Lack of cytokine gene upregulation in PHH treated with REP 2055.

<p>The expression levels of <i>TNF</i> (a), <i>IL6</i> (b), <i>IL10</i> (c), <i>IFNA4</i> (d) and <i>IFNB1</i> (e) genes were assessed by quantitative RT-PCR 6 hr after treatment with the NAP REP 2055 (0.01–10 μM) or without treatment (w/o), ODN 2216 (a CpG oligonucleotide TLR-9 agonist; 2 μM), poly I:C (a double stranded RNA TLR-3 agonist; 25 μg/ml) or Pam3CK4 (a TLR-1/2 agonist; 1 μM). Values represent mean ± SEM (normalized to 100,000 copies of beta actin mRNA). Statistically significant changes compared to untreated controls are reported for p< 0.05 (*) and p< 0.01 (**).</p

<i>In vivo</i> Experiment 1: Detection of DHBsAg and DHBcAg positive hepatocytes in liver tissue.

<p>^a--- = no liver tissue harvested</p><p>^bLower limit of detection of DHBsAg positive hepatocytes is 0.001%.</p><p>^cLower limit of detection of DHBcAg positive hepatocytes is 0.006%.</p><p>^dThe liver of duck 296 contained amyloid deposits</p><p>^eND = not done due to sample exhaustion</p><p><i>In vivo</i> Experiment 1: Detection of DHBsAg and DHBcAg positive hepatocytes in liver tissue.</p

<i>In vivo</i> Experiment 1.

<p><b>Response to various REP 2055 treatment regimens in Groups 1–4</b>. Individual duck data for serum DHBsAg (top row) and serum DHBV DNA (bottom row). Days of REP 2055 treatment are indicated at the top of each graph by inverted triangles (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140909#pone.0140909.g001" target="_blank">Fig 1A</a>). LLOQ for DHBsAg (0.88 μg/ml) and DHBV DNA (24000 copies/ml) are shown as dashed lines.</p

Experimental design of REP 2055 and NS treatment in <i>in vivo</i> Experiment 1 and 2.

<p>Fourteen-day-old ducks were infected with 5x10⁸ DHBV genome equivalents via the jugular vein. In Experiment 1 (a) all ducks were treated by IP injection with REP 2055. Group 1 received 10 mg/kg/day from 1 day prior to DHBV infection to 14 dpi; Group 2 received 10 mg/kg/day from 12–19 dpi and 10 mg/kg once weekly for 49 days; Group 3 received 10 mg/kg/day from 4–18 dpi and; Group 4 received 2 mg/kg/day from 4–18 dpi. After treatment, ducks in Groups 1, 3 and 4 were followed for an additional 49 days, from 19–68 dpi. Liver biopsies were performed prior to treatment in Group 2 and at the end of treatment in Groups 1, 3 and 4. Autopsies were performed at 68 dpi at the end of treatment in Group 2 and the end of follow-up in Groups 1, 3 and 4. In Experiment 2 (b), 14 DHBV-infected ducks were treated by IP injection with 10 mg/kg/day of REP 2055 from 12–40 dpi. A control group of 14 DHBV-infected ducks received daily IP injections of NS. Blood samples were collected during treatment and from 41–103 dpi during the first 9 weeks of follow-up. Based on interim analysis of serum DHBV DNA, 7 REP 2055-treated ducks that maintained control of their infection and 7 randomly selected NS-treated ducks, were followed from 103–155 dpi (total 16 weeks of follow-up). Liver biopsies were performed in all animals at 12 dpi prior to treatment. Biopsies or autopsies were performed at 103 dpi (9 weeks follow-up) and additional autopsies were performed in 7 animals per group at 155 dpi (16 weeks follow-up).</p

<i>In vivo</i> Experiment 2: Detection of DHBsAg and DHBcAg positive hepatocytes in liver tissue.

<p>^aNS or REP 2055 was administered via IP injection.</p><p>^b--- = no liver tissue harvested (ducks autopsied at 103 dpi, 9 weeks of follow-up)</p><p>^cLower limit of detection of DHBsAg positive hepatocytes is 0.001%.</p><p>^dLower limit of detection of DHBcAg positive hepatocytes is 0.006%.</p><p><i>In vivo</i> Experiment 2: Detection of DHBsAg and DHBcAg positive hepatocytes in liver tissue.</p

Quantitative analysis of the purities of cultured cell populations.

<p>Abbreviations: Primary human hepatocytes (PHH); Kupffer cells (KC); Liver sinusoidal endothelial cells (LSEC); Hepatic stellate cells (HSC); Cluster of differentiation (CD); α-smooth muscle actin (α-SMA); Standard error of mean (SEM).</p><p>Quantitative analysis of the purities of cultured cell populations.</p

Functional activity of cultured cells.

<p>In PHH activity of CYP3A4 was determined after stimulation with 25μM dexamethasone for 6-48h. In addition CYP3A4 was inhibited by treatment with 2% DMSO. Cells were treated with ETOH or DMSO as negative controls. Relative light units (RLU) are given as mean±SEM (n = 3). Furthermore, CYP3A4 and albumin fluorescence imaging revealed a heterogeneous pattern of cultured PHH (A). KC exhibited strong phagocytic activity by the uptake of 1μm fluorescent latex beads (green) in a time-dependent manner (B). LSEC exhibited efficient endocytic capability, shown by the efficient incorporation of AcLDL (green) after 1h of incubation which was metabolized after incubation for 6h (C). HSC, cultured for a short time, were visualized by retinol (vitamin A) autofluorescence signals. Brightness reinforcement (BrightR) detection mode was used to amplify dim structures and make them accessible. To distinguish HSC from myofibroblasts, CYGB (green) and α-SMA (red) were fluorescently stained in the HSC population. Nuclei were stained with DAPI (blue). Images were taken at 40× magnification. Scale bar, 50μm. Furthermore, RNA was extracted from freshly isolated (uncultured) HSC and HSC cultured for 10 days (n = 6). Gene expression of <i>ACTA2</i>, <i>COL1A1</i> and <i>LOXL2</i> was determined by RT-qPCR. Data represent mean of copy numbers (mean±SEM) normalized to the reference gene <i>ACTB</i> (D). Asterisks indicate significant results (* p<0.05; ** p<0.01; *** p<0.001).</p

Identification of PHH and NPC.

<p>Cell morphology was examined by phase contrast microscopy using an EVOS^TM XL Core Imaging System (AMG). Isolated cell populations were identified by immunofluorescence staining of cell type—specific markers (n = 5). Nuclei were counterstained with DAPI (blue). Scale bar, 50μm. In addition, RNA was extracted from liver cells (n = 10) and gene expression of cell type-specific markers was determined by RT-qPCR. Copy numbers were normalized to the reference gene <i>ACTB</i>. The expression of cell markers in the reference cell type was defined as 100% (e.g. APOB in PHH). The expression of these markers in the other cell types is expressed as fold change in %. PHH exhibited a binucleated, polygonal shape (A) and strongly expressed albumin (B, red). In addition, PHH showed <i>APOB</i> and <i>ASGR1</i> gene expression (C). KC exhibited an irregular morphology (D) and expressed CD68 on protein level (E, red). Furthermore KC showed <i>CD163</i> and <i>CD14</i> gene expression (F). LSEC formed their unique morphology (G) and were identified by staining of CD146 (H, red). LSEC expressed the markers <i>PECAM1</i>, <i>VWF</i> and <i>LYVE-1</i> (I). HSC changed into myofibroblast-like cells (J) and were identified with anti-α-SMA antibody (K, green). Moreover, HSC were characterized by the gene expression of the markers <i>VCL</i>, <i>DES</i> and <i>CYGB</i> (L).</p