Publications by authors named "Frederik Dagnaes-Hansen"

96 Publications

FRMD6 has tumor suppressor functions in prostate cancer.

Oncogene 2021 Jan 28;40(4):763-776. Epub 2020 Nov 28.

Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.

Available tools for prostate cancer (PC) prognosis are suboptimal but may be improved by better knowledge about genes driving tumor aggressiveness. Here, we identified FRMD6 (FERM domain-containing protein 6) as an aberrantly hypermethylated and significantly downregulated gene in PC. Low FRMD6 expression was associated with postoperative biochemical recurrence in two large PC patient cohorts. In overexpression and CRISPR/Cas9 knockout experiments in PC cell lines, FRMD6 inhibited viability, proliferation, cell cycle progression, colony formation, 3D spheroid growth, and tumor xenograft growth in mice. Transcriptomic, proteomic, and phospho-proteomic profiling revealed enrichment of Hippo/YAP and c-MYC signaling upon FRMD6 knockout. Connectivity Map analysis and drug repurposing experiments identified pyroxamide as a new potential therapy for FRMD6 deficient PC cells. Finally, we established orthotropic Frmd6 and Pten, or Pten only (control) knockout in the ROSA26 mouse prostate. After 12 weeks, Frmd6/Pten double knockouts presented high-grade prostatic intraepithelial neoplasia (HG-PIN) and hyperproliferation, while Pten single-knockouts developed only regular PIN lesions and displayed lower proliferation. In conclusion, FRMD6 was identified as a novel tumor suppressor gene and prognostic biomarker candidate in PC.
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http://dx.doi.org/10.1038/s41388-020-01548-wDOI Listing
January 2021

FcRn overexpression in human cancer drives albumin recycling and cell growth; a mechanistic basis for exploitation in targeted albumin-drug designs.

J Control Release 2020 06 4;322:53-63. Epub 2020 Mar 4.

Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark. Electronic address:

Albumin accumulation in tumours could reflect a role of albumin in transport of endogenous nutrient cargos required for cellular growth and not just a suggested source of amino acids; a role driven by albumin engagement with its cognate cellular recycling neonatal Fc receptor. We investigate the hypothesis that albumin cellular recruitment is increased by higher human FcRn (hFcRn) expression in human cancer tissue that provides the mechanistic basis for exploitation in albumin-based drug designs engineered to optimise this process. Eight out of ten different human cancer tissue types screened for hFcRn expression by immunohistochemistry (310 samples) exhibited significantly higher hFcRn expression compared to healthy tissues. Accelerated tumour growth over 28 days in mice inoculated with hFcRn-expressing HT-29 human colorectal cancer cell xenografts, compared to CRISPR/Cas9 hFcRn-knockout HT-29, suggests a hFcRn-mediated tumour growth effect. Direct correlation between hFcRn expression and albumin recycling supports hFcRn-mediated diversion of albumin from lysosomal degradation. Two-fold increase in accumulation of fluorescent labelled high-binding hFcRn albumin, compared to wild type albumin, in luciferase MDA-MB-231-Luc-D3H2LN breast cancer xenografts was shown. This work identifies overexpression of hFcRn in several human cancer types with mechanistic data suggesting hFcRn-driven albumin recruitment for increased cellular growth that has the potential to be exploited with high hFcRn-binding albumin variants for targeted therapies.
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http://dx.doi.org/10.1016/j.jconrel.2020.03.004DOI Listing
June 2020

Molecular, Macromolecular, and Supramolecular Glucuronide Prodrugs: Lead Identified for Anticancer Prodrug Monotherapy.

Angew Chem Int Ed Engl 2020 05 11;59(19):7390-7396. Epub 2020 Mar 11.

Department of Chemistry, Aarhus University, Aarhus, Denmark.

In this work, a tumor growth intervention by localized drug synthesis within the tumor volume, using the enzymatic repertoire of the tumor itself, is presented. Towards the overall success, molecular, macromolecular, and supramolecular glucuronide prodrugs were designed for a highly potent toxin, monomethyl auristatin E (MMAE). The lead candidate exhibited a fold difference in toxicity between the prodrug and the drug of 175, had an engineered mechanism to enhance the deliverable payload to tumours, and contained a highly potent toxin such that bioconversion of only a few prodrug molecules created a concentration of MMAE sufficient enough for efficient suppression of tumor growth. Each of these points is highly significant and together afford a safe, selective anticancer measure, making tumor-targeted glucuronides attractive for translational medicine.
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http://dx.doi.org/10.1002/anie.201916124DOI Listing
May 2020

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection.

J Vis Exp 2020 01 31(155). Epub 2020 Jan 31.

Department of Clinical Medicine, Aarhus University; Department of Infectious Diseases, Aarhus University Hospital.

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%-50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.
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http://dx.doi.org/10.3791/60723DOI Listing
January 2020

Imaging Rheumatoid Arthritis in Mice Using Combined Near Infrared and F Magnetic Resonance Modalities.

Sci Rep 2019 10 4;9(1):14314. Epub 2019 Oct 4.

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark.

Rheumatoid arthritis (RA) is an autoimmune disease that causes pain and tissue destruction in people worldwide. An accurate diagnosis is paramount in order to develop an effective treatment plan. This study demonstrates that combining near infrared (NIR) imaging and F MRI with the injection of labelled nanoparticles provides high diagnostic specificity for RA. The nanoparticles were made from poly(ethylene glycol)-block-poly(lactic-co-glycolic acid) (NP) or PLGA-PEG-Folate (Folate-NP), loaded with perfluorooctyl bromide (PFOB) and indocyanine green (ICG) and evaluated in vitro and in a collagen-induced arthritic (CIA) mouse model. The different particles had a similar size and a spherical shape according to dynamic light scattering (DLS) and transmission electron microscopy (TEM). Based on flow cytometry and F MRI analysis, Folate-NP yielded a higher uptake than NP in activated macrophages in vitro. The potential RA-targeting ability of the particles was studied in CIA mice using NIR and F MRI analysis. Both NP and Folate-NP accumulated in the RA tissues, where they were visible in NIR and F MRI for up to 24 hours. The presence of folate as a targeting ligand significantly improved the NIR signal from inflamed tissue at the early time point (2 hours), but not at later time points. Overall, these results suggest that our nanoparticles can be applied for combined NIR and F MRI imaging for improved RA diagnosis.
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http://dx.doi.org/10.1038/s41598-019-50043-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778085PMC
October 2019

Lipidoid-siRNA Nanoparticle-Mediated IL-1β Gene Silencing for Systemic Arthritis Therapy in a Mouse Model.

Mol Ther 2019 08 15;27(8):1424-1435. Epub 2019 May 15.

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, 8000 Aarhus C, Denmark; Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark. Electronic address:

Interleukin-1 beta (IL-1β) plays a central role in the induction of rheumatoid arthritis (RA). In the present study, we demonstrated that lipidoid-polymer hybrid nanoparticle (FS14-NP) can efficiently deliver siRNA against IL-1β (siIL-1β) to macrophages and effectively suppress the pathogenesis of experimental arthritis induced by collagen antibody (CAIA mice). FS14-NP/siIL-1β achieved approximately 70% and 90% gene-silencing efficiency in the RAW 264.7 cell line and intraperitoneal macrophages, respectively. Intravenous administration of FS14-NP/siRNA led to rapid accumulation of siRNA in macrophages within the arthritic joints. Furthermore, FS14-NP/siIL-1β treatment lowered the expression of pro-inflammatory cytokines in arthritic joints and dramatically attenuated ankle swelling, bone erosion, and cartilage destruction. These results demonstrate that FS14-NP/siIL-1β may represent an effective therapy for systemic arthritis and other inflammatory disorders.
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http://dx.doi.org/10.1016/j.ymthe.2019.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697342PMC
August 2019

Targeted lipid nanoparticle delivery of calcitriol to human monocyte-derived macrophages in vitro and in vivo: investigation of the anti-inflammatory effects of calcitriol.

Int J Nanomedicine 2019 23;14:2829-2846. Epub 2019 Apr 23.

Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark,

Background: Vitamin D possesses anti-inflammatory and modulatory properties in addition to its role in calcium and phosphate homeostasis. Upon activation, macrophages (M) can initiate and sustain pro-inflammatory cytokine production in inflammatory disorders and play a pathogenic role in certain cancers.

Purpose: The main purpose of this study was to encapsulate and specifically target calcitriol to macrophages and investigate the anti-inflammatory properties of calcitriol in vitro and in vivo.

Methods: In this study we have designed and developed near-infrared calcitriol PEGylated nanoparticles (PEG-LNP(Cal)) using a microfluidic mixing technique and modified lipid nanoparticles (LNPs) to target the M specific endocytic receptor CD163. We have investigated LNP cellular uptake and anti-inflammatory effect in LPS-induced M in vitro by flow cytometry, confocal microscopy and gene expression analyses. LNP pharmacodynamics, bio-distribution and organ specific LNP accumulation was also investigated in mice in vivo.

Results: In vitro, we observed the specific uptake of PEG-LNP(Cal)-hCD163 in human M, which was significantly higher than the non-specific uptake of control PEG-LNP(Cal)-IgG(h) in M. Pretreatment with encapsulated calcitriol was able to attenuate intracellular TNF-expression, and M surface marker HLA-DR expression more efficiently than free calcitriol in LPS-induced M in vitro. Encapsulated calcitriol diminished mRNA gene levels of TNF-, NF-B, MCP-1 and IL-6, while upregulating IL-10. TNF- and IL-6 protein secretion also decreased. In mice, an in vivo pharmacodynamic study of PEG-LNP(Cal) showed a rapid clearance of IgG and CD163 modified LNPs compared to PEG-LNP(Cal). Antibody modified PEG-LNP(Cal) accumulated in the liver, spleen and kidney, whereas unmodified PEG-LNP(Cal) accumulation was only observed in the liver.

Conclusion: Our results show that calcitriol can be effectively targeted to M. Our data confirms the anti-inflammatory properties of calcitriol and this may be a potential way to deliver high dose bioactive calcitriol to M during inflammation in vivo.
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http://dx.doi.org/10.2147/IJN.S192113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488164PMC
July 2019

cAIMP administration in humanized mice induces a chimerization-level-dependent STING response.

Immunology 2019 06 6;157(2):163-172. Epub 2019 May 6.

Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.

It is well understood that the STING signalling pathway is critical for generating a robust innate immune response to pathogens. Human and mouse STING signalling pathways are not identical, however. For example, mice lack IFI16, which has been proven important for the human STING pathway. Therefore, we investigated whether humanized mice are an appropriate experimental platform for exploring the human STING signalling cascade in vivo. We found that NOG mice reconstituted with human cord blood haematopoietic stem cells (humanized NOG mice) exhibit human STING signalling responses to an analogue of the cyclic di-nucleotide cGAMP. There was an increase in the proportions of monocytes in the lungs of mice receiving cGAMP analogue. The most robust levels of STING expression and STING-induced responses were observed in mice exhibiting the highest levels of human chimerization. Notably, differential levels of STING in lung versus spleen following cGAMP analogue treatment suggest that there are tissue-specific kinetics of STING activation and/or degradation in effector versus inductive sites. We also examined the mouse innate immune response to cGAMP analogue treatment. We detected that mouse cells in the immunodeficient NOG mice responded to the cGAMP analogue and they do so with distinct kinetics from the human response. In conclusion, humanized NOG mice represent a valuable experimental model for examining in vivo human STING responses.
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http://dx.doi.org/10.1111/imm.13061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6526644PMC
June 2019

In Vivo Editing of the Adult Mouse Liver Using CRISPR/Cas9 and Hydrodynamic Tail Vein Injection.

Methods Mol Biol 2019 ;1961:329-341

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.

CRISPR/Cas9 technology allows facile modification of the genome in virtually any desired way through the use of easily designed plasmid constructs that express a gRNA targeting a genomic site-of-interest and Cas9. Hydrodynamic tail vein injection, on the other hand, is a simple method to deliver "naked" plasmid DNA to 5-40% of the hepatocytes of the liver of adult mice. Here, we describe how these two techniques can be combined to create a workflow for fast, easy, and cost-efficient in vivo genome editing of the adult mouse liver. Using this method, large cohorts of mice with genetically modified livers can be established within 3 weeks to generate models for gene function in normal physiology and diseases of the liver.
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http://dx.doi.org/10.1007/978-1-4939-9170-9_20DOI Listing
July 2019

Synchronous delivery of hydroxyapatite and connective tissue growth factor derived osteoinductive peptide enhanced osteogenesis.

J Control Release 2019 05 14;301:129-139. Epub 2019 Mar 14.

Department of Engineering, Aarhus University, DK-8000 Aarhus C, Denmark; Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark. Electronic address:

In bone tissue engineering, electrospun fibrous scaffolds can provide excellent mechanical support, extracellular matrix mimicking components, such as 3D spacial fibrous environment for cell growth and controlled release of signaling molecules for osteogenesis. Here, a facile strategy comprising the incorporation of an osteogenic inductive peptide H1, derived from the cysteine knot (CT) domain of connective tissue growth factor (CTGF), in the core of Silk Fibroin (SF) was developed for osteogenic induction, synergistically with co-delivering hydroxyapatite (HA) from the shell of poly(l-lactic acid-co-ε-caprolactone) (PLCL). The core-shell nanofibrous structure was confirmed by transmission electron microscopy (TEM). Furthermore, the sustained released H1 has effectively promoted proliferation and osteoblastic differentiation of human induced pluripotent stem cells-derived mesenchymal stem cells (hiPS-MSCs). Moreover, after 8 weeks implantation in mice, this SF-H1/PLCL-HA composite induced bone tissue formation significantly faster than SF/PLCL as indicated by μCT. The present study is the first to demonstrate that release of short hydrophilic peptides derived from CTGF combined with HA potentiated the regenerative capacity for healing critical sized calvarial defect in vivo.
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http://dx.doi.org/10.1016/j.jconrel.2019.02.037DOI Listing
May 2019

A new class of recombinant human albumin with multiple surface thiols exhibits stable conjugation and enhanced FcRn binding and blood circulation.

J Biol Chem 2019 03 2;294(10):3735-3743. Epub 2019 Jan 2.

From the Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, and

Human serum albumin is an endogenous ligand transport protein whose long circulatory half-life is facilitated by engagement with the human cellular recycling neonatal Fc receptor (hFcRn). The single free thiol located at Cys-34 in domain I of albumin has been exploited for monoconjugation of drugs. In this work, we increased the drug-to-albumin ratio potential by engineering recombinant human albumin (rHSA) variants with varying hFcRn affinity to contain three free, conjugation-competent cysteines. Structural analysis was used to identify positions for cysteine introduction to maximize rHSA stability and formation of the conjugated product without affecting hFcRn binding. The thiol rHSA variants exhibited up to 95% monomeric stability over 24 months and retained hFcRn engagement compared with a WT unconjugated control demonstrated by Biolayer Interferometry. The additional cysteines were further introduced into a panel of rHSA variants engineered with different affinities for hFcRn. After conjugation with three Alexa Fluor 680 (AF680) fluorophores, hFcRn binding was similar to that of the original triple-thiol nonconjugated rHSA variants (0.88 and 0.25 μm for WT albumin with or without 3xAF680 respectively, and 0.04 and 0.02 μm for a high hFcRn-binding variant with or without 3xAF680, respectively). We also observed a 1.3-fold increase in the blood circulatory half-life of a high hFcRn-binding triple-thiol variant conjugated with AF680 ( = 22.4 h) compared with its WT counterpart ( = 17.3 h) in mice. Potential high drug-to-albumin ratios combined with high hFcRn engagement are attractive features of this new class of albumins that offer a paradigm shift for albumin-based drug delivery.
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http://dx.doi.org/10.1074/jbc.RA118.005870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416450PMC
March 2019

Up-Regulated FGFR1 Expression as a Mediator of Intrinsic TKI Resistance in EGFR-Mutated NSCLC.

Transl Oncol 2019 Mar 16;12(3):432-440. Epub 2018 Dec 16.

Department of Biomedicine, Aarhus University, Aarhus, Denmark. Electronic address:

Non-small cell lung carcinoma patients with epidermal growth factor receptor (EGFR) mutations are offered EGFR tyrosine kinase inhibitors (TKI) as first line treatment, but 20-40% of these patients do not respond. High expression of alternative receptor tyrosine kinases, such as Fibroblast growth factor receptor 1 (FGFR1), potentially mediates intrinsic EGFR TKI resistance. To study this in molecular detail, we used CRISPR-dCas9 Synergistic Activation Mediator (SAM) for up-regulation of FGFR1 in physiological relevant levels in the EGFR mutated NSCLC cell lines HCC827 and PC9 thereby generating HCC827 and PC9. The sensitivity to the TKI erlotinib was investigated in vitro and in a BALBc nu/nu mouse xenograft model. FGFR1 up-regulation decreased TKI-sensitivity in both NSCLC cell lines in the presence of the ligand fibroblast growth factor 2 (FGF2). Xenografts were established with PC9 cells and it was demonstrated that there was no significant difference in tumor size between TKI- and vehicle-treated PC9 tumors. This supports decreased TKI-sensitivity in NSCLC cells with FGFR1 up-regulation. Our study points to FGFR1 signaling being an intrinsic resistance mechanism abolishing TKI response in EGFR mutated NSCLC.
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http://dx.doi.org/10.1016/j.tranon.2018.11.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297127PMC
March 2019

Non-covalent hitchhiking on endogenous carriers as a protraction mechanism for antiviral macromolecular prodrugs.

J Control Release 2019 01 12;294:298-310. Epub 2018 Dec 12.

Department of Chemistry, Aarhus University, 8000 Aarhus C, Denmark. Electronic address:

Albumin is a highly successful tool of drug delivery providing drastically extended body and blood residence time for the associated cargo, but it only traffics single drug copies at a time. In turn, macromolecular prodrugs (MP) are advantaged in carrying a high drug payload but offering only a modest extension of residence time to the conjugated drugs. In this work, we engineer MP to contain terminal groups that bind to albumin via non-covalent association and reveal that this facile measure affords a significant protraction for the associated polymers. This methodology is applied to MP of acyclovir, a successful drug against herpes simplex virus infection but with poor pharmacokinetics. Resulting albumin-affine MP were efficacious agents against herpes simplex virus type 2 (HSV-2) both in vitro and in vivo. In the latter case, sub-cutaneous administration of MP resulted in local (vaginal) antiviral effects and a systemic protection. Presented benefits of non-covalent association with albumin are readily transferrable to a wide variety of MP in development for drug delivery as anticancer, anti-inflammatory, and anti-viral measures.
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http://dx.doi.org/10.1016/j.jconrel.2018.12.016DOI Listing
January 2019

Improved Lentiviral Gene Delivery to Mouse Liver by Hydrodynamic Vector Injection through Tail Vein.

Mol Ther Nucleic Acids 2018 Sep 6;12:672-683. Epub 2018 Aug 6.

Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark. Electronic address:

Delivery of genes to mouse liver is routinely accomplished by tail-vein injections of viral vectors or naked plasmid DNA. While viral vectors are typically injected in a low-pressure and -volume fashion, uptake of naked plasmid DNA to hepatocytes is facilitated by high pressure and volumes, also known as hydrodynamic delivery. In this study, we compare the efficacy and specificity of delivery of vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentiviral vectors to mouse liver by a number of injection schemes. Exploiting in vivo bioluminescence imaging as a readout after lentiviral gene transfer, we compare delivery by (1) "conventional" tail-vein injections, (2) "primed" injections, (3) "hydrodynamic" injections, or (4) direct "intrahepatic" injections into exposed livers. Reporter gene activity demonstrate potent and targeted delivery to liver by hydrodynamic injections. Enhanced efficacy is confirmed by analysis of liver sections from mice treated with GFP-encoding vectors, demonstrating 10-fold higher transduction rates and gene delivery to ∼80% of hepatocytes after hydrodynamic vector delivery. In summary, lentiviral vector transfer to mouse liver can be strongly augmented by hydrodynamic tail-vein injections, resulting in both reduced off-target delivery and transduction of the majority of hepatocytes. Our findings pave the way for more effective use of lentiviral gene delivery in the mouse.
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http://dx.doi.org/10.1016/j.omtn.2018.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6083003PMC
September 2018

Cellular recycling-driven in vivo half-life extension using recombinant albumin fusions tuned for neonatal Fc receptor (FcRn) engagement.

J Control Release 2018 10 29;287:132-141. Epub 2018 Aug 29.

Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark. Electronic address:

Recombinant albumin-drug genetic fusions are an effective technology to prolong the serum half-life of therapeutics that has resulted in marketed products. Indirect evidence suggests albumin fusions' long circulation is controlled by engagement with the cellular recycling neonatal Fc receptor (FcRn) in addition to reduced kidney filtration. In this work, we have used a panel of recombinant fusions, engineered with different human FcRn (hFcRn) affinity, including a novel high binding albumin variant (HBII), to directly define and importantly, control the intracellular mechanism as a half-life extension tuning method. mNeonGreen or mCherry fusion to the N-terminal of the recombinant human albumin (rHA) variants null-binder (rHA NB), wild-type (rHA WT), high-binder I (rHA HBI), and high-binder II (rHA HBII) did not generally interfere with hFcRn interaction determined by Biolayer Interferometry. Co-localisation of the albumins with endosomal, but not lysosomal, markers was shown by confocal microscopy for high, but not low, hFcRn binders in a human microvascular endothelial hFcRn overexpressing cell line (HMEC-1 FcRn) suggestive of endosomal compartmentalisation. Furthermore, a cellular recycling assay revealed increased recycling of albumin fusions for the high binding variants (mNeonGreen WT; ~1, mNeonGreen HBI; 5.26-fold higher, and mNeonGreen HBII; 5.77-fold higher) in the hFcRn overexpressing cell line. In vivo experiments demonstrated a direct in vitro recycling/in vivo half-life correlation with a longer circulation for the mCherry fusions engineered with high hFcRn affinity that was highest with the HBII variant of 30.1 h compared to 18.2 h for the mCherry WT. This work gives the first direct evidence for an FcRn-driven endosomal cellular recycling pathway for recombinant albumin fusions that correlates with half-life extension controlled by the affinity to hFcRn; promoting a versatile method to tune the pharmacokinetics of albumin fusion-based therapeutics not met by current technologies.
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http://dx.doi.org/10.1016/j.jconrel.2018.07.023DOI Listing
October 2018

Theranostic Niosomes for Efficient siRNA/MicroRNA Delivery and Activatable Near-Infrared Fluorescent Tracking of Stem Cells.

ACS Appl Mater Interfaces 2018 Jun 29;10(23):19494-19503. Epub 2018 May 29.

RNA interference-mediated gene regulation in stem cells offers great potential in regenerative medicine. In this study, we developed a theranostic platform for efficient delivery of small RNAs [small interfering RNA (siRNA)/microRNA (miRNA)] to human mesenchymal stem cells (hMSCs) to promote differentiation, and meanwhile, to specifically label the transfected cells for the in vivo tracking purpose. We encapsulated indocyanine green (ICG) in a nonionic surfactant vesicle, termed "niosome", that is mainly composed of a nonionic surfactant sorbitan monooleate (Span 80) and a cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). This novel ICG-containing niosome system (iSPN) demonstrated highly efficient siRNA and miRNA delivery in hMSCs. Specific inhibition of miR-138, a negative regulator of osteoblast differentiation, was achieved by iSPN/miR-138, which significantly promoted osteogenesis of hMSCs. Furthermore, iSPN exhibited OFF/ON activatable fluorescence upon cellular internalization, resulting in efficient near-infrared labeling and the capability to dynamically monitor stem cells in mice. In addition, iSPN/siRNA achieved simultaneous long-term cell tracking and in vivo gene silencing after implantation in mice. These results indicate that our theranostic niosomes could represent a promising platform for future development of stem cell-based therapy.
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http://dx.doi.org/10.1021/acsami.8b05513DOI Listing
June 2018

Fibrogenic and angiogenic commitments of human induced pluripotent stem cells derived mesenchymal stem cells in connective tissue growth factor-delivering scaffold in an immune-deficient mice model.

J Biomed Mater Res B Appl Biomater 2018 08 31;106(6):2266-2274. Epub 2017 Oct 31.

Department of Engineering, Aarhus University, Aarhus C, Denmark.

Compared to terminal differentiated cells, stem cells play important roles in the maintenance and regeneration, and thus have been intensively researched as the most promising cell based therapy. In order to maximize the effectiveness of stem cell based therapies, it is essential to understand the environmental (niche) signals that regulate stem cell behavior. Recent findings suggest that fibroblasts have a mesenchymal origin and that mesenchymal stem cells (MSCs) demonstrate proangiogenic function, where both fibrogenic and angiogenic activities are associated with connective tissue growth factor (CTGF), a matricellular protein that serves as an essential mediator of skeletogenesis in development and vascular remodeling. Here, for the first time, we demonstrate that upon local delivery of CTGF from a three dimensional (3D) nanocomposite scaffold, human induced pluripotent stem cells derived MSCs can be directed to differentiate toward fibroblasts in a 3D nanocomposite scaffold in female nonobese diabetic CB-17/Icr-severe combined immunodeficient mice. The stem cell-scaffold constructs present not only intriguingly strong fibroblastic commitments but also angiogenic induction in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2266-2274, 2018.
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http://dx.doi.org/10.1002/jbm.b.34030DOI Listing
August 2018

CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1-Prkaca Gene Fusion Is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma.

Gastroenterology 2017 12 18;153(6):1662-1673.e10. Epub 2017 Sep 18.

Biotech Research and Innovation Centre, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Background & Aims: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects children and young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1-Prkaca fusion and monitored the mice for liver tumor development.

Methods: We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1-Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8-week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1-Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing.

Results: Livers from 12 of the 15 mice given the vectors to induce the Dnajb1-Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1-Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by the Dnajb1-Prkaca fusion revealed a lack of mutations in genes commonly associated with liver cancers, as observed in human FL-HCC.

Conclusions: Using CRISPR/Cas9 technology, we found generation of the Dnajb1-Prkaca fusion gene in wild-type mice to be sufficient to initiate formation of tumors that have many features of human FL-HCC. Strategies to block DNAJB1-PRKACA might be developed as therapeutics for this form of liver cancer.
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http://dx.doi.org/10.1053/j.gastro.2017.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801691PMC
December 2017

A genetically inducible porcine model of intestinal cancer.

Mol Oncol 2017 11 10;11(11):1616-1629. Epub 2017 Oct 10.

Department of Molecular Medicine, Aarhus University Hospital, Denmark.

Transgenic porcine cancer models bring novel possibilities for research. Their physical similarities with humans enable the use of surgical procedures and treatment approaches used for patients, which facilitates clinical translation. Here, we aimed to develop an inducible oncopig model of intestinal cancer. Transgenic (TG) minipigs were generated using somatic cell nuclear transfer by handmade cloning. The pigs encode two TG cassettes: (a) an Flp recombinase-inducible oncogene cassette containing KRAS-G12D, cMYC, SV40LT - which inhibits p53 - and pRB and (b) a 4-hydroxytamoxifen (4-OHT)-inducible Flp recombinase activator cassette controlled by the intestinal epithelium-specific villin promoter. Thirteen viable transgenic minipigs were born. The ability of 4-OHT to activate the oncogene cassette was confirmed in vitro in TG colonic organoids and ex vivo in tissue biopsies obtained by colonoscopy. In order to provide proof of principle that the oncogene cassette could also successfully be activated in vivo, three pigs were perorally treated with 400 mg tamoxifen for 2 × 5 days. After two months, one pig developed a duodenal neuroendocrine carcinoma with a lymph node metastasis. Molecular analysis of the carcinoma and metastasis confirmed activation of the oncogene cassette. No tumor formation was observed in untreated TG pigs or in the remaining two treated pigs. The latter indicates that tamoxifen delivery can probably be improved. In summary, we have generated a novel inducible oncopig model of intestinal cancer, which has the ability to form metastatic disease already two months after induction. The model may be helpful in bridging the gap between basic research and clinical usage. It opens new venues for longitudinal studies of tumor development and evolution, for preclinical assessment of new anticancer regimens, for pharmacology and toxicology assessments, as well as for studies into biological mechanisms of tumor formation and metastasis.
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http://dx.doi.org/10.1002/1878-0261.12136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664002PMC
November 2017

Analysis of Factor D Isoforms in Malpuech-Michels-Mingarelli-Carnevale Patients Highlights the Role of MASP-3 as a Maturase in the Alternative Pathway of Complement.

J Immunol 2017 Aug 9. Epub 2017 Aug 9.

Department of Biomedicine, Aarhus University, DK-8000 Aarhus, Denmark.

Factor D (FD), which is also known as adipsin, is regarded as the first-acting protease of the alternative pathway (AP) of complement. It has been suggested that FD is secreted as a mature enzyme that does not require subsequent activation. This view was challenged when it was shown that mice lacking mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1) and MASP-3 contain zymogenic FD (pro-FD), and it is becoming evident that MASP-3 is implicated in pro-FD maturation. However, the necessity of MASP-3 for pro-FD cleavage has been questioned, because AP activity is still observed in sera from MASP-1/3-deficient Malpuech-Michels-Mingarelli-Carnevale (3MC) patients. The identification of a novel 3MC patient carrying a previously unidentified MASP-3 G665S mutation prompted us to develop an analytical isoelectric focusing technique that resolves endogenous FD variants in complex samples. This enabled us to show that although 3MC patients predominantly contain pro-FD, they also contain detectable levels of mature FD. Moreover, using isoelectric focusing analysis, we show that both pro-FD and FD are present in the circulation of healthy donors. We characterized the naturally occurring 3MC-associated MASP-3 mutants and found that they all yielded enzymatically inactive proteins. Using MASP-3-depleted human serum, serum from 3MC patients, and mice, we found that lack of enzymatically active MASP-3, or complete MASP-3 deficiency, compromises the conversion of pro-FD to FD. In summary, our observations emphasize that MASP-3 acts as an important maturase in the AP of complement, while also highlighting that there exists MASP-3-independent pro-FD maturation in 3MC patients.
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http://dx.doi.org/10.4049/jimmunol.1700518DOI Listing
August 2017

Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and β1.

Dis Model Mech 2017 07;10(7):869-880

Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark

Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and β1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology.
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http://dx.doi.org/10.1242/dmm.028662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536904PMC
July 2017

Pancreas specific expression of oncogenes in a porcine model.

Transgenic Res 2017 10 29;26(5):603-612. Epub 2017 Jun 29.

Department of Clinical Medicine, Aarhus University, 8200, Aarhus N, Denmark.

Pancreatic cancer is the fourth leading course of cancer death and early detection of the disease is crucial for successful treatment. However, pancreatic cancer is difficult to detect in its earliest stages and once symptoms appear, the cancer has often progressed beyond possibility for curing. Research into the disease has been hampered by the lack of good models. We have generated a porcine model of pancreatic cancer with use of transgenic overexpression of an oncogene cassette containing MYC, KRAS and SV40 LT. The expression was initiated from a modified Pdx-1 promoter during embryogenesis in a subset of pancreatic epithelial cells. Furthermore, cells expressing the oncogenes also expressed a yellow fluorescent protein (mVenus) and an inducible negative regulator protein (rtTR-KRAB). Cells where the Pdx-1 promoter had not been activated, expressed a red fluorescent protein (Katushka). In vitro analyses of cells obtained from the transgenic pigs showed increased proliferation and expression of the transgenes when activated. Induction of the repressor protein eliminated the oncogene expression and decreased cell proliferation. In vivo analysis identified foci of pancreatic cells expressing the oncogenes at day zero post farrowing. These populations expanded and formed hyperplastic foci, with beginning abnormality at day 45. Cells in the foci expressed the oncogenic proteins and the majority of the cells were positive for the proliferation marker, Ki67. We predict that this model could be used for advanced studies in pancreatic cancer in a large animal model with focus on early detection, treatment, and identification of new biomarkers.
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http://dx.doi.org/10.1007/s11248-017-0031-4DOI Listing
October 2017

Fatty Acid-Modified Gapmer Antisense Oligonucleotide and Serum Albumin Constructs for Pharmacokinetic Modulation.

Mol Ther 2017 07 20;25(7):1710-1717. Epub 2017 Jun 20.

The Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark. Electronic address:

Delivery technologies are required for realizing the clinical potential of molecular medicines. This work presents an alternative technology to preformulated delivery systems by harnessing the natural transport properties of serum albumin using endogenous binding of gapmer antisense oligonucleotides (ASOs)/albumin constructs. We show by an electrophoretic mobility assay that fatty acid-modified gapmer and human serum albumin (HSA) can self-assemble into constructs that offer favorable pharmacokinetics. The interaction was dependent on fatty acid type (either palmitic or myristic acid), number, and position within the gapmer ASO sequence, as well as phosphorothioate (PS) backbone modifications. Binding correlated with increased blood circulation in mice (t increased from 23 to 49 min for phosphodiester [PO] gapmer ASOs and from 28 to 66 min for PS gapmer ASOs with 2× palmitic acid modification). Furthermore, a shift toward a broader biodistribution was detected for PS compared with PO gapmer ASOs. Inclusion of 2× palmitoyl to the ASOs shifted the biodistribution to resemble that of natural albumin. This work, therefore, presents a novel strategy based on the proposed endogenous assembly of gapmer ASOs/albumin constructs for increased circulatory half-life and modulation of the biodistribution of gapmer ASOs that offers tunable pharmacokinetics based on the gapmer modification design.
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http://dx.doi.org/10.1016/j.ymthe.2017.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5498835PMC
July 2017

Clonal outbreaks of [ Pasteurella] pneumotropica biovar Heyl in two mouse colonies.

Lab Anim 2017 Dec 30;51(6):613-621. Epub 2017 Mar 30.

1 Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

The aim of this study was to document the pathogenic role of biovar Heyl of [ Pasteurella] pneumotropica in mouse colonies. Fifty-three isolates associated with mastitis and orbital, cutaneous and vaginal abscesses as well as isolates from the nose and vagina of healthy mice were investigated. According to phenotypic characteristics and rpoB sequencing, the isolates were identified as [ P.] pneumotropica biovar Heyl. Pulsed-field gel electrophoresis (PFGE) revealed five closely related profiles separated by only one to four fragments. The outbreak strains diverged from epidemiologically unrelated strains with the same rpoB sequence type, as shown by the PFGE profiles. The investigation documented that members of biovar Heyl of [ P.] pneumotropica caused disease outbreaks in mouse colonies since the clonality indicated a primary role of [ P.] pneumotropica biovar Heyl in the infections observed.
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http://dx.doi.org/10.1177/0023677217698503DOI Listing
December 2017

Impact of PEG Chain Length on the Physical Properties and Bioactivity of PEGylated Chitosan/siRNA Nanoparticles in Vitro and in Vivo.

ACS Appl Mater Interfaces 2017 Apr 3;9(14):12203-12216. Epub 2017 Apr 3.

Interdisciplinary Nanoscience Center (iNANO), Aarhus University , DK-8000 Aarhus C, Denmark.

PEGylation of cationic polyplexes is a promising approach to enhance the stability and reduce unspecific interaction with biological components. Herein, we systematically investigate the impact of PEGylation on physical and biological properties of chitosan/siRNA polyplexes. A series of chitosan-PEG copolymers (CS-PEG2k, CS-PEG5k and CS-PEG10k) were synthesized with similar PEG mass content but with different molecular weight. PEGylation with higher molecular weight and less grafting degree resulted in smaller and more compacted nanoparticles with relatively higher surface charge. PEGylated polyplexes showed distinct mechanism of endocytosis, which was macropinocytosis and caveolae-dependent and clathrin-independent. In vitro silencing efficiency in HeLa and H1299 cells was significantly improved by PEGylation and CS-PEG5k/siRNA achieved the highest knockdown efficiency. Efficient silence of ribonucleotide reductase subunit M2 (RRM2) in HeLa cells by CS-PEG5k/siRRM2 significantly induced cell cycle arrest and inhibited cell proliferation. In addition, PEGylation significantly inhibited macrophage phagocytosis and unspecific interaction with red blood cells (RBCs). Significant extension of in vivo circulation was achieved only with high molecular weight PEG modification (CS-PEG10k), whereas all CS/siRNA and CS-PEG/siRNA nanoparticles showed similar pattern of biodistribution with major accumulation in liver and kidney. These results imply that PEGylation with higher molecular weight PEG and less grafting rate is a promising strategy to improve chitosan/siRNA nanocomplexes performance both in vitro and in vivo.
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http://dx.doi.org/10.1021/acsami.6b16556DOI Listing
April 2017

Endogenous Natural Complement Inhibitor Regulates Cardiac Development.

J Immunol 2017 04 3;198(8):3118-3126. Epub 2017 Mar 3.

Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark;

Congenital heart defects are a major cause of perinatal mortality and morbidity, affecting >1% of all live births in the Western world, yet a large fraction of such defects have an unknown etiology. Recent studies demonstrated surprising dual roles for immune-related molecules and their effector mechanisms during fetal development and adult homeostasis. In this article, we describe the function of an endogenous complement inhibitor, mannan-binding lectin (MBL)-associated protein (MAp)44, in regulating the composition of a serine protease-pattern recognition receptor complex, MBL-associated serine protease (MASP)-3/collectin-L1/K1 hetero-oligomer, which impacts cardiac neural crest cell migration. We used knockdown and rescue strategies in zebrafish, a model allowing visualization and assessment of heart function, even in the presence of severe functional defects. Knockdown of embryonic expression of MAp44 caused impaired cardiogenesis, lowered heart rate, and decreased cardiac output. These defects were associated with aberrant neural crest cell behavior. We found that MAp44 competed with MASP-3 for pattern recognition molecule interaction, and knockdown of endogenous MAp44 expression could be rescued by overexpression of wild-type MAp44. Our observations provide evidence that immune molecules are centrally involved in the orchestration of cardiac tissue development.
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http://dx.doi.org/10.4049/jimmunol.1601958DOI Listing
April 2017

Cholinergic PET imaging in infections and inflammation using C-donepezil and F-FEOBV.

Eur J Nucl Med Mol Imaging 2017 Mar 26;44(3):449-458. Epub 2016 Oct 26.

Department of Nuclear Medicine and PET Centre, Aarhus University Hospital, Noerrebrogade 44, bygn. 10G, kaelderen, DK-8000, Aarhus C, Denmark.

Introduction: Immune cells utilize acetylcholine as a paracrine-signaling molecule. Many white blood cells express components of the cholinergic signaling pathway, and these are up-regulated when immune cells are activated. However, in vivo molecular imaging of cholinergic signaling in the context of inflammation has not previously been investigated.

Methods: We performed positron emission tomography (PET) using the glucose analogue 18F-FDG, and 11C-donepezil and 18F-FEOBV, markers of acetylcholinesterase and the vesicular acetylcholine transporter, respectively. Mice were inoculated subcutaneously with Staphylococcus aureus, and PET scanned at 24, 72, 120, and 144 h post-inoculation. Four pigs with post-operative abscesses were also imaged. Finally, we present initial data from human patients with infections, inflammation, and renal and lung cancer.

Results: In mice, the FDG uptake in abscesses peaked at 24 h and remained stable. The 11C-donepezil and 18F-FEOBV uptake displayed progressive increase, and at 120-144 h was nearly at the FDG level. Moderate 11C-donepezil and slightly lower 18F-FEOBV uptake were seen in pig abscesses. PCR analyses suggested that the 11C-donepezil signal in inflammatory cells is derived from both acetylcholinesterase and sigma-1 receptors. In humans, very high 11C-donepezil uptake was seen in a lobar pneumonia and in peri-tumoral inflammation surrounding a non-small cell lung carcinoma, markedly superseding the 18F-FDG uptake in the inflammation. In a renal clear cell carcinoma no 11C-donepezil uptake was seen.

Discussion: The time course of cholinergic tracer accumulation in murine abscesses was considerably different from 18F-FDG, demonstrating in the 11C-donepezil and 18F-FEOBV image distinct aspects of immune modulation. Preliminary data in humans strongly suggest that 11C-donepezil can exhibit more intense accumulation than 18F-FDG at sites of chronic inflammation. Cholinergic PET imaging may therefore have potential applications for basic research into cholinergic mechanisms of immune modulation, but also clinical applications for diagnosing infections, inflammatory disorders, and cancer inflammation.
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http://dx.doi.org/10.1007/s00259-016-3555-6DOI Listing
March 2017

Lack of immunological DNA sensing in hepatocytes facilitates hepatitis B virus infection.

Hepatology 2016 09 26;64(3):746-59. Epub 2016 Jul 26.

Department of Biomedicine and, University of Aarhus, Aarhus, Denmark.

Unlabelled: Hepatitis B virus (HBV) is a major human pathogen, and about one third of the global population will be exposed to the virus in their lifetime. HBV infects hepatocytes, where it replicates its DNA and infection can lead to acute and chronic hepatitis with a high risk of liver cirrhosis and hepatocellular carcinoma. Despite this, there is limited understanding of how HBV establishes chronic infections. In recent years it has emerged that foreign DNA potently stimulates the innate immune response, particularly type 1 interferon (IFN) production; and this occurs through a pathway dependent on the DNA sensor cyclic guanosine monophosphate-adenosine monophosphate synthase and the downstream adaptor protein stimulator of IFN genes (STING). In this work we describe that human and murine hepatocytes do not express STING. Consequently, hepatocytes do not produce type 1 IFN in response to foreign DNA or HBV infection and mice lacking STING or cyclic guanosine monophosphate-adenosine monophosphate synthase exhibit unaltered ability to control infection in an adenovirus-HBV model. Stimulation of IFN production in the murine liver by administration of synthetic RNA decreases virus infection, thus demonstrating that IFN possesses anti-HBV activity in the liver. Importantly, introduction of STING expression specifically in hepatocytes reconstitutes the DNA sensing pathway, which leads to improved control of HBV in vivo.

Conclusion: The lack of a functional innate DNA-sensing pathway in hepatocytes hampers efficient innate control of HBV infection; this may explain why HBV has adapted to specifically replicate in hepatocytes and could contribute to the weak capacity of this cell type to clear HBV infection. (Hepatology 2016;64:746-759).
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http://dx.doi.org/10.1002/hep.28685DOI Listing
September 2016

An albumin-mediated cholesterol design-based strategy for tuning siRNA pharmacokinetics and gene silencing.

J Control Release 2016 06 12;232:143-51. Epub 2016 Apr 12.

Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark. Electronic address:

Major challenges for the clinical translation of small interfering RNA (siRNA) include overcoming the poor plasma half-life, site-specific delivery and modulation of gene silencing. In this work, we exploit the intrinsic transport properties of human serum albumin to tune the blood circulatory half-life, hepatic accumulation and gene silencing; based on the number of siRNA cholesteryl modifications. We demonstrate by a gel shift assay a strong and specific affinity of recombinant human serum albumin (rHSA) towards cholesteryl-modified siRNA (Kd>1×10(-7)M) dependent on number of modifications. The rHSA/siRNA complex exhibited reduced nuclease degradation and reduced induction of TNF-α production by human peripheral blood mononuclear cells. The increased solubility of heavily cholesteryl modified siRNA in the presence of rHSA facilitated duplex annealing and consequent interaction that allowed in vivo studies using multiple cholesteryl modifications. A structural-activity-based screen of in vitro EGFP-silencing was used to select optimal siRNA designs containing cholesteryl modifications within the sense strand that were used for in vivo studies. We demonstrate plasma half-life extension in NMRI mice from t1/2 12min (naked) to t1/2 45min (single cholesteryl) and t1/2 71min (double cholesteryl) using fluorescent live bioimaging. The biodistribution showed increased accumulation in the liver for the double cholesteryl modified siRNA that correlated with an increase in hepatic Factor VII gene silencing of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing.
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http://dx.doi.org/10.1016/j.jconrel.2016.04.013DOI Listing
June 2016

Rifampicin-containing combinations are superior to combinations of vancomycin, linezolid and daptomycin against Staphylococcus aureus biofilm infection in vivo and in vitro.

Pathog Dis 2016 Jun 1;74(4):ftw019. Epub 2016 Apr 1.

Department of Infectious Diseases, Institute of Clinical Medicine, Aarhus University Hospital, Palle Juul-Jensens bvld 99, 8200 Aarhus, Denmark.

Susceptibility to antibiotics is dramatically reduced when bacteria form biofilms. In clinical settings this has a profound impact on treatment of implant-associated infections, as these are characterized by biofilm formation. Current routine susceptibility testing of microorganisms from infected implants does not reflect the actual susceptibility, and the optimal antibiotic strategy for treating implant-associated infections is not established. In this study of biofilm formation in implant-associated osteomyelitis, we compared thein vitroandin vivoefficacy of selected antibiotics alone and in combination againstStaphylococcus aureus.We tested vancomycin, linezolid, daptomycin and tigecycline alone and in combination with rifampicin, vancomycin, linezolid and daptomycin againstS. aureusIn vitro, biofilm formation dramatically reduced susceptibility by a factor of 500-2000.In vivo, antibiotic combinations were tested in a murine model of implant-associated osteomyelitis. Mice were infected by inserting implants colonized withS. aureustrough their tibia. After 11 days, the animals were divided into different groups (five animals/group) and given 14 days of antibiotic therapy. All antibiotics resulted in a reduced bacterial load in the infected bone surrounding the implant. Overall, the most effective antibiotic combinations contained rifampicin. Combinations containing two non-rifampicin antibiotics were not more active than single drugs.
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http://dx.doi.org/10.1093/femspd/ftw019DOI Listing
June 2016