Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.

BACKGROUND & AIMS: Hepatitis B and D viruses (HBV and HDV) are human pathogens with restricted host ranges and high selectivity for hepatocytes; the HBV L-envelope protein interacts specifically with a receptor on these cells. We aimed to identify this receptor and analyze whether it is the recently described sodium-taurocholate co-transporter polypeptide (NTCP), encoded by the SLC10A1 gene. METHODS: To identify receptor candidates, we compared gene expression patterns between differentiated HepaRG cells, which express the receptor, and naïve cells, which do not. Receptor candidates were evaluated by small hairpin RNA silencing in HepaRG cells; the ability of receptor expression to confer binding and infection were tested in transduced hepatoma cell lines. We used interspecies domain swapping to identify motifs for receptor-mediated host discrimination of HBV and HDV binding and infection. RESULTS: Bioinformatic analyses of comparative expression arrays confirmed that NTCP, which was previously identified through a biochemical approach is a bona fide receptor for HBV and HDV. NTCPs from rat, mouse, and human bound Myrcludex B, a peptide ligand derived from the HBV L-protein. Myrcludex B blocked NTCP transport of bile salts; small hairpin RNA-mediated knockdown of NTCP in HepaRG cells prevented their infection by HBV or HDV. Expression of human but not mouse NTCP in HepG2 and HuH7 cells conferred a limited cell-type-related and virus-dependent susceptibility to infection; these limitations were overcome when cells were cultured with dimethyl sulfoxide. We identified 2 short-sequence motifs in human NTCP that were required for species-specific binding and infection by HBV and HDV. CONCLUSIONS: Human NTCP is a specific receptor for HBV and HDV. NTCP-expressing cell lines can be efficiently infected with these viruses, and might be used in basic research and high-throughput screening studies. Mapping of motifs in NTCPs have increased our understanding of the species specificities of HBV and HDV, and could lead to small animal models for studies of viral infection and replication.

Cyclosporin A inhibits hepatitis B and hepatitis D virus entry by cyclophilin-independent interference with the NTCP receptor.

BACKGROUND & AIMS: Chronic hepatitis B and hepatitis D are global health problems caused by the human hepatitis B and hepatitis D virus. The myristoylated preS1 domain of the large envelope protein mediates specific binding to hepatocytes by sodium taurocholate co-transporting polypeptide (NTCP). NTCP is a bile salt transporter known to be inhibited by cyclosporin A. This study aimed to characterize the effect of cyclosporin A on HBV/HDV infection. METHODS: HepaRG cells, primary human hepatocytes, and susceptible NTCP-expressing hepatoma cell lines were applied for infection experiments. The mode of action of cyclosporin A was studied by comparing the effect of different inhibitors, cyclophilin A/B/C-silenced cell lines as well as NTCP variants and mutants. Bile salt transporter and HBV receptor functions were investigated by taurocholate uptake and quantification of HBVpreS binding. RESULTS: Cyclosporin A inhibited hepatitis B and D virus infections during and--less pronounced--prior to virus inoculation. Binding of HBVpreS to NTCP was blocked by cyclosporin A concentrations at 8 µM. An NTCP variant deficient in HBVpreS binding but competent for bile salt transport showed resistance to cyclosporin A. Silencing of cyclophilins A/B/C did not abrogate transporter and receptor inhibition. In contrast, tacrolimus, a cyclophilin-independent calcineurin inhibitor, was inactive. CONCLUSIONS: HBV and HDV entry via sodium taurocholate co-transporting polypeptide is inhibited by cyclosporin A. The interaction between the drug and the viral receptor is direct and overlaps with a functional binding site of the preS1 domain, which mediates viral entry.

Myristoylated PreS1-domain of the hepatitis B virus L-protein mediates specific binding to differentiated hepatocytes.

UNLABELLED: Chronic infection with the human hepatitis B virus (HBV) is a global health problem and a main cause of progressive liver diseases. HBV exhibits a narrow host range, replicating primarily in hepatocytes. Both host and hepatocyte specificity presumably involve specific receptor interactions on the target cell; however, direct evidence for this hypothesis is missing. Following the observation that HBV entry is specifically blocked by L-protein-derived preS1-lipopeptides, we visualized specific HBV receptor/ligand complexes on hepatic cells and quantified the turnover kinetics. Using fluorescein isothiocyanate-labeled, myristoylated HBV preS1-peptides we demonstrate (1) the presence of a highly specific HBV receptor on the plasma membrane of HBV-susceptible primary human and tupaia hepatocytes and HepaRG cells but also on hepatocytes from the nonsusceptible species mouse, rat, rabbit and dog; (2) the requirement of a differentiated state of the hepatocyte for specific preS1-binding; (3) the lack of detectable amounts of the receptor on HepG2 and HuH7 cells; (4) a slow receptor turnover at the hepatocyte membrane; and (5) an association of the receptor with actin microfilaments. The presence of the preS1-receptor in primary hepatocytes from some non-HBV-susceptible species indicates that the lack of susceptibility of these cells is owed to a postbinding step. CONCLUSION: These findings suggest that HBV hepatotropism is mediated by the highly selective expression of a yet unknown receptor* on differentiated hepatocytes, while species specificity of the HBV infection requires selective downstream events, e.g., the presence of host dependency or the absence of host restriction factors. The criteria defined here will allow narrowing down reasonable receptor candidates and provide a binding assay for HBV-receptor expression screens in hepatic cells.

Liver imaging with a novel hepatitis B surface protein derived SPECT-tracer.

Diagnostic imaging of the liver by ultrasound, computed tomography (CT) and magnetic resonance tomography (MRT) is generally limited to the visualization of the morphology. In order to exploit the intriguing liver tropism of the human hepatitis B virus (HBV) for molecular imaging of the liver, peptidic tracers derived from the HBV large envelope protein (L) were studied. An N-terminally stearoylated tracer comprising amino acids 2-48 of the PreS1-domain of the L protein was synthesized by solid phase peptide synthesis. Mercaptoacetyltriglycerin (MAG3) was linked to this peptide to enable (99m)Tc labeling. Biodistribution studies in mice showed an excellent liver accumulation of this novel class of radiotracer with 84%, 84%, 65%, and 16% of the injected dose in the liver after 10 min, 1 h, 4 h, and 24 h, respectively. Imaging studies on a gamma camera showed a clear visualization of the liver already 10 min post intravenous injection. These studies confirmed the exclusive accumulation of the tracer in the liver with negligible background in other organs. Owing to a significant biliary clearance rate of the (99m)Tc bound via a linker, the tracer enabled imaging of the bile ducts starting 30 min after injection. Analysis of the route of excretion revealed complete clearance within 24 h post injection. Clearance was predominantly via renal secretion. In conclusion, the novel class of tracers shows excellent pharmacokinetic, biodistribution, and clearance kinetics. It provides unique biological information different from the current imaging modalities. Its primary application is likely to be the evaluation of liver cancer patients. Specific indications may include tumor staging, the differentiation of malignant versus benign hepatic lesions, and liver tumors of nonhepatocellular origin.

Correction: Oweira et al. Kynurenine Is the Main Metabolite of Tryptophan Degradation by Tryptophan 2,3-Dioxygenase in HepaRG Tumor Cells. J. Clin. Med. 2022, 11, 4794.

There was an error in the original publication [...].

Kynurenine Is the Main Metabolite of Tryptophan Degradation by Tryptophan 2,3-Dioxygenase in HepG2 Tumor Cells.

There are two main enzymes that convert tryptophan (Trp) to kynurenine (Kyn): tryptophan-2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). Kyn accumulation can promote immunosuppression in certain cancers. In this study, we investigated Trp degradation to Kyn by IDO and TDO in primary human hepatocytes (PHH) and tumoral HepG2 cells. To quantify Trp-degradation and Kyn-accumulation, using reversed-phase high-pressure liquid chromatography, the levels of Trp and Kyn were determined in the culture media of PHH and HepG2 cells. The role of IDO in Trp metabolism was investigated by activating IDO with IFN-γ and inhibiting IDO with 1-methyl-tryptophan (1-DL-MT). The role of TDO was investigated using one of two TDO inhibitors: 680C91 or LM10. Real-time PCR was used to measure TDO and IDO expression. Trp was degraded in both PHH and HepG2 cells, but degradation was higher in PHH cells. However, Kyn accumulation was higher in the supernatants of HepG2 cells. Stimulating IDO with IFN-γ did not significantly affect Trp degradation and Kyn accumulation, even though it strongly upregulated IDO expression. Inhibiting IDO with 1-DL-MT also had no effect on Trp degradation. In contrast, inhibiting TDO with 680C91 or LM10 significantly reduced Trp degradation. The expression of TDO but not of IDO correlated positively with Kyn accumulation in the HepG2 cell culture media. Furthermore, TDO degraded L-Trp but not D-Trp in HepG2 cells. Kyn is the main metabolite of Trp degradation by TDO in HepG2 cells. The accumulation of Kyn in HepG2 cells could be a key mechanism for tumor immune resistance. Two TDO inhibitors, 680C91 and LM10, could be useful in immunotherapy for liver cancers.

Enhancement of anti-tumor activity in vitro and in vivo by CD150 and SAP.

Signaling lymphocyte activation molecule (SLAM, CD150) is a co-stimulatory receptor involved in T cell activation. The activity of CD150 is dependent on the intracellular signaling molecule SAP. Here, we investigated anti-CD3 activated human lymphocytes, transfected either with CD150-plasmid or with CD150- or SAP-siRNA in cytotoxicity assays against human colon cancer cells in vitro and in a xenograft model (CB/Scid/CrL mice) in vivo. Up-regulation or silencing of CD150 was accompanied by increased or decreased cytotoxic activity, respectively. Similar effects could also be shown in an IFN-gamma ELISpot assay. Furthermore, CD150 co-localized after activation with lipid rafts in specific membrane compartments on CD8 T cells. Treatment of xenografted mice with CD150 over-expressing lymphocytes decelerated tumor growth significantly. Lymphocytes were detectable in spleen 18 days after injection and expressed mainly CD8, CD45RO and CD150 above average. In conclusion, over-expression of CD150 in lymphocytes is accompanied with enhanced cytotoxic activity and IFN-gamma secretion in vitro and anti-tumor activity in vivo, whereas silencing of CD150 down-regulates effector functions. Adoptive cell transfer of CD150 over-expressing lymphocytes results in an accumulation of CD8, CD45RO and CD150 cells in tumor and spleen indicating together with the observed CD150 co-localization with lipid rafts that CD150 mediates a Th1 response.

Bortezomib is ineffective in an orthotopic mouse model of pancreatic adenocarcinoma.

The purpose of the present study was to evaluate the potency of the proteasome inhibitor bortezomib +/- gemcitabine in vitro and in vivo in pancreatic carcinoma. It could be shown that bortezomib induced apoptosis and inhibited proliferation of pancreatic carcinoma very efficiently in vitro. In contrast, in an orthotopic pancreatic adenocarcinoma mouse model, gemcitabine treatment inhibited tumor growth, whereas bortezomib promoted it. Bortezomib-treated animals showed significantly higher tumor burden compared with gemcitabine-treated and control animals, although bortezomib was locally active and induced a decrease of proteasome activity, which was most pronounced following the simultaneous administration of gemcitabine. Also, tumor progression was not caused by immunosuppression as a result of proteasome inhibition. Interestingly, anti-CD31 staining of tumors showed that angiogenesis was significantly increased in the tumors of bortezomib-treated mice compared with the tumors of control animals. In addition, bortezomib resulted an increase of pericytes, vascular endothelial growth factor, RGS-5, and hypoxia-inducible factor-1alpha in the tumor. Although this study supports efficacy of bortezomib against pancreatic carcinoma in vitro, it strongly indicates that bortezomib therapy has a significant tumor-promoting effect in vivo by induction of angiogenesis. The data are in accordance with the complete failure of bortezomib in a phase II trial for this indication. Choosing the right schedule of gemcitabine and bortezomib showed some synergistic effects, but the gain might not be big enough to compensate the potentially detrimental effects.

Interferon-alpha restitutes the chemosensitivity in pancreatic cancer.

BACKGROUND: Multidrug resistance is a major obstacle in the treatment of pancreatic cancer. Immunochemotherapy including interferon-alpha increases response rates and survival. MATERIALS AND METHODS: Pancreatic cancer was induced in an orthotopic mouse model. Animals received standard chemotherapy or combinative treatment with interferon-alpha. Expression and function of drug-resistance proteins were analyzed. Immunological phenotyping, cytotoxic activity assays and analysis of T-cell activation status were performed. RESULTS: Addition of interferon-alpha to chemotherapeutic regimes significantly reduced chemotherapy-induced expression of multidrug resistance proteins and drug efflux activity of cancer cells. Tumor size and metastatic seeding decreased significantly upon combination therapy and survival was prolonged. A significantly higher proportion of activated and cytotoxic active CD8+ tumor infiltrating lymphocytes was detectable after induction of drug resistance. CONCLUSION: Restitution of chemosensitivity by the addition of interferon alpha to chemotherapy was demonstrated in experimental pancreatic cancer for the first time. Since drug-resistance proteins may function as tumor antigens, our data support immunochemotherapy as an encouraging new approach.

Role of human corneal endothelial cells in T-cell-mediated alloimmune attack in vitro.

PURPOSE: Human corneal endothelial cells (HCEC) are a potential target of immune attack after corneal transplantation. The aim of this in vitro study was to investigate the role of HCEC during the alloimmune response of T-cells by examining cytokine profiles, function of the immunosuppressive enzyme indoleamine 2,3-dioxigenase (IDO), major histocompatibility complex (MHC-I/-II), T-cell proliferation, and the induction of cell death. METHODS: Real-time PCR and RP-HPLC were used to determine IDO expression and activity. Multiplex assay was performed for quantification of cytokine levels. T-cell proliferation was assessed by thymidine incorporation, and HCEC cell death was measured by flow cytometry. RESULTS: Human corneal endothelial cells induce strong proliferation of allogeneic T-cells and an increase of proinflammatory cytokines such as interleukin-1α (IL-1α), IL-1ß, IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). Tumor necrosis factor-alpha (and to a lesser extent IFN-γ) induces apoptosis. Moreover, IFN-γ strongly upregulates MHC-II molecules and IDO activity in HCEC as reflected by high kynurenine (Kyn) concentrations. Interestingly, the T-cell response was not affected by increased IDO activity, since blocking of IDO did not affect the proliferation rate. Indoleamine 2,3-dioxigenase-induced Kyn levels did not exceed concentrations of 175 ± 20 µM. Concentrations of ≥400 µM Kyn were required to suppress T-cell proliferation. CONCLUSIONS: Our data show that T-cell attack on HCEC leads to increased concentrations of proinflammatory cytokines. Inflammatory cytokines induce apoptosis and upregulate MHC-II molecules and IDO in HCEC. Although increased IDO activity does not influence the T-cell response, it constitutes an inflammatory marker of the alloimmune response toward HCEC.

Comparison of phenotype of gammadelta T cells generated using various cultivation methods.

It has been demonstrated, that gammadelta T cells play an important role in the development of immune responses to many pathogens. gammadelta T cells play a role in the clearance of viral and microbiological infections, anti-tumor responses, but also in autoimmune diseases. Many different protocols for the isolation and cultivation of gammadelta T cells can be found in the literature. Here we compare three common cultivation protocols for gammadelta T cells derived from peripheral blood with a newly developed protocol depending on SLAM (Signaling Lymphocyte Activation Molecule) stimulation. We demonstrate that the cultivation protocol chosen to raise gammadelta T cells has direct impact on the resulting gammadelta T cell phenotype. We show differences in gammadelta TCR composition, memory phenotype formation, CD8 receptor expression and the expression of NK cell markers depending on the stimulation protocol used. As such, the cultivation protocol chosen for a series of experiments might have significant impact on the outcome of the experiments and should be considered carefully.

SAP and SLAM expression in anti-CD3 activated lymphocytes correlates with cytotoxic activity.

Signalling lymphocyte activation molecule (SLAM)-associated protein (SAP) is a small protein that is mutant in humans with X-linked lymphoproliferative (XLP) disease. Patients with XLP disease are affected by fatal EBV infection and malignant B-cell lymphomas. The increased risk for B-cell lymphomas is suggested to result from impaired immunosurveillance of B-cell proliferation by T cells. In this study, we investigated the role of SLAM and SAP in activation of effector cells with cytotoxic activity, cytokine-induced killer (CIK) cells, which are generated by non-specific stimulation of the TCR and addition of exogenous IL-2. Agonistic TCR activation 1 day after preparation (day +1) resulted in cell activation, with a peak of SLAM on day +6 visible at both the protein and mRNA level as well as membrane detectable SLAM. This increase in SLAM expression correlated significantly with SAP expression at the mRNA level as well as at the protein level. Cytotoxic activity peaked 1 day after the observed SAP and SLAM peaks. At that point in time, IL-10 secretion, which was high during the early days of culture, decreased. In conclusion, activation of peripheral blood cells with agonistic anti-CD3 antibody and exogenous IL-2, as used for generation of CIK cells, results in significant SLAM and SAP activation 5 days after TCR stimulation. This peak correlates with cytotoxic activity against tumour cells. Expression of SLAM and SAP seems to be important in the activation of cytotoxic effector cells.

Evidence of chromosomal integration of AAV DNA in human testis tissue.

Persistent infection with adeno-associated virus (AAV) has been demonstrated in human tissues, most frequently in the female and male genital tract. The clinical significance of latent AAV infection remains, however, uncertain to date. The mode of latency of AAV is not known, i.e., it is unclear whether the viral genome is integrated in the cellular genome, and if integration occurs site-specifically in chromosome 19 as has been observed in cell culture. Therefore we investigated if viral DNA in AAV DNA-positive human testis samples from two patients, is integrated in the cellular genome. Using two different molecular approaches, uni-directional PCR and Walking Primer PCR, we could demonstrate that AAV DNA is present in an integrated form in testis tissue. Virus-cell DNA junction fragments were cloned and sequenced. A detailed analysis revealed integration within sequences of the so-called AAVS1 region on chromosome 19. These data demonstrate that AAV DNA can integrate also after natural infection, and that integration occurs within the AAVS1 region, at least in some cases.