J M Pollok

University Medical Center Hamburg - Eppendorf, Hamburg, Hamburg, Germany

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Publications (47)173.69 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Chronic hepatitis B virus (HBV) infection has been associated with alterations in lipid metabolism. Moreover, the sodium-taurocholate cotransporting polypeptide (NTCP), responsible for bile acid uptake into hepatocytes, was identified as the functional cellular receptor mediating HBV entry. Aim of the study was to determine whether HBV alters the liver metabolic profile by employing HBV-infected and uninfected human liver-chimeric mice. Methods: Humanized uPA/SCID mice were used to establish chronic HBV infection. Gene expression profiles were determined by RT-PCR using primers specifically recognizing transcripts of either human or murine origin. Liver biopsy samples obtained from HBV-chronic individuals were used to validate changes determined in mice. Results: Besides modest changes in lipid metabolism, HBV-infected mice displayed a significant enhancement of human cholesterol 7α-Hydroxylase (hCYP7A1; median 12-fold induction; p<0.0001), the rate-limiting enzyme promoting the conversion of cholesterol to bile acids, and of genes involved in transcriptional regulation, biosynthesis and uptake of cholesterol (hSREBP2, hHMG-CoA reductase, hLDL receptor) compared to uninfected controls. Significant hCYP7A1 induction and reduction of hSHP, the co-repressor of hCYP7A1 transcription, was also confirmed in liver biopsies from HBV-infected patients. Notably, administration of Myrcludex-B, an entry inhibitor derived from the preS1-domain of HBV envelope, provoked a comparable mCYP7A1 induction in uninfected mice, thus designating the preS1-domain as the viral component triggering such metabolic alterations. Conclusion: Binding of HBV to NTCP limits its function, thus promoting compensatory bile acid synthesis and cholesterol provision. The intimate link determined between HBV and liver metabolism underlines the importance to exploit further metabolic pathways, as well as possible NTCP-related viral-drug interactions. (Hepatology 2014)
    Hepatology 04/2014; · 12.00 Impact Factor
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    ABSTRACT: Clinical studies have shown that hepatitis Delta virus (HDV) infection can persist for years and intrahepatic latency of large Delta antigen (HDAg) has been detected following liver transplantation. However, large HDAg arising via RNA-editing is associated with increasing amounts of non-infectious HDV quasi-species. This study investigated whether HDV could persist intrahepatically in the absence of HBV in vivo and whether infectious HDV could subsequently be released following HBV super-infection. Humanized mice were infected with HDV particles lacking HBV. To test for rescue of latent HDV infection 3 and 6 weeks HDV mono-infected mice were super-infected with HBV. Viral loads and cell toxicity were determined by qRT-PCR and immunohistochemistry. The presence of HDAg-positive human hepatocytes determined after 2, 3 and 6 weeks of HDV inoculation demonstrated establishment and maintenance of intrahepatic HDV mono-infection. Although intrahepatic amounts of large HDAg and edited HDV-RNA forms increased over time in HDV mono-infected livers, HBV super-infection led to prompt viremia development (up to 10(8) HDV-RNA and 10(7) HBV-DNA copies/ml) even after 6 weeks of latent mono-infection. Concurrently, the number of HDAg-positive human hepatocytes increased, demonstrating intrahepatic HDV spreading. The infectivity of the rescued HDV virions was verified by serial passage in naive chimeric mice. HDV mono-infection can persist intrahepatically for at least 6 weeks before being rescued by HBV. Conversion of a latent HDV infection to a productive HBV/HDV co-infection may contribute to HDV persistence even in patients with low HBV replication and in the setting of liver transplantation.
    Journal of Hepatology 11/2013; · 9.86 Impact Factor
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    ABSTRACT: Although donor-specific lymphocytotoxic antibodies are regarded as a contraindication for kidney transplantation (KTx), the data available for liver or combined liver or kidney transplantation (cLKTx) are scarce. Here, we report a case of a highly sensitized young man receiving his sixth liver and second kidney graft. Multiple anti-HLA antibodies were present at the time of transplantation. As a result of suspected antibody-mediated graft damage, the patient was treated with rituximab, plasmapheresis, intravenous immunoglobulins, splenectomy, and bortezomib to decrease the antibody production. So far, patient and allograft survival has reached 4 years despite failure to achieve a permanent reduction of anti-HLA antibodies, and particularly nondonor directed antibodies.
    Transplant International 04/2013; · 3.16 Impact Factor
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    ABSTRACT: Currently approved antivirals rarely cure hepatitis B virus (HBV) infection. Therefore additional therapeutic strategies interfering with other viral replication steps are needed. Using synthetic lipopeptides derived from the HBV envelope protein we previously demonstrated prevention of de novo HBV infection in vivo. Aim of this study was to investigate the ability of the lipopeptide Myrcludex-B to block HBV spreading post-infection. METHODS: uPA/SCID mice reconstituted with human hepatocytes were infected with HBV. Daily subcutaneous Myrcludex-B administration was initiated either 3 days, 3 weeks or 8 weeks post HBV-inoculation. Viral loads were quantitated in serum, liver and visualized by immunohistochemistry. RESULTS: Myrcludex-B efficiently prevented viral spreading from the initially infected human hepatocytes, as demonstrated by the lack of increase in viremia, antigen levels and amount of HBcAg-positive human hepatocytes determined 6 weeks after treatment. Myrcludex-B efficiently blocked HBV dissemination also when treatment was started in the ramp-up phase of infection in mice displaying moderate levels of circulating virions (median 3x10E6 HBV-DNA copies/ml). Notably, after 6-weeks of treatment not only the amount of HBcAg-positive hepatocytes but also intrahepatic cccDNA loads remained comparable to values found in mice sacrificed 3-weeks post-infection. In none of the experimental settings drug administration affected human hepatocyte half-life or altered virion productivity. CONCLUSIONS: Myrcludex-B efficiently not only prevented HBV spreading from infected human hepatocytes in vivo but also hindered amplification of the cccDNA pool in initially infected hepatocytes. Administration of an entry inhibitor, possibly used in combination with current HBV-drugs, may improve patients' treatment outcome.
    Journal of Hepatology 12/2012; · 9.86 Impact Factor
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    ABSTRACT: No specific drugs are currently available against hepatitis delta virus (HDV), a defective virus leading to the most severe form of chronic viral hepatitis in man. The lack of convenient HDV infection models has hampered the development of effective therapeutics. In this study, naïve and hepatitis B virus (HBV) chronically infected humanized uPA/SCID mice were employed to establish a small animal model of HBV/HDV coinfection and superinfection. For preclinical antiviral drug evaluation, the GMP version of the myristoylated preS-peptide (Myrcludex-B), a lipopeptide derived from the pre-S1 domain of the HBV envelope, was applied to prevent de novo HBV/HDV coinfection in vivo. Virological parameters were determined at serological and intrahepatic level both by real-time polymerase chain reaction (PCR) and by immunohistochemistry. Establishment of HDV infection was highly efficient in both HBV-infected and naïve chimeric mice with HDV titers rising up to 1 × 10E9 copies/mL. Notably, HDV superinfection led to a median 0.6log reduction of HBV viremia, which although not statistically significant suggests that HDV may hinder HBV replication. In the setting of HBV/HDV simultaneous infection, a majority of human hepatocytes stained HDAg-positive long before HBV spreading was completed, confirming that HDV can replicate intrahepatically also in the absence of HBV infection. Furthermore, the increase of HBV viremia and intrahepatic cccDNA loads was significantly slower than in HBV mono-infected mice. Treatment with the HBV entry inhibitor Myrcludex-B, efficiently hindered the establishment of HDV infection in vivo. CONCLUSION: We established an efficient model of HBV/HDV infection to exploit mechanisms of viral interference in human hepatocytes and to test the efficacy of an HDV-entry inhibitor in vivo.
    Hepatology 03/2012; 55(3):685-94. · 12.00 Impact Factor
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    ABSTRACT: Suppression of viral replication with nucleoside/nucleotide inhibitors has been shown to greatly improve the outcome of chronic HBV infection. β-l-nucleoside analogues, especially β-l-deoxycytidine derivatives represent one of the most efficient groups of antiretroviral compounds. We recently described that hydroxylation of the amino group of these β-l-deoxycytidine derivatives preserved their strong HBV inhibitory activity in vitro, but strongly reduced their cytotoxicity. From this new group of compounds we selected β-l-2',3'-didehydro-2',3'-dideoxy-N(4)-hydroxy-5-fluorocytidine (l-Hyd4FC) for a first in vivo investigation. The aim of this study was to determine the antiviral activity of l-Hyd4FC in HBV-infected human liver chimeric urokinase plasminogen activator (uPA)/SCID mice. Stably infected animals (median 6×10(7) HBV DNA/ml) were injected daily with either l-Hyd4FC (50 mg/kg) or saline as controls. Mice treated with lamivudine served to compare the in vivo antiviral potency of l-Hyd4FC. Virological changes were determined by quantitative PCR. Treatment with l-Hyd4FC for 4 weeks induced a 2-log reduction of viraemia, while a median 1.5-log decline was achieved with lamivudine. Intrahepatically, l-Hyd4FC induced a median eightfold decline of viral activity (relaxed circular DNA/covalently closed circular DNA), and threefold reduction of pregenomic RNA/GAPDH levels. No significant decline of subgenomic HBV transcripts, as well as of circulating hepatitis B e antigen and hepatitis B surface antigen was detected. Maintenance of human serum albumin concentrations throughout the study, negative TUNEL staining and occurrence of viral rebound after drug withdrawal indicated that l-Hyd4FC was not toxic in human hepatocytes. Administration of l-Hyd4FC in uPA/SCID mice harbouring HBV-infected human hepatocytes demonstrated the high antiviral potency of this drug in vivo. Such characteristics make l-Hyd4FC a good candidate for further investigations a as potential HBV therapeutic agent.
    Antiviral therapy 02/2012; 17(4):623-31. · 3.07 Impact Factor
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    ABSTRACT: Interferon (IFN)-α therapy is not effective for most patients with chronic hepatitis B virus (HBV) infection for reasons that are not clear. We investigated whether HBV infection reduced IFN-α-mediated induction of antiviral defense mechanisms in human hepatocytes. Human hepatocytes were injected into severe combined immune-deficient mice (SCID/beige) that expressed transgenic urokinase plasminogen activator under control of the albumin promoter. Some mice were infected with HBV; infected and uninfected mice were given injections of human IFN-α. Changes in viral DNA and expression of human interferon-stimulated genes (ISGs) were measured by real-time polymerase chain reaction, using human-specific primers, and by immunohistochemistry. Median HBV viremia (0.8log) and intrahepatic loads of HBV RNA decreased 3-fold by 8 or 12 hours after each injection of IFN-α, but increased within 24 hours. IFN-α activated expression of human ISGs and nuclear translocation of signal transducers and activators of transcription-1 (STAT1) in human hepatocytes that repopulated the livers of uninfected mice. Although baseline levels of human ISGs were slightly increased in HBV-infected mice, compared with uninfected mice, IFN-α failed to increase expression of the ISGs OAS-1, MxA, MyD88, and TAP-1 (which regulates antigen presentation) in HBV-infected mice. IFN-α did not induce nuclear translocation of STAT1 in HBV-infected human hepatocytes. Administration of the nucleoside analogue entecavir (for 20 days) suppressed HBV replication but did not restore responsiveness to IFN-α. HBV prevents induction of IFN-α signaling by inhibiting nuclear translocation of STAT1; this can interfere with transcription of ISGs in human hepatocytes. These effects of HBV might contribute to the limited effectiveness of endogenous and therapeutic IFN-α in patients and promote viral persistence.
    Gastroenterology 03/2011; 140(7):2074-83, 2083.e1-2. · 12.82 Impact Factor
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    ABSTRACT: Liver transplantation is an established treatment for acute and chronic liver disease. However, because of the shortage of donor organs, it does not fulfill the needs of all patients. Hepatocyte transplantation is promising as an alternative method for the treatment of end-stage liver disease and as bridging therapy until liver transplantation. Our group has been working on the optimization of matrix-based hepatocyte transplantation. In order to increase cell survival after transplantation, freshly isolated human hepatocytes were seeded onto biodegradable poly(l-lactic acid) (PLLA) polymer scaffolds and were cultured in a flow bioreactor. PLLA discs were seeded with human hepatocytes and exposed to a recirculated medium flow for 6 days. Human hepatocytes formed spheroidal aggregates with a liver-like morphology and active metabolic function. Phase contrast microscopy showed increasing numbers of spheroids of increasing diameter during the culture period. Hematoxylin and eosin histology showed viable and intact hepatocytes inside the spheroids. Immunohistochemistry confirmed sustained hepatocyte function and a preserved hepatocyte-specific cytoskeleton. Albumin, alpha-1-antitrypsin, and urea assays showed continued production during the culture period. Northern blot analysis demonstrated increasing albumin signals. Scanning electron micrographs showed hepatocyte spheroids with relatively smooth undulating surfaces and numerous microvilli. Transmission electron micrographs revealed intact hepatocytes and junctional complexes with coated pits and vesicles inside the spheroids. Therefore, we conclude that primary human hepatocytes, precultured in a flow bioreactor on a PLLA scaffold, reorganize to form morphologically intact liver neotissue, and this might offer an optimized method for hepatocyte transplantation because of the expected reduction of the initial cell loss, the high regenerative potential in vivo, and the preformed functional integrity.
    Liver Transplantation 02/2011; 17(2):104-14. · 3.94 Impact Factor
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    ABSTRACT: Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two-dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell-specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three-dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen-coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver-cell-specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver-specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real-time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well-preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF-4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re-established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO-1-positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes.
    Biotechnology and Bioengineering 01/2011; 108(1):141-50. · 4.16 Impact Factor
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    ABSTRACT: Background and Aims: For many liver-based metabolic diseases no curative therapies exist. Ex vivo gene therapy represents a possible alternative to liver transplantation. Apart from successful engraftment of transplanted cells in the liver, this approach requires suitable therapeutic gene transfer vectors. Usage of adenoviral vectors is limited because of the transient nature of this vector system. In contrast, lentiviral vectors can stably transduce differentiated non-dividing cells such as hepatocytes. We investigated the capability of newly developed lentiviral gene ontology (LeGO) vectors to transduce hepatocytes efficiently with marker genes and a therapeutic gene. Methods: Freshly isolated hepatocytes from mice and humans or cultured human cells were transduced with LeGO vectors under various MOIs, transduction conditions, and culture media. Transduced cells were analyzed by microscopy and FACS, and transplanted intrasplenically in recipient mice. Results: After establishing lentiviral marking of a human tumor cell line and human fetal liver cells with multiple fluorescent marker proteins, cells were transplanted into immunodeficient mice. Engrafted cells or resulting tumors were detected in recipient livers. Subsequently, conditions were optimized for murine hepatocytes, resulting in transduction rates of approximately 50% after vector incubation for only 1 h with MOIs of 10–50, not requiring spininoculation, without relevant loss of viability. We next transduced hepatocytes from phenylketonuria mice with a LeGO vector for phenylalanine hydroxylase, which resulted in an enzyme activity in diseased cells, comparable to wildtype hepatocytes in a newly developed metabolic rate assay. The same transduction protocol also enabled lentiviral GFP-marking of human hepatocytes with up to 80%. Finally, we showed that freshly isolated and transduced hepatocytes engrafted in livers of NOD-SCID mice after intrasplenic injection. Following transplantation in a liver regeneration model (Alb-uPA mice), transduced mouse hepatocytes replenished large areas of the recipient liver with stable expression of the marker gene despite cell proliferation. Conclusion: Efficient transduction of hepatocytes with LeGO vectors and stable gene expression after cell transplantation could make this technique suitable for ex-vivo gene therapy of liver based metabolic diseases.
    Journal of Hepatology - J HEPATOL. 01/2011; 54.
  • Transplantation 01/2010; 90. · 3.78 Impact Factor
  • Transplantation 01/2010; 90. · 3.78 Impact Factor
  • Transplantation 01/2010; 90. · 3.78 Impact Factor
  • Transplantation 01/2010; 90. · 3.78 Impact Factor
  • Zeitschrift Fur Gastroenterologie - Z GASTROENTEROL. 01/2007; 45(08).
  • Journal of Hepatology - J HEPATOL. 01/2007; 46.
  • Journal of Hepatology - J HEPATOL. 01/2006; 44.
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    ABSTRACT: Recent studies indicate that after transplantation, circulating bone marrow-derived stem cells migrate into the liver and contribute to liver regeneration. Whether such cells are actively recruited from the bone marrow for liver repair remains to be determined. In this regard, we investigated whether liver resection leads to a release of stem cell marker-positive (+) cells into the peripheral circulation. Peripheral blood samples from 11 living liver donors were analyzed by flow cytometry one day before and 12h after partial hepatectomy (PH) using antibodies against CD133, CD34, CD45, CD14, c-kit, bcrp-1. Immunomagnetic separation was performed to select CD133+ cells for functional assays in vitro. A significant increase in the percentage of CD133+ cells could be demonstrated in all donors studied. Unexpectedly, virtually all CD133+ cells coexpressed CD45 and CD14, whereas only a small subset expressed CD34. No significant staining was observed for c-kit and bcrp-1. In culture, immunoselected CD133+ cells displayed characteristics of myelomonocytic precursors. In addition, enriched CD133+ generated an adherent cell population that expressed CK8, alpha-fetoprotein, and human albumin. PH induces mobilization of a distinct population of myelomonocytic progenitor cells, which have hepatic differentiation potential in vitro, and might play a role in liver regeneration after PH in humans.
    Journal of Hepatology 12/2005; 43(5):845-53. · 9.86 Impact Factor
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    ABSTRACT: Transplantation of primary human hepatocytes and establishment of hepatitis B virus (HBV) infection in immunodeficient urokinase plasminogen activator (uPA) transgenic mice was shown. However, the availability of usable primary human hepatocytes is very limited. Therefore, alternative and more accessible sources of hepatocytes permissive for HBV infection are highly desirable. Here we investigated the potential of primary hepatocytes from the tree shrew Tupaia belangeri that were shown to be susceptible to HBV infection. Freshly isolated or cryopreserved primary tupaia hepatocytes were transplantated via intrasplenic injection into immunodeficient uPA/RAG-2 mice. Engrafted mice were then infected with HBV and woolly monkey (WM)-HBV positive sera. Extensive proliferation of xenografted cells was demonstrated by the stable production of tupaia alpha1-antitrypsin in serum and liver of transplanted mice. Quantitative PCR assays demonstrated the presence of circulating viral particles as well as intracellular viral DNA, including covalently closed circular (ccc) DNA, in transplanted mice. Viral infection could be serially passaged in mice. Furthermore, viral replication was strongly inhibited by treating mice with adefovir dipivoxil. uPA mice repopulated with tupaia hepatocytes represent a useful and more accessible model for HBV infection studies, including the evaluation of antiviral therapy and cccDNA.
    Journal of Hepatology 02/2005; 42(1):54-60. · 9.86 Impact Factor
  • Transplantation 01/2004; 78. · 3.78 Impact Factor

Publication Stats

612 Citations
173.69 Total Impact Points

Institutions

  • 2000–2012
    • University Medical Center Hamburg - Eppendorf
      • Department of Hepatobiliary and Transplant Surgery
      Hamburg, Hamburg, Germany
  • 1999–2012
    • University of Hamburg
      • • II. Department of Internal Medicine
      • • Department of Hepatobiliary and Transplant Surgery
      • • Center for Internal Medicine
      • • Department of General, Visceral and Thoracic Surgery Department and Clinic
      Hamburg, Hamburg, Germany
  • 1997–1998
    • Harvard Medical School
      • Department of Surgery
      Boston, MA, United States
  • 1996–1998
    • Boston Children's Hospital
      Boston, Massachusetts, United States