) cases by c-TEE. Among 330 patients with cryptogenic stroke, PFO-RLS was detected in 198 cases; according to the c-TTE method (198/330, 60.0%), concurrently, 83 participants suffered from PFO-RLS and P-RSL (83/330, 25.1%), including 1 case with PFO and pulmonary arteriovenous fistula. Among 330 patients with migraine, PFO-RLS was detected in 194 cases; according to the c-TTE method (194/330, 58.7%), specifically, 90 participants suffered from PFO-RLS and P-RSL (90/330, 27.2%). There was no statistical significance between the two groups. P-RLS singly was detected in 28 cases with cryptogenic stroke, while in 38 cases with migraine, excluding from pulmonary arteriovenous fistula by CTA examination. In addition, semiquantitative results on c-TTE grading of RLS were compared between the two groups: grade 1 RLS in the migraine group (144/322) was significantly higher than that in the cryptogenic stroke group (71/309) (P < 0.05). Grade 3 RLS in the cryptogenic stroke group (113/309) was significantly higher than that in the migraine group (67/322) (P < 0.05). For grade 2 RLS, there was no statistical difference between the two groups (P = 0.12). CONCLUSIONS: c-TEE can increase the positive rate of PFO diagnosis compared with TEE color Doppler. There is no significant difference in the incidence of PFO-PLS and P-RLS between the cryptogenic stroke group and the migraine group. The grades 2-3 RLS are mainly detected in the cryptogenic stroke group, while grades 1-2 RLS are mostly detected in the migraine group.

Multiple leadless pacemakers implanted in the right ventricle of swine.

AIMS: The miniaturized leadless pacemaker has recently emerged as a bradycardia therapy in humans, and many patients may need at least two pacemakers in their lifetime. Thus, the present study assessed the effect of two leadless pacemakers in the right ventricle (RV) on cardiac function in a swine model. METHODS AND RESULTS: Fourteen mini pigs were chronically studied. Each animal received two sequential leadless pacemakers in the RV with 1 month between two separate implantations. All animals were then followed for 6 months. Cardiac function was assessed with echocardiography at baseline, the second implantation, and the end of the 6-month follow-up. A necropsy at the end of the study was performed to measure the length of the fibrous tissue covering the devices and assess tricuspid valve integrity. Four animals died during the surgical procedure, and one animal died of infection during the follow-up period. In the remaining nine animals, there were no significant changes in left ventricular ejection fraction, aortic time integral, cardiac output, and left ventricular size from baseline to the end of the study. The mean length of fibrous tissue covering Micra devices was 14.3 ± 7.8 mm. No tricuspid valve injury was observed. CONCLUSION: Multiple leadless pacemakers implanted in the RV do not appear to impact cardiac function. Therefore, it appears safe to implant multiple leadless pacemakers in the same heart.

Quantification of mitral regurgitation by general imaging three-dimensional quantification: feasibility and accuracy.

BACKGROUND: Mitral regurgitant volume (MRvol) is an important index of the severity of mitral regurgitation (MR), but MRvol measurement remains challenging. With the development of probe technology and software, General Imaging 3D Quantification (GI 3DQ) allows the direct measurement of MR jet volume. The aim of this study was to evaluate the feasibility and accuracy of MRvol by quantification of MR jet volume using GI 3DQ. METHODS: Ninety-three patients were included, 61 with functional MR and 32 with mitral valve prolapse. Patients with MR were also divided into those with central MR (n = 41) and those with eccentric MR (n = 52). MRvol was assessed using GI 3DQ and the proximal isovelocity surface area (PISA) method. MRvol using effective regurgitant orifice area by real-time three-dimensional echocardiography multiplied by the MR time-velocity integral was used as the reference method. RESULTS: MRvol measured by GI 3DQ and the PISA method had good correlation with MRvol by the reference method. A significant underestimation of MRvol using GI 3DQ and the PISA method was observed in the assessment of eccentric MR, but without a significant difference in the assessment of central MR. CONCLUSIONS: Quantification of MRvol with GI 3DQ was feasible. Quantification of central MRvol using this methodology is accurate compared with the reference method. Quantification of MRvol with GI 3DQ has no significant difference from the currently recommended PISA method.