Benefit and harm of waiting time in liver transplantation for HCC.

Liver transplantation is the most successful treatment for limited-stage HCC. The waiting time for liver transplantation (LT) can be a critical factor affecting the oncological prognosis and outcome of patients with HCC. Efficient strategies to optimize waiting time are essential to maximize the benefits of LT and to reduce the harm of delay in transplantation. The ever-increasing demand for donor livers emphasizes the need to improve the organization of the waiting list for transplantation and to optimize organ availability for patients with and without HCC. Current progress in innovations to expand the donor pool includes the implementation of living donor LT and the use of grafts from extended donors. By expanding selection criteria, an increased number of patients are eligible for transplantation, which necessitates criteria to prevent futile transplantations. Thus, the selection criteria for LT have evolved to include not only tumor characteristics but biomarkers as well. Enhancing our understanding of HCC tumor biology through the analysis of subtypes and molecular genetics holds significant promise in advancing the personalized approach for patients. In this review, the effect of waiting time duration on outcome in patients with HCC enlisted for LT is discussed.

The Yield of Routine Post-Operative Doppler Ultrasound to Detect Early Post-Liver Transplantation Vascular Complications.

Early detection of liver transplantation (LT) vascular complications enables timely management. Our aim was to assess if routine Doppler ultrasound (rDUS) improves the detection of hepatic artery thrombosis (HAT), portal vein thrombosis (PVT) and hepatic venous outflow obstruction (HVOO). We retrospectively analysed timing and outcomes, number needed to diagnose one complication (NND) and positive predictive value (PPV) of rDUS on post-operative day (POD) 0,1 and 7 in 708 adult patients who underwent primary LT between 2010-2022. We showed that HAT developed in 7.1%, PVT in 8.2% and HVOO in 3.1% of patients. Most early complications were diagnosed on POD 0 (26.9%), 1 (17.3%) and 5 (17.3%). rDUS correctly detected 21 out of 26 vascular events during the protocol days. PPV of rDUS was 53.8%, detection rate 1.1% and NND was 90.5. Median time to diagnosis was 4 days for HAT and 47 days for PVT and 21 days for HVOO. After intervention, liver grafts were preserved in 57.1%. In conclusion, rDUS protocol helps to detect first week's vascular events, but with low PPV and a high number of ultrasounds needed.

Immune suppressive checkpoint interactions in the tumour microenvironment of primary liver cancers.

Liver cancer is one of the most prevalent cancers, and the third most common cause of cancer-related mortality worldwide. The therapeutic options for the main types of primary liver cancer-hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA)-are very limited. HCC and CCA are immunogenic cancers, but effective immune-mediated tumour control is prevented by their immunosuppressive tumour microenvironment. Despite the critical involvement of key co-inhibitory immune checkpoint interactions in immunosuppression in liver cancer, only a minority of patients with HCC respond to monotherapy using approved checkpoint inhibitor antibodies. To develop effective (combinatorial) therapeutic immune checkpoint strategies for liver cancer, in-depth knowledge of the different mechanisms that contribute to intratumoral immunosuppression is needed. Here, we review the co-inhibitory pathways that are known to suppress intratumoral T cells in HCC and CCA. We provide a detailed description of insights from preclinical studies in cellular crosstalk within the tumour microenvironment that results in interactions between co-inhibitory receptors on different T-cell subsets and their ligands on other cell types, including tumour cells. We suggest alternative immune checkpoints as promising targets, and draw attention to the possibility of combined targeting of co-inhibitory and co-stimulatory pathways to abrogate immunosuppression.

Modelling immune cytotoxicity for cholangiocarcinoma with tumour-derived organoids and effector T cells.

BACKGROUND: Immunotherapy with immune checkpoint inhibitors (ICIs) is being explored to improve cholangiocarcinoma (CCA) therapy. However, it remains difficult to predict which ICI will be effective for individual patients. Therefore, the aim of this study is to develop a co-culture method with patient-derived CCA organoids and immune cells, which could represent anti-cancer immunity in vitro. METHODS: CCA organoids were co-cultured with peripheral blood mononuclear cells or T cells. Flow cytometry, time-lapse confocal imaging for apoptosis, and quantification of cytokeratin 19 fragment (CYFRA) release were applied to analyse organoid and immune cell behaviour. CCA organoids were also cultured in immune cell-conditioned media to analyse the effect of soluble factors. RESULTS: The co-culture system demonstrated an effective anti-tumour organoid immune response by a decrease in live organoid cells and an increase in apoptosis and CYFRA release. Interpatient heterogeneity was observed. The cytotoxic effects could be mediated by direct cell-cell contact and by release of soluble factors, although soluble factors only decreased viability in one organoid line. CONCLUSIONS: In this proof-of-concept study, a novel CCA organoid and immune cell co-culture method was established. This can be the first step towards personalised immunotherapy for CCA by predicting which ICIs are most effective for individual patients.

Activated CD4+ T Cells and Highly Differentiated Alloreactive CD4+ T Cells Distinguish Operationally Tolerant Liver Transplantation Recipients.

Spontaneous operational tolerance to the allograft develops in a proportion of liver transplantation (LT) recipients weaned off immunosuppressive (IS) drugs. Several studies have investigated whether peripheral blood circulating T cells could play a role in the development or identify operational tolerance, but never characterized alloreactive T cells in detail due to the lack of a marker for these T cells. In this study, we comprehensively investigated phenotypic and functional characteristics of alloreactive circulating T cell subsets in tolerant LT recipients (n = 15) using multiparameter flow cytometry and compared these with LT recipients on IS drugs (n = 23) and healthy individuals (n = 16). Activation-induced CD137 was used as a marker for alloreactive T cells upon allogenic stimulation. We found that central and effector memory CD4+ T cells were hyporesponsive against donor and third-party splenocyte stimulation in tolerant LT recipients, whereas an overall hyperresponsiveness was observed in alloreactive terminally differentiated effector memory CD4+ T cells. In addition, elevated percentages of circulating activated T helper cells were observed in these recipients. Lastly, tolerant and control LT recipients did not differ in donor-specific antibody formation. In conclusion, a combination of circulating hyperresponsive highly differentiated alloreactive CD4+ T cells and circulating activated T helper cells could discriminate tolerant recipients from a larger group of LT recipients.

Histopathological growth patterns modify the prognostic impact of microvascular invasion in non-cirrhotic hepatocellular carcinoma.

BACKGROUND: Microvascular invasion (MVI) is an established prognosticator in hepatocellular carcinoma (HCC). Histopathological growth patterns (HGPs) classify the invasive margin of hepatic tumors, with superior survival observed for the desmoplastic HGP. Our aim was to investigate non-cirrhotic HCC in light of MVI and the HGP. METHODS: A retrospective cohort study was performed in resected non-cirrhotic HCC. MVI was assessed prospectively. The HGP was determined retrospectively, blinded, and according to guidelines. Overall and disease-free survival (OS, DFS) were evaluated by Kaplan-Meier and multivariable Cox regression. RESULTS: The HGP was determined in 155 eligible patients, 55 (35%) featured a desmoplastic HGP. MVI was observed in 92 (59%) and was uncorrelated with HGP (64% vs 57%, p = 0.42). On multivariable analysis, non-desmoplastic and MVI-positive were associated with an adjusted HR [95%CI] of 1.61 [0.98-2.65] and 3.22 [1.89-5.51] for OS, and 1.59 [1.05-2.41] and 2.30 [1.52-3.50] for DFS. Effect modification for OS existed between HGP and MVI (p < 0.01). Non-desmoplastic MVI-positive patients had a 5-year OS of 36% (HR: 5.21 [2.68-10.12]), compared to 60% for desmoplastic regardless of MVI (HR: 2.12 [1.08-4.18]), and 86% in non-desmoplastic MVI-negative. CONCLUSION: HCCs in non-cirrhotic livers display HGPs which may be of prognostic importance, especially when combined with MVI.

HHLA2 is expressed in pancreatic and ampullary cancers and increased expression is associated with better post-surgical prognosis.

BACKGROUND: HHLA2 is a recently discovered member of the B7-family of immune checkpoint molecules with limited expression in normal tissues but overexpression in several types of cancer. The aim was to determine the expression, prevalence and biological relevance of HHLA2 protein expression in two closely related human cancer types, namely pancreatic cancer and ampullary cancer. METHODS: HHLA2 expression levels were retrospectively determined by immunohistochemistry in tissue micro-arrays of surgically resected tumours of 122 pancreatic cancer patients and 72 patients with ampullary cancer of the pancreato-biliary subtype. RESULTS: HHLA2 was expressed at variable levels by tumour cells in 67% of pancreatic tumours and 93% of ampullary tumours. In the combined cohort high tumoural HHLA2 expression levels were significantly associated with delayed cancer recurrence and improved post-operative cancer-specific survival. The association of HHLA2 expression with cancer-specific survival and recurrence was statistically significant for the pancreatic cancer subgroup while a similar trend was found for the ampullary cancer subgroup. In multivariable analysis together with clinicopathologic characteristics, higher HHLA2 expression was an independent predictor of cancer-specific survival. CONCLUSION: The wide expression of HHLA2 in tumour cells and its association with cancer recurrence and patient survival suggest that HHLA2 represents a relevant immune checkpoint molecule in pancreatic and ampullary cancers.

Enrichment of the tumour immune microenvironment in patients with desmoplastic colorectal liver metastasis.

BACKGROUND: Patients with resected colorectal liver metastasis (CRLM) who display only the desmoplastic histopathological growth pattern (dHGP) exhibit superior survival compared to patients with any non-desmoplastic growth (non-dHGP). The aim of this study was to compare the tumour microenvironment between dHGP and non-dHGP. METHODS: The tumour microenvironment was investigated in three cohorts of chemo-naive patients surgically treated for CRLM. In cohort A semi-quantitative immunohistochemistry was performed, in cohort B intratumoural and peritumoural T cells were counted using immunohistochemistry and digital image analysis, and in cohort C the relative proportions of individual T cell subsets were determined by flow cytometry. RESULTS: One hundred and seventeen, 34, and 79 patients were included in cohorts A, B, and C, with dHGP being observed in 27%, 29%, and 15% of patients, respectively. Cohorts A and B independently demonstrated peritumoural and intratumoural enrichment of cytotoxic CD8+ T cells in dHGP, as well as a higher CD8+/CD4+ ratio (cohort A). Flow cytometric analysis of fresh tumour tissues in cohort C confirmed these results; dHGP was associated with higher CD8+ and lower CD4+ T cell subsets, resulting in a higher CD8+/CD4+ ratio. CONCLUSION: The tumour microenvironment of patients with dHGP is characterised by an increased and distinctly cytotoxic immune infiltrate, providing a potential explanation for their superior survival.

Modeling liver cancer and therapy responsiveness using organoids derived from primary mouse liver tumors.

The current understanding of cancer biology and development of effective treatments for cancer remain far from satisfactory. This in turn heavily relies on the availability of easy and robust model systems that resemble the architecture/physiology of the tumors in patients to facilitate research. Cancer research in vitro has mainly been based on the use of immortalized 2D cancer cell lines that deviate in many aspects from the original primary tumors. The recent development of the organoid technology allowing generation of organ-buds in 3D culture from adult stem cells has endowed the possibility of establishing stable culture from primary tumors. Although culturing organoids from liver tumors is thought to be difficult, we now convincingly demonstrate the establishment of organoids from mouse primary liver tumors. We have succeeded in culturing 91 lines from 129 liver tissue/tumors. These organoids can be grown in long-term cultures in vitro. About 20% of these organoids form tumors in immunodeficient mice upon (serial) transplantation, confirming their tumorigenic and self-renewal properties. Interestingly, single cells from the tumor organoids have high efficiency of organoid initiation, and a single organoid derived from a cancer cell is able to initiate a tumor in mice, indicating the enrichment of tumor-initiating cells in the tumor organoids. Furthermore, these organoids recapitulate, to some extent, the heterogeneity of liver cancer in patients, with respect to phenotype, cancer cell composition and treatment response. These model systems shall provide enormous opportunities to advance our research on liver cancer (stem cell) biology, drug development and personalized medicine.

Action and clinical significance of CCAAT/enhancer-binding protein delta in hepatocellular carcinoma.

CCAAT/enhancer-binding protein delta (CEBPD) is associated with the regulation of apoptosis and cell proliferation and is a candidate tumor suppressor gene. Here, we investigated its role in hepatocellular carcinoma (HCC). We observe that CEBPD mRNA expression is significantly downregulated in HCC tumors as compared with adjacent tissues. Protein levels of CEBPD are also lower in tumors relative to adjacent tissues. Reduced expression of CEBPD in the tumor correlates with worse clinical outcome. In both Huh7 and HepG2 cells, shRNA-mediated CEBPD knockdown significantly reduces cell proliferation, single cell colony formation and arrests cells in the G0/G1 phase. Subcutaneous xenografting of Huh7 in nude mice show that CEBPD knockdown results in smaller tumors. Gene expression analysis shows that CEBPD modulates interleukin-1 signaling. We conclude that CEBPD expression uncouples cancer compartment expansion and clinical outcome in HCC, potentially by modulating interleukin-1 signaling. Thus, although our results support the notion that CEBPD acts as a tumor suppressor in HCC, its action does not involve impairing compartment expansion per se but more likely acts through improving anticancer immunity.

GITR ligation enhances functionality of tumor-infiltrating T cells in hepatocellular carcinoma.

No curative treatment options are available for advanced hepatocellular carcinoma (HCC). Anti-PD1 antibody therapy can induce tumor regression in 20% of advanced HCC patients, demonstrating that co-inhibitory immune checkpoint blockade has therapeutic potential for this type of cancer. However, whether agonistic targeting of co-stimulatory receptors might be able to stimulate anti-tumor immunity in HCC is as yet unknown. We investigated whether agonistic targeting of the co-stimulatory receptor GITR could reinvigorate ex vivo functional responses of tumor-infiltrating lymphocytes (TIL) freshly isolated from resected tumors of HCC patients. In addition, we compared GITR expression between TIL and paired samples of leukocytes isolated from blood and tumor-free liver tissues, and studied the effects of combined GITR and PD1 targeting on ex vivo TIL responses. In all three tissue compartments, CD4+ FoxP3+ regulatory T cells (Treg) showed higher GITR- expression than effector T-cell subsets. The highest expression of GITR was found on CD4+ FoxP3hi CD45RA- activated Treg in tumors. Recombinant GITR-ligand as well as a humanized agonistic anti-GITR antibody enhanced ex vivo proliferative responses of CD4+ and CD8+ TIL to tumor antigens presented by mRNA-transfected autologous B-cell blasts, and also reinforced proliferation, IFN-γ secretion and granzyme B production in stimulations of TIL with CD3/CD28 antibodies. Combining GITR ligation with anti-PD1 antibody nivolumab further enhanced tumor antigen-specific responses of TIL in some, but not all, HCC patients, compared to either single treatment. In conclusion, agonistic targeting of GITR can enhance functionality of HCC TIL, and may therefore be a promising strategy for single or combinatorial immunotherapy in HCC.

Reduction of immunosuppressive tumor microenvironment in cholangiocarcinoma by ex vivo targeting immune checkpoint molecules.

BACKGROUND & AIMS: Cholangiocarcinoma is an aggressive hepatobiliary malignancy originating from biliary tract epithelium. Whether cholangiocarcinoma is responsive to immune checkpoint antibody therapy is unknown, and knowledge of its tumor immune microenvironment is limited. We aimed to characterize tumor-infiltrating lymphocytes (TILs) in cholangiocarcinoma and assess functional effects of targeting checkpoint molecules on TILs. METHODS: We isolated TILs from resected tumors of patients with cholangiocarcinoma and investigated their compositions compared with their counterparts in tumor-free liver (TFL) tissues and blood, by flow cytometry and immunohistochemistry. We measured expression of immune co-stimulatory and co-inhibitory molecules on TILs, and determined whether targeting these molecules improved ex vivo functions of TILs. RESULTS: Proportions of cytotoxic T cells and natural killer cells were decreased, whereas regulatory T cells were increased in tumors compared with TFL. While regulatory T cells accumulated in tumors, the majority of cytotoxic and helper T cells were sequestered at tumor margins, and natural killer cells were excluded from the tumors. The co-stimulatory receptor GITR and co-inhibitory receptors PD1 and CTLA4 were over-expressed on tumor-infiltrating T cells compared with T cells in TFL and blood. Antagonistic targeting of PD1 or CTLA4 or agonistic targeting of GITR enhanced effector molecule production and T cell proliferation in ex vivo stimulation of TILs derived from cholangiocarcinoma. The inter-individual variations in TIL responses to checkpoint treatments were correlated with differences in TIL immune phenotype. CONCLUSIONS: Decreased numbers of cytotoxic immune cells and increased numbers of suppressor T cells that over-express co-inhibitory receptors suggest that the tumor microenvironment in cholangiocarcinoma is immunosuppressive. Targeting GITR, PD1 or CTLA4 enhances effector functions of tumor-infiltrating T cells, indicating that these molecules are potential immunotherapeutic targets for patients with cholangiocarcinoma. LAY SUMMARY: The defense functions of immune cells are suppressed in cholangiocarcinoma tumors. Stimulating or blocking "immune checkpoint" molecules expressed on tumor-infiltrating T cells can enhance the defense functions of these cells. Therefore, these molecules may be promising targets for therapeutic stimulation of immune cells to eradicate the tumors and prevent cancer recurrence in patients with cholangiocarcinoma.

Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas.

BACKGROUND & AIMS: Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), down-regulates the T-cell-mediated immune response (called immune checkpoints). Antibodies that block these receptors increase antitumor immunity in patients with melanoma, non-small-cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4+ and CD8+ T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their antitumor functions. METHODS: We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8+ and CD4+ T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays. RESULTS: Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8+ and CD4+ T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8+ TIL, compared with other CD8+ TIL. Compared with TIL that did not express these inhibitory receptors, CD8+ and CD4+ TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-ligand1 [PD-L1]), TIM3, or LAG3 increased proliferation of CD8+ and CD4+ TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TIL functions. CONCLUSIONS: The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared with T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer.

Dynamics of Proliferative and Quiescent Stem Cells in Liver Homeostasis and Injury.

BACKGROUND & AIMS: Adult liver stem cells are usually maintained in a quiescent/slow-cycling state. However, a proliferative population, marked by leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), was recently identified as an important liver stem cell population. We aimed to investigate the dynamics and functions of proliferative and quiescent stem cells in healthy and injured livers. METHODS: We studied LGR5-positive stem cells using diphtheria toxin receptor and green fluorescent protein (GFP) knock-in mice. In these mice, LGR5-positive cells specifically coexpress diphtheria toxin receptor and the GFP reporter. Lineage-tracing experiments were performed in mice in which LGR5-positive stem cells and their daughter cells expressed a yellow fluorescent protein/mTmG reporter. Slow-cycling stem cells were investigated using GFP-based, Tet-on controlled transgenic mice. We studied the dynamics of both stem cell populations during liver homeostasis and injury induced by carbon tetrachloride. Stem cells were isolated from mouse liver and organoid formation assays were performed. We analyzed hepatocyte and cholangiocyte lineage differentiation in cultured organoids. RESULTS: We did not detect LGR5-expressing stem cells in livers of mice at any stage of a lifespan, but only following liver injury induced by carbon tetrachloride. In the liver stem cell niche, where the proliferating LGR5+ cells are located, we identified a quiescent/slow-cycling cell population, called label-retaining cells (LRCs). These cells were present in the homeostatic liver, capable of retaining the GFP label over 1 year, and expressed a panel of progenitor/stem cell markers. Isolated single LRCs were capable of forming organoids that could be carried in culture, expanded for months, and differentiated into hepatocyte and cholangiocyte lineages in vitro, demonstrating their bona fide stem cell properties. More interestingly, LRCs responded to liver injury and gave rise to LGR5-expressing stem cells, as well as other potential progenitor/stem cell populations, including SOX9- and CD44-positive cells. CONCLUSIONS: Proliferative LGR5 cells are an intermediate stem cell population in the liver that emerge only during tissue injury. In contrast, LRCs are quiescent stem cells that are present in homeostatic liver, respond to tissue injury, and can give rise to LGR5 stem cells, as well as SOX9- and CD44-positive cells.

Prognostic value of intra-tumoral CD8+ /FoxP3+ lymphocyte ratio in patients with resected colorectal cancer liver metastasis.

BACKGROUND AND OBJECTIVES: Patients with isolated colorectal-cancer-liver-metastases (CRCLM) frequently undergo metastatectomy. Tumor-infiltrating-lymphocytes (TILs) have prognostic potential in the setting of primary colorectal cancer, however, their role in CRCLM is less studied. We aimed to study the spatial distribution and prognostic role of tumor-infiltrating CD8+ cytotoxic T-cells and FoxP3+ regulatory T-cells at the metastatic site of CRCLM patients. METHODS: TILs were isolated from fresh metastatic tissues of 47 patients with CRCLM. Archived paraffin-embedded tissue, from the same patients, was retrieved. CD8+ and FoxP3+ cells, both in the intra-tumoral and the peri-tumoral compartments, were measured by immunohistochemistry on full tissue sections. Proportions of cytotoxic T-cells (CD8+ ) and regulatory T-cells (CD4+ CD25+ FoxP3+ ), within CD45+ TILs, were measured by flow-cytometry. RESULTS: By immunohistochemistry, individual densities of intra-tumoral or peri-tumoral CD8+ and FoxP3+ cells were not prognostic of survival. However, the intra-tumoral, but not the peri-tumoral, CD8+ /FoxP3+ ratio was an independent predictor of survival (HR 0.43, 95%CI 0.19-0.95, P = 0.032). By flow cytometry, the intra-tumoral CD8+ /regulatory T-cell ratio was also an independent predictor of survival (HR 0.45, 95%CI 0.20-0.99, P = 0.044). CONCLUSIONS: The ratio of cytotoxic (CD8+ ) to regulatory (FoxP3+ ) T-cells, in the intra-tumoral compartment, but not in the peri-tumoral compartment, can predict survival after resection of CRCLM.

Hepatitis E Virus Infects Neurons and Brains.

Hepatitis E virus (HEV), as a hepatotropic virus, is supposed to exclusively infect the liver and only cause hepatitis. However, a broad range of extrahepatic manifestations (in particular, idiopathic neurological disorders) have been recently reported in association with its infection. In this study, we have demonstrated that various human neural cell lines (embryonic stem cell-derived neural lineage cells) induced pluripotent stem cell-derived human neurons and primary mouse neurons are highly susceptible to HEV infection. Treatment with interferon-α or ribavirin, the off-label antiviral drugs for chronic hepatitis E, exerted potent antiviral activities against HEV infection in neural cells. More importantly, in mice and monkey peripherally inoculated with HEV particles, viral RNA and protein were detected in brain tissues. Finally, patients with HEV-associated neurological disorders shed the virus into cerebrospinal fluid, indicating a direct infection of their nervous system. Thus, HEV is neurotropic in vitro, and in mice, monkeys, and possibly humans. These results challenge the dogma of HEV as a pure hepatotropic virus and suggest that HEV infection should be considered in the differential diagnosis of idiopathic neurological disorders.

Inhibition of Calcineurin or IMP Dehydrogenase Exerts Moderate to Potent Antiviral Activity against Norovirus Replication.

Norovirus is a major cause of acute gastroenteritis worldwide and has emerged as an important issue of chronic infection in transplantation patients. Since no approved antiviral is available, we evaluated the effects of different immunosuppressants and ribavirin on norovirus and explored their mechanisms of action by using a human norovirus (HuNV) replicon-harboring model and a surrogate murine norovirus (MNV) infectious model. The roles of the corresponding drug targets were investigated by gain- or loss-of-function approaches. We found that the calcineurin inhibitors cyclosporine (CsA) and tacrolimus (FK506) moderately inhibited HuNV replication. Gene silencing of their cellular targets, cyclophilin A, FKBP12, and calcineurin, significantly inhibited HuNV replication. A low concentration, therapeutically speaking, of mycophenolic acid (MPA), an uncompetitive IMP dehydrogenase (IMPDH) inhibitor, potently and rapidly inhibited norovirus replication and ultimately cleared HuNV replicons without inducible resistance following long-term drug exposure. Knockdown of the MPA cellular targets IMPDH1 and IMPDH2 suppressed HuNV replication. Consistent with the nucleotide-synthesizing function of IMPDH, exogenous guanosine counteracted the antinorovirus effects of MPA. Furthermore, the competitive IMPDH inhibitor ribavirin efficiently inhibited norovirus and resulted in an additive effect when combined with immunosuppressants. The results from this study demonstrate that calcineurin phosphatase activity and IMPDH guanine synthase activity are crucial in sustaining norovirus infection; thus, they can be therapeutically targeted. Our results suggest that MPA shall be preferentially considered immunosuppressive medication for transplantation patients at risk of norovirus infection, whereas ribavirin represents as a potential antiviral for both immunocompromised and immunocompetent patients with norovirus gastroenteritis.

IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes.

IFN regulatory factor 1 (IRF1) is one of the most important IFN-stimulated genes (ISGs) in cellular antiviral immunity. Although hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide, how ISGs counteract HEV infection is largely unknown. This study was conducted to investigate the effect of IRF1 on HEV replication. Multiple cell lines were used in 2 models that harbor HEV. In different HEV cell culture systems, IRF1 effectively inhibited HEV replication. IRF1 did not trigger IFN production, and chromatin immunoprecipitation sequencing data analysis revealed that IRF1 bound to the promoter region of signal transducers and activators of transcription 1 (STAT1). Functional assay confirmed that IRF1 could drive the transcription of STAT1, resulting in elevation of total and phosphorylated STAT1 proteins and further activating the transcription of a panel of downstream antiviral ISGs. By pharmacological inhibitors and RNAi-mediated gene-silencing approaches, we revealed that antiviral function of IRF1 is dependent on the JAK-STAT cascade. Furthermore, induction of ISGs and the anti-HEV effect of IRF1 overlapped that of IFNα, but was potentiated by ribavirin. We demonstrated that IRF1 effectively inhibits HEV replication through the activation of the JAK-STAT pathway, and the subsequent transcription of antiviral ISGs, but independent of IFN production.-Xu, L., Zhou, X., Wang, W., Wang, Y., Yin, Y., van der Laan, L. J. W., Sprengers, D., Metselaar, H. J., Peppelenbosch, M. P., Pan, Q. IFN regulatory factor 1 restricts hepatitis E virus replication by activating STAT1 to induce antiviral IFN-stimulated genes.

Evidence of good prognosis of hepatocellular adenoma in post-menopausal women.

BACKGROUND & AIMS: Hepatocellular adenoma (HCA) is a rare benign liver tumor, which typically develops in women in their reproductive phase and is associated with the use of oral contraceptives. The aim of this study was to evaluate whether follow-up of HCA can be safely terminated after the occurrence of menopause. Secondary, we studied the impact of the diagnosis HCA on health-related quality of life (HRQoL). METHODS: This was a cross-sectional cohort study, including 48 post-menopausal women with HCA. Patients underwent ultrasound examination and the size of HCA was compared to size at the last follow-up imaging (CT, MRI or ultrasound). HRQoL was evaluated by the Liver Disease Symptom Index 2.0 and Short Form 12. RESULTS: Median time since last follow-up was 60.5months. In 44 patients 43.5% of the lesions were undetectable, 32.6% were stable in size and 19.6% became smaller. Mean diameter of HCA was 17.2mm compared to 35.9mm at last follow-up (p<0.001). There was a positive correlation between difference in size and time since last follow-up (p<0.001). No significant effect of HCA subtype on difference in size was found. Regarding HRQoL, study patients scored significantly lower on the mental component summary score compared to the general female Dutch population. CONCLUSIONS: HCA diameter became significantly smaller after the occurrence of menopause and as time progresses, this regression increased. This suggests that routine follow-up of HCA <5cm in post-menopausal women after subsequent follow-up is not required. Notably we found that patient's mental HRQoL was inferior to that of the general population. LAY SUMMARY: In this study we investigated if hepatocellular adenoma, a benign tumor of the liver that is found mostly in women and is associated with female hormones, regresses in size after the occurrence of menopause in female patients over 50years of age. We made an ultrasound of the liver lesion and found that the average size of the adenomas becomes significantly smaller. This could mean that female patients with a small (<5cm) hepatocellular adenoma who are post-menopausal do not have to remain in follow-up. CLINICAL TRIAL NUMBER: MEC-2015-385.

Cross Talk between Nucleotide Synthesis Pathways with Cellular Immunity in Constraining Hepatitis E Virus Replication.

Viruses are solely dependent on host cells to propagate; therefore, understanding virus-host interaction is important for antiviral drug development. Since de novo nucleotide biosynthesis is essentially required for both host cell metabolism and viral replication, specific catalytic enzymes of these pathways have been explored as potential antiviral targets. In this study, we investigated the role of different enzymatic cascades of nucleotide biosynthesis in hepatitis E virus (HEV) replication. By profiling various pharmacological inhibitors of nucleotide biosynthesis, we found that targeting the early steps of the purine biosynthesis pathway led to the enhancement of HEV replication, whereas targeting the later step resulted in potent antiviral activity via the depletion of purine nucleotide. Furthermore, the inhibition of the pyrimidine pathway resulted in potent anti-HEV activity. Interestingly, all of these inhibitors with anti-HEV activity concurrently triggered the induction of antiviral interferon-stimulated genes (ISGs). Although ISGs are commonly induced by interferons via the JAK-STAT pathway, their induction by nucleotide synthesis inhibitors is completely independent of this classical mechanism. In conclusion, this study revealed an unconventional novel mechanism of cross talk between nucleotide biosynthesis pathways and cellular antiviral immunity in constraining HEV infection. Targeting particular enzymes in nucleotide biosynthesis represents a viable option for antiviral drug development against HEV. HEV is the most common cause of acute viral hepatitis worldwide and is also associated with chronic hepatitis, especially in immunocompromised patients. Although often an acute and self-limiting infection in the general population, HEV can cause severe morbidity and mortality in certain patients, a problem compounded by the lack of FDA-approved anti-HEV medication available. In this study, we have investigated the role of the nucleotide synthesis pathway in HEV infection and its potential for antiviral drug development. We show that targeting the later but not the early steps of the purine synthesis pathway exerts strong anti-HEV activity. In particular, IMP dehydrogenase (IMPDH) is the most important anti-HEV target of this cascade. Importantly, the clinically used IMPDH inhibitors, including mycophenolic acid and ribavirin, have potent anti-HEV activity. Furthermore, targeting the pyrimidine synthesis pathway also exerts potent antiviral activity against HEV. Interestingly, antiviral effects of nucleotide synthesis pathway inhibitors appear to depend on the medication-induced transcription of antiviral interferon-stimulated genes. Thus, this study reveals an unconventional novel mechanism as to how nucleotide synthesis pathway inhibitors can counteract HEV replication.