Qiuwei Pan

Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells

Recent evidence indicates there is a role for small membrane vesicles, including exosomes, as vehicles for intercellular communication. Exosomes secreted by most cell types can mediate transfer of proteins, mRNAs, and microRNAs, but their role in the transmission of infectious agents is less established. Recent studies have shown that hepatocyte-derived exosomes containing hepatitis C virus (HCV) RNA can activate innate immune cells, but the role of exosomes in the transmission of HCV between hepatocytes remains unknown. In this study, we investigated whether exosomes transfer HCV in the presence of neutralizing antibodies. Purified exosomes isolated from HCV-infected human hepatoma Huh7.5.1 cells were shown to contain full-length viral RNA, viral protein, and particles, as determined by RT-PCR, mass spectrometry, and transmission electron microscopy. Exosomes from HCV-infected cells were capable of transmitting infection to naive human hepatoma Huh7.5.1 cells and establishing a productive infection. Even with subgenomic replicons, lacking structural viral proteins, exosome-mediated transmission of HCV RNA was observed. Treatment with patient-derived IgGs showed a variable degree of neutralization of exosome-mediated infection compared with free virus. In conclusion, this study showed that hepatic exosomes can transmit productive HCV infection in vitro and are partially resistant to antibody neutralization. This discovery sheds light on neutralizing antibodies resistant to HCV transmission by exosomes as a potential immune evasion mechanism.

Hepatocyte‐derived microRNAs as serum biomarkers of hepatic injury and rejection after liver transplantation

Recent animal and human studies have highlighted the potential of hepatocyte‐derived microRNAs (HDmiRs) in serum as early, stable, sensitive, and specific biomarkers of liver injury. Their usefulness in human liver transplantation, however, has not been addressed. The aim of this study was to investigate serum HDmiRs as markers of hepatic injury and rejection in liver transplantation. Serum samples from healthy controls and liver transplant recipients (n = 107) and peritransplant liver allograft biopsy samples (n = 45) were analyzed via the real‐time polymerase chain reaction quantification of HDmiRs (miR‐122, miR‐148a, and miR‐194). The expression of miR‐122 and miR‐148a in liver tissue was significantly reduced with prolonged graft warm ischemia times. Conversely, the serum levels of these HDmiRs were elevated in patients with liver injury and positively correlated with aminotransferase levels. HDmiRs appear to be very sensitive because patients with normal aminotransferase values (<50 IU/L) had 6‐ to 17‐fold higher HDmiR levels in comparison with healthy controls (P < 0.005). During an episode of acute rejection, serum HDmiRs were elevated up to 20‐fold, and their levels appeared to rise earlier than aminotransferase levels. HDmiRs in serum were stable during repeated freezing and thawing. In conclusion, this study shows that liver injury is associated with the release of HDmiRs into the circulation. HDmiRs are promising candidates as early, stable, and sensitive biomarkers of rejection and hepatic injury after liver transplantation. Liver Transpl 18:290–297, 2012.

Hepatic cell-to-cell transmission of small silencing RNA can extend the therapeutic reach of RNA interference (RNAi)

Background/aims RNA interference (RNAi), a sequence-specific gene silencing technology triggered by small interfering RNA (siRNA), represents promising new avenues for treatment of various liver diseases including hepatitis C virus (HCV) infection. In plants and invertebrates, RNAi provides an important mechanism of cellular defence against viral pathogens and is dependent on the spread of siRNA to neighbouring cells. A study was undertaken to investigate whether vector-delivered RNAi can transfer between hepatic cells in vitro and in mice, and whether this exchange could extend the therapeutic effect of RNAi against HCV infection. Methods Transmission of RNAi was investigated in culture by assessing silencing of HCV replication and expression of viral entry receptor CD81 using a human hepatic cell line and primary B lymphocytes transduced with siRNA-expressing vectors. In vivo transmission between hepatic cells was investigated in NOD/SCID mice. Involvement of exosomes was demonstrated by purification, uptake and mass spectrometric analysis. Results Human and mouse liver cells, as well as primary human B cells, were found to have the ability to exchange small RNAs, including cellular endogenous microRNA and delivered siRNA targeting HCV or CD81. The transmission of RNAi was largely independent of cell contact and partially mediated by exosomes. Evidence of RNAi transmission in vivo was observed in NOD/SCID mice engrafted with human hepatoma cells producing CD81 siRNA, causing suppression of CD81 expression in mouse hepatocytes. Conclusion Both human and mouse hepatic cells exchange small silencing RNAs, partially mediated by shuttling of exosomes. Transmission of siRNA potentially extends the therapeutic reach of RNAi-based therapies against HCV as well as other liver diseases.

Ribavirin Inhibits In Vitro Hepatitis E Virus Replication through Depletion of Cellular GTP Pools and Is Moderately Synergistic with Alpha Interferon

ABSTRACT Hepatitis E virus (HEV) is a common cause of acute hepatitis that results in high mortality in pregnant women and may establish chronic infections in immunocompromised patients. We demonstrate for the first time that alpha interferon (IFN-α) and ribavirin inhibit in vitro HEV replication in both a subgenomic replicon and an infectious culture system based on a genotype 3 strain. IFN-α showed a moderate but significant synergism with ribavirin. These findings corroborate the reported clinical effectiveness of both drugs. In addition, the antiviral activity of ribavirin against wild-type genotype 1, 2, and 3 strains was confirmed by immunofluorescence staining. Furthermore, the in vitro activity of ribavirin depends on depletion of intracellular GTP pools, which is evident from the facts that (i) other GTP-depleting agents (5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide [EICAR] and mycophenolic acid) inhibit viral replication, (ii) exogenously added guanosine reverses the antiviral effects, and (iii) a strong correlation (R2 = 0.9998) exists between the antiviral activity and GTP depletion of ribavirin and other GTP-depleting agents.

Secreted Factors of Human Liver-Derived Mesenchymal Stem Cells Promote Liver Regeneration Early After Partial Hepatectomy

Rapid liver regeneration is required after living-donor liver transplantation and oncologic liver resections to warrant sufficient liver function and prevent small-for-size syndrome. Recent evidence highlights the therapeutic potential of mesenchymal stem cells (MSC) for treatment of toxic liver injury, but whether MSC and their secreted factors stimulate liver regeneration after surgical injury remains unknown. Therefore, the aim of this study is to investigate the effect of human liver-derived MSC-secreted factors in an experimental liver resection model. C57BL/6 mice were subjected to a 70% partial hepatectomy and treated with either concentrated MSC-conditioned culture medium (MSC-CM) or vehicle control. Animals were analyzed for liver and body weight, hepatocyte proliferation, and hepatic gene expression. Effects of MSC-CM on gene expression in a human hepatocyte-like cell line (Huh7 cells) were analyzed using genome-wide gene expression arrays. Liver regeneration was significantly stimulated by MSC-CM as shown by an increase in liver to body weight ratio and hepatocyte proliferation. MSC-CM upregulated hepatic gene expression of cytokines and growth factors relevant for cell proliferation, angiogenesis, and anti-inflammatory responses. In vitro, treatment of Huh7 cells with MSC-CM significantly altered expression levels of ~3,000 genes. Functional analysis revealed strong effects on networks associated with protein synthesis, cell survival, and cell proliferation. This study shows that treatment with MSC-derived factors can promote hepatocyte proliferation and regenerative responses in the early phase after surgical resection. MSC-CM may represent a feasible new strategy to promote liver regeneration in patients undergoing extensive liver resection or after transplantation of small liver grafts.

Mycophenolic acid augments interferon‐stimulated gene expression and inhibits hepatitis C Virus infection in vitro and in vivo

Mycophenolic acid (MPA) is a highly effective immunosuppressant that has broad antiviral activity against different viruses and can act in synergy with interferon‐α (IFN‐α) on hepatitis C virus (HCV) replication. MPA is a potent inosine monophosphate dehydrogenase (IMPDH) inhibitor but the antiviral mechanisms are less understood. The aim of this study was to investigate the inhibition of HCV infection by MPA and the molecular basis for its synergy with IFN‐α. The role of IMPDH and interferon‐stimulated genes (ISGs) was investigated in two HCV models using gain‐ or loss‐of‐function approaches. The in vivo effect of MPA treatment was studied in NOD/SCID mice engrafted with HCV replicon cells. Potent antiviral effects of MPA at clinically relevant concentrations were observed with both the subgenomic and JFH1‐derived infectious HCV models. MPA treatment in mice resulted in a specific and robust inhibition of HCV replication. Ectopic expression of an MPA‐resistant IMPDH2 mutant in HCV host cells completely reversed the antiproliferative effect of MPA but only partially affected the antiviral potency. However, similar to ribavirin, MPA induced expression of multiple antiviral ISGs, including interferon regulatory factor 1 (IRF1). Cotreatment of MPA with IFN‐α resulted in additive effects on ISG expression and enhanced IFN‐induced luciferase reporter activity. Knockdown of IRF1, but not IFITM3, significantly attenuated the inhibition of HCV replication by MPA. Conclusion: MPA exerts a potent anti‐HCV effect in vitro and in mice and acts in synergy with IFN‐α. MPAs antiviral activity partially depends on IMPDH but also involves stimulation of ISGs, providing a molecular basis for its synergy with IFN‐α. (HEPATOLOGY 2012;55:1673–1683)

Excretion of infectious hepatitis E virus into milk in cows imposes high risks of zoonosis

Hepatitis E virus (HEV) represents the main cause of acute hepatitis worldwide. HEV infection in immunocompromised patients involves a high risk for the development of chronic hepatitis. Because HEV is recognized as a zoonotic pathogen, it is currently believed that swine is the primary reservoir. However, this is not sufficient to justify the strikingly high seroprevalence of HEV in both developing and Western countries. Thus, this study aimed to identify new zoonotic sources that bear a high risk of transmission to humans. We collected fecal, blood, and milk samples of cows in a typical rural region of Yunnan Province in southwest China, where mixed farming of domestic animals is a common practice. HEV RNA was quantified by quantitative real‐time polymerase chain reaction, and the whole genome was sequenced. HEV infectivity was assessed in rhesus macaques. We found a high prevalence of active HEV infection in cows as determined by viral RNA positivity in fecal samples. Surprisingly, we discovered that HEV is excreted into milk that is produced by infected cows. Phylogenetic analysis revealed that all HEV isolates from cow/milk belong to genotype 4 and subtype 4h. Gavage with HEV‐contaminated raw and even pasteurized milk resulted in active infection in rhesus macaques. Importantly, a short period of boiling, but not pasteurization, could completely inactivate HEV. Conclusion: Infectious HEV‐contaminated cow milk is recognized as a new zoonotic source that bears a high risk of transmission to humans; these results call attention to understanding and establishing proper measurement and control of HEV zoonotic transmission, particularly in the setting of mixed farming of domestic animals. (Hepatology 2016;64:350‐359)

Rapamycin and everolimus facilitate hepatitis e virus replication: Revealing a basal defense mechanism of PI3K-PKB-mTOR pathway

BACKGROUND & AIMS Humans are frequently exposed to hepatitis E virus (HEV). Nevertheless, the disease mainly affects pregnant women and immunocompromised individuals. Organ recipients receiving immunosuppressants, such as rapalogs, to prevent rejection have a high risk for developing chronic hepatitis following HEV infection. Rapalogs constitute potent inhibitors of mTOR including rapamycin and everolimus. As a master kinase, the mechanism-of-action of mTOR is not only associated with the immunosuppressive capacity of rapalogs but is also tightly regulated during pregnancy because of increased nutritional demands. METHODS We thus investigated the role of mTOR in HEV infection by using two state-of-the-art cell culture models: a subgenomic HEV containing luciferase reporter and a full-length HEV infectious cell culture system. RESULTS In both subgenomic and full-length HEV models, HEV infection was aggressively escalated by treatment of rapamycin or everolimus. Inhibition of mTOR was confirmed by Western blot showing the inhibition of its downstream target, S6 phosphorylation. Consistently, stable silencing of mTOR by lentiviral RNAi resulted in a significant increase in intracellular HEV RNA, suggesting an antiviral function of mTOR in HEV infection. By targeting a series of other up- and downstream elements of mTOR signaling, we further revealed an effective basal defense mechanism of the PI3K-PKB-mTOR pathway against HEV, which is through the phosphorylated eIF4E-binding protein 1 (4E-BP1), however independent of autophagy formation. CONCLUSIONS The discovery that PI3K-PKB-mTOR pathway limits HEV infection through 4E-BP1 and acts as a gate-keeper in human HEV target cells bears significant implications in managing immunosuppression in HEV-infected organ transplantation recipients.

Detection of spontaneous tumorigenic transformation during culture expansion of human mesenchymal stromal cells

Human mesenchymal stem/stromal cells (MSCs) have been explored in a number of clinical trials as a possible method of treating various diseases. However, the effect of long-term cell expansion in vitro on physiological function and genetic stability is still poorly understood. In this study, MSC cultures derived from bone marrow and liver were evaluated for the presence of aberrant cells following long-term expansion. In 46 independent cultures, four batches of transformed MSCs (TMCs) were found, which were all beyond the culture period of five weeks. These aberrant cells were first identified based on the appearance of abnormal cytology and the acquirement of growth advantage. Despite common MSC markers being diminished or absent, TMCs remain highly susceptible to lysis by allogenic natural killer (NK) cells. When transplanted into immunodeficient mice, TMCs formed sarcoma-like tumors, whereas parental MSCs did not form tumors in mice. Using a combination of high-resolution genome-wide DNA array and short-tandem repeat profiling, we confirmed the origin of TMCs and excluded the possibility of human cell line contamination. Additional genomic duplication and deletions were observed in TMCs, which may be associated with the transformation event. Using gene and microRNA expression arrays, a number of genes were identified that were differentially expressed between TMCs and their normal parental counterparts, which may potentially serve as biomarkers to screen cultures for evidence of early transformation events. In conclusion, the spontaneous transformation of MSCs resulting in tumorigenesis is rare and occurs after relatively long-term (beyond five weeks) culture. However, as an added safety measure, cultures of MSCs can potentially be screened based on a novel gene expression signature.

Modeling rotavirus infection and antiviral therapy using primary intestinal organoids.

Despite the introduction of oral vaccines, rotavirus still kills over 450,000 children under five years of age annually. The absence of specific treatment prompts research aiming at further understanding of pathogenesis and the development of effective antiviral therapy, which in turn requires advanced experimental models. Given the intrinsic limitations of the classical rotavirus models using immortalized cell lines infected with laboratory-adapted strains in two dimensional cultures, our study aimed to model infection and antiviral therapy of both experimental and patient-derived rotavirus strains using three dimensional cultures of primary intestinal organoids. Intestinal epithelial organoids were successfully cultured from mouse or human gut tissues. These organoids recapitulate essential features of the in vivo tissue architecture, and are susceptible to rotavirus. Human organoids are more permissive to rotavirus infection, displaying an over 10,000-fold increase in genomic RNA following 24h of viral replication. Furthermore, infected organoids are capable of producing infectious rotavirus particles. Treatment of interferon-alpha or ribavirin inhibited viral replication in organoids of both species. Importantly, human organoids efficiently support the infection of patient-derived rotavirus strains and can be potentially harnessed for personalized evaluation of the efficacy of antiviral medications. Therefore, organoids provide a robust model system for studying rotavirus-host interactions and assessing antiviral medications.