Persistent and productive infection of DHHs by HCVcc.

<p>(<i>A</i>) Continuous replication of HCVcc in DHHs. Day-10 DHHs were exposed to Jc1/GLuc2A for 9 h before the inoculum was removed and the cells were changed to medium E with or without cyclophilin inhibitor CsA at 1 µg/ml. Culture supernatants were collected daily for measurement of luciferase activity. The culture medium was replaced with thorough washing every 48 h, and CsA was included every time fresh medium was used. Error bars represent standard deviations from triplicate experiments. (<i>B</i>) Secretion of HCV core antigen into the culture medium by infected DHHs. Day-13 DHHs were exposed to HCVcc for 9 h before the inoculum was removed, and the cells washed and changed to medium E, then immediately collected as the 0-h samples. The infected cells were then incubated for an additional 48 h in medium E with or without IFN-α (50 units/ml) before the culture supernatants were collected as the 48-h samples. Error bars represent standard deviations from replicate experiments. (<i>C</i>) Reinfection of Huh-7.5 cells by HCV particles produced from DHHs. The 48-h media from (<i>B</i>) were used to infect Huh-7.5 cells, which were then fixed for NS3 staining four days after infection. The infectious titer of the HCVcc produced by DHHs is shown. FFU: focus-forming units.</p

Cellular determinants of HCV susceptibility.

<p>(<i>A</i>) Induction of microRNA miR-122 expression by FGF-10 during hepatic specification. Equal amounts of total cellular RNA from various cells at the indicated days were subjected to a real-time RT-PCR assay for detection of miR-122 expression. (<i>B</i>) Microarray heat map of gene expression levels in day-10 versus day-7 cells. Two independent RNA samples were processed for each time point. The numbers represent the average values and standard deviations. The conventional color spectrum with green representing downregulation and red representing upregulation was adopted. Fold of changes were also listed next to the name of the gene. (<i>C</i>) Quantitative RT-PCR results of EGFR and EphA2 induction. (<i>D</i>) Upregulation of PI4KIIIα protein during the differentiation process. The levels of CyPA and DDX-3 remained unchanged in the same samples. (<i>E</i>) Quantitative RT-PCR results of IFITM1 and IFI30 expression induction.</p

Genetic modification of hESCs and HCV-resistant DHHs.

<p>(<i>A</i>) Suppression of CyPA expression by shRNA in WA09 cells and day-21 DHHs. (<i>B</i>) CyPA knockdown did not affect the expression of pluripotency marker Oct-4 in WA09 cells. (<i>C</i>) Modified DHHs were resistant to wildtype HCV infection. Infection of both the wildtype and CyPA-KD (LA) DHHs were done at day 13 and allowed to proceed for 48 h. Luciferase in the culture supernatant for monitored. Wildtype HCVcc (Jc1/GLuc2A) infected unmodified DHHs but not CyPA-KD DHHs (redlines), and the DEYN mutant infected both cell types (blue lines). Error bars represent standard deviations of replicate experiments.</p

Hepatic differentiation from human embryonic stems cells (hESCs).

<p>(<i>A</i>) Representative images of cell morphology and protein marker expression of hESCs (day 0), definitive endoderm (day 4), hepatic progenitor cells (days 8–10), and hepatocyte-like cells (both immature and mature, days 11–21). For day-10 cells, double-staining of AFP and CK-7 (middle panel, 40×) showed mutually exclusive expression in the cell population. (<i>B</i>) Reciprocal expression of pluripotent marker Nanog and liver-specific marker AFP during differentiation. RT: reverse transcriptase. (<i>C</i>) Expression of mRNAs of ALB and AAT during differentiation. PHH: primary human hepatocytes; (<i>D</i>) Albumin secretion by differentiated human hepatocyte-like cells (DHHs). Culture media were collected at the indicated time points during differentiation and subjected to albumin detection with an ELISA kit. Error bars represent standard deviation from replicate experiments. (<i>E</i>) Periodic acid-Schiff staining of stem cells (WA09), DHHs, and PHHs.</p

Time course of infection for determination of the transition point at which the differentiating cells became permissive for HCV.

<p>(<i>A</i>) List of growth factors in media used in the various stages of differentiation. (<i>B</i>) Time course of DHH infection. Cells were exposed for 6 h on the indicated days before the inoculum was removed. The cells were then cultured in the appropriate medium for an additional 48 h before the cell lysates were collected for detection of NS3 expression. (<i>C</i>) Secreted luciferase activities were monitored in the same experiments described in (B). Error bars represent standard deviation of triplicate experiments. (<i>D</i>) Hepatic maturation was not required for HCV infection of day-10 cells. Day-10 DHHs were infected and then either kept in medium D (hepatic specification medium) or changed to HGF-containing Medium E (hepatic maturation medium) until day 21, when all cells were collected for western blotting. The anti-NS3 antibody also recognized a nonspecific band in the mock-infected sample. (<i>E</i>) A diagram indicating the time point for transition of DHHs to HCV permissiveness on the basis of results shown in (<i>B</i>) and (<i>C</i>).</p

Metabolism of a Phenylarsenical in Human Hepatic Cells and Identification of a New Arsenic Metabolite

Environmental contamination and human consumption of chickens could result in potential exposure to Roxarsone (3-nitro-4-hydroxyphenylarsonic acid), an organic arsenical that has been used as a chicken feed additive in many countries. However, little is known about the metabolism of Roxarsone in humans. The objective of this research was to investigate the metabolism of Roxarsone in human liver cells and to identify new arsenic metabolites of toxicological significance. Human primary hepatocytes and hepatocellular carcinoma HepG2 cells were treated with 20 or 100 μM Roxarsone. Arsenic species were characterized using a strategy of complementary chromatography and mass spectrometry. The results showed that Roxarsone was metabolized to more than 10 arsenic species in human hepatic cells. A new metabolite was identified as a thiolated Roxarsone. The 24 h IC₅₀ values of thiolated Roxarsone for A549 lung cancer cells and T24 bladder cancer cells were 380 ± 80 and 42 ± 10 μM, respectively, more toxic than Roxarsone, whose 24 h IC₅₀ values for A549 and T24 were 9300 ± 1600 and 6800 ± 740 μM, respectively. The identification and toxicological studies of the new arsenic metabolite are useful for understanding the fate of arsenic species and assessing the potential impact of human exposure to Roxarsone

GCV induced histopathological changes.

<p><b>A.</b> vTK+FVB/N mice that did not receive GCV showed normal liver histology. <b>B–C.</b> vTK+FVB/N mice that received 50 mg/kg GCV showed increased cytoplasmic and nuclear enlargement with increased acidophilic bodies (B, arrowhead), apoptotic bodies (B, arrow), and areas of confluent necrosis (C, arrows) (A–C:Hematoxylin and eosin, original magnification ×200). <b>D.</b> TUNEL immunostaining showing increased apoptotic nuclei (arrows) in livers post GCV (Immunoperoxidase, original magnification ×200).</p

Histology of chimeric SCID/uPA liver at 12 weeks old.

<p>Engrafted human hepatocytes (HH, brown nuclei) in paraffin embedded liver sections were distinguished from mouse hepatocytes (MH, blue nuclei) and other murine constituents (M, blue nuclei) by hybridization with fluoresceinated Alu probe (i, left). Serial sections were analyzed by PAS staining (right) without (ii) or with (iii) prior diastase treatment. Ceroid macrophages (iii, arrowheads). (Original magnification ×50).</p

Impact of vTK/GCV on non transplanted SCID/uPA mouse liver.

<p>Gross Appearance (upper panels) and H&E staining (lower panels) of livers from non-transplanted and age-matched vTK+SCID/uPA mice (3 months old) treated with 0, 25, 50 and 100 mg/kg GCV (i.p. every 48 h for 10 days). RN, regenerative nodules; PL, pale liver.</p

Functional expression of vTK in Huh7 cells.

<p><b>A.</b> Huh7 cells were transfected with pCI-vTK and G418-resistant Huh cellular clones (cl) (1–6) were evaluated for vTK expression (∼43 KDa, arrowheads) by immunoblot analysis. Lmw, low molecular weight markers; 293TvTK, 293T cells transiently transfected with pCI-vTK. <b>B.</b> In vitro cytotoxicity of GCV in vTK expressing cells. HuhvTKcl1 and HuhvTKcl6 cells were incubated with various concentrations of GCV for 72 hours, followed by cell survival quantitation by MTT assay. Data represent the Mean +/− S.D from quadruplicate cell cultures for each GCV dose. Mock, Huh cells that underwent stable selection after transfection with empty pCIneo vector.</p