A targeted, stimuli-responsive, polymeric drug delivery vehicle has been developed to help alleviate the severe side-effects caused by narrow therapeutic window drugs. Doxorubicin, a commonly used chemotherapeutic agent has been conjugated to dextran by two different techniques. In the first method, doxorubicin and hepatocyte-targeting galactosamine were attached to dextran through amine bonds. Conjugation efficiency based on the amount loaded of each reactant varied from 1% to 50% for doxorubicin and from 2% to 20% for galactosamine, depending on various synthesis parameters. For the second conjugate, doxorubicin was attached to dextran through an acid-labile hydrazide bond. Fluorescence quenching indicated that all our conjugates can bind to DNA. The degree of binding was improved with increasing polymer molecular weight and substitution of doxorubicin, and also with hydrazide-bonded conjugate. In cell culture experiments, we have found that the uptake of conjugates was much lower than that of free doxorubicin. Lower uptake of conjugates decreased the toxicity of doxorubicin. Also, the uptake of non-galactosylated conjugate was lower than that of the galactosylated conjugate. Microscopy studies indicated that doxorubicin was localized almost exclusively at the nucleus, whereas the amine-bonded conjugates were present throughout the cell. Targeted amine-linked conjugates and hydrazide-bonded conjugates achieved greatly improved cytotoxicity. Following uptake, the doxorubicin was dissociated from the hydrazide conjugate in an endosomal compartment and diffused to the nucleus. The LC₅₀ values of non-targeted amine-linked, targeted amine-linked, and hydrazide-linked doxorubicin were 19.81 μg/mL, 7.33 μg/mL and 4.39 μg/mL, respectively. The amine-linked conjugates were also tested on a multidrug-resistant cell line; the LC₅₀ values of doxorubicin and the non-targeted amine-linked conjugate were 8.60 μg/mL and 36.02 μg/mL, respectively. / Singapore-MIT Alliance (SMA)

Primary human hepatocytes are a scare resource with limited lifespan and variable function which diminishes with time in culture. As a consequence, their use in tissue modelling and therapy is restricted. Human embryonic stem cells (hESC) could provide a stable source of human tissue due to their self-renewal properties and their ability to give rise to all the cell types of the human body. Therefore, hESC have the potential to provide an unlimited supply of hepatocytes. To date, the use of hESCs-derived somatic cells is limited due to the undefined, variable and xeno-containing microenvironment that influences the cell performance and life span, limiting scale-up and downstream application. Therefore, the development of highly defined cell based systems is required if the true potential of stem cell derived hepatocytes is to be realised. In order to replace the use of animal derived culture substrates to differentiate and maintain hESCs-derived hepatocytes, an interdisciplinary approach was employed to define synthetic materials, which maintain hepatocyte-like cell phenotype in culture. A simple polyurethane, PU134, was identified which improved hepatocyte performance and stability when compared to biological matrices. Moreover, the synthetic polymer was amenable to scale up and demonstrated batch-to-batch consistency. I subsequently used the synthetic polymer surface to probe the underlying biology, identifying key modulators of hepatocyte-like cell phenotype. This resulted in the identification of a novel genetic signature, MMP13, CTNND2 and THBS2, which was associated with stable hepatocyte performance. Importantly, those findings could be translated to two hESC lines derived at GMP. In conclusion, hepatocyte differentiation of pluripotent stem cells requires a defined microenvironment. The novel gene signature identified in this study represents an example of how to deliver stable hESCs-derived hepatocytes.

Brominated flame retardants (BFR) such as hexachlorocyclopentadienyl-dibromocyclooctane (HCDBCO), bis(2-ethylhexyl)tetrabromophthalate (BEHTBP), 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE) and decabromodiphenylethane (DBDPE) are contaminants of environmental concern. These BFRs are replacement alternatives for some of the major production BFRs, which have been restricted from the marketplace due to their adverse health effects. Their presence in environmental matrices, including wild birds, suggests they should be tested for possible toxic effects. BFR alternatives have been detected in the eggs of colonial fish-eating birds, suggesting maternal transfer during ovogenesis and the potential for these chemicals to bioaccumulate through the food chain. However, information regarding the toxicity of HCDBCO, BEHTBP, BTBPE and DBDPE exposure in birds is lacking. This thesis consisted of a combined in vitro/in ovo approach to determine: 1) the concentration-dependent effects of these four BFR alternatives in chicken embryonic hepatocytes (CEH), and 2) the dose-dependent effects of HCDBCO and BTBPE in chicken embryos following injection into the air cell of eggs prior to incubation. Changes in the mRNA expression levels of genes previously found to be responsive to other BFRs were assessed in CEH and liver tissue, in addition to examining overt toxicity (i.e. cytotoxicity, pipping success). None of the BFRs tested were cytotoxic up to 60 µM HCDBCO, 60 µM BEHTBP, 1.4 µM BTBPE or 0.2 µM DBDPE in CEH. Injection doses up to 50 µg/g egg HCDBCO and 10 µg/g egg BTBPE had no effect on embryonic pipping success. The accumulation of HCDBCO and BTBPE was variable in liver and did not follow a linear uptake pattern with respect to injection dose, due in part to difficulties with the solubility of these chemicals in the dimethyl sulfoxide (DMSO) vehicle. In, CEH, HCDBCO caused a decrease in CYP1A4/5 mRNA at all concentrations tested, while CYP2H1 and CYP3A37 were induced only at 10 µM. In contrast, only TTR mRNA was down-regulated in hepatic tissue at all injection concentrations of HCDBCO. The highest concentration of BTBPE induced CYP1A4/5 mRNA to 115- and 18-fold in CEH, and 6.5- and 1.8-fold in liver tissue. In vitro and in ovo exposure to BTBPE caused a concentration-dependent decrease in DIO3 mRNA, while CYP3A37 was down-regulated 2-fold at 10 µg/g in liver tissue. In CEH, DBDPE induced CYP1A4/5 mRNA to a maximum of 29- and 59-fold at 0.2 µM, and increases in DIO1 mRNA and decreases in CYP3A37 mRNA were also observed. None of the gene targets were responsive to BEHTBP exposure in CEH. This is the first study to report on the toxicological and molecular effects of HCDBCO, BEHTBP, BTBPE and DBDPE in an avian species. Using this combined in vitro/in ovo approach has permitted the characterization of these four BFR alternatives by defining possible mechanisms of biological action in a model avian species, the chicken.

Mouse hepatocytes respond to osmotic stress with adaptive changes in transmembrane potential, Vm, such that hypotonic stress hyperpolarizes cells and hypertonic stress depolarizes them. These changes in Vm provide electromotive force for redistribution of ions such as CI−, and this comprises part of the mechanism of hepatocyte volume regulation. We conducted the present study to determine whether ethanol administered in vitro to mouse liver slices increases hepatocyte water volume, and whether this swelling triggers adaptive changes in the Vm. Cells in mouse liver slices were loaded with tetramethylammonium ion (TMA). Changes in hepatocyte water volume were computed from measurements with Ion sensitive micro‐electrodes of changes in intracellular activity of TMA (a1TMA) that resulted from water fluxes. Ethanol (70 mM) increased hepatocyte water volume Immediately, and this peaked at 17% by 7 to 8 min, by which time a plateau was reached. Liver slices also were obtained from mice treated 12 hr prior with 4‐methylpyrazole (4 mM). The effect of ethanol on their hepatocyte water volume was identical to that from untreated mice, except that the onset and peak were delayed 2 min. Hepatocyte Vm showed no differences between control or ethanol‐treated cells during the course of volume changes. In contrast, hyposmotic stress, created by dropping external osmolality 50 mosm, increased Vm from –30 mV to –46 mV. Ethanol did not inhibit this osmotic stress‐induced hyperpolarization, except partially at high concentrations of 257 mM or greater. We infer that ethanol‐induced swelling of hepatocytes differs from that resulting from hyposmotic stress. Cellular events associated with increased activity of intracellular water most likely trigger the hyperpolarization of Vm that accompanies the latter. We conclude, therefore, that ethanol‐induced swelling occurs without change in cell water activity. This may result from the retention of macromolecules by ethanol in cells that constitutively secrete protein.

HCV is remarkable at disrupting human immunity to establish chronic infection. The accumulation of Treg cells at the site of infection and upregulation of inhibitory signaling pathways (such as T-cell Ig and mucin domain protein-3 (Tim-3) and galectin-9 (Gal-9)) play pivotal roles in suppressing antiviral effector T (Teff) cells that are essential for viral clearance. While Tim-3/Gal-9 interactions have been shown to negatively regulate Teff cells, their role in regulating Treg cells is poorly understood. To explore how Tim-3/Gal-9 interactions regulate HCV-mediated Treg-cell development, here we provide pilot data showing that HCV-infected human hepatocytes express higher levels of Gal-9 and TGF-β, and upregulate Tim-3 expression and regulatory cytokines TGF-β/IL-10 in co-cultured human CD4+ T cells, driving conventional CD4+ T cells into CD25+Foxp3+ Treg cells. Additionally, recombinant Gal-9 protein can transform TCR-activated CD4+ T cells into Foxp3+ Treg cells in a dose-dependent manner. Importantly, blocking Tim-3/Gal-9 ligations abrogates HCV-mediated Treg-cell induction by HCV-infected hepatocytes, suggesting that Tim-3/Gal-9 interactions may regulate human Foxp3+ Treg-cell development and function during HCV infection.

HCV is remarkable at disrupting human immunity to establish chronic infection. The accumulation of Treg cells at the site of infection and upregulation of inhibitory signaling pathways (such as T-cell Ig and mucin domain protein-3 (Tim-3) and galectin-9 (Gal-9)) play pivotal roles in suppressing antiviral effector T (Teff) cells that are essential for viral clearance. While Tim-3/Gal-9 interactions have been shown to negatively regulate Teff cells, their role in regulating Treg cells is poorly understood. To explore how Tim-3/Gal-9 interactions regulate HCV-mediated Treg-cell development, here we provide pilot data showing that HCV-infected human hepatocytes express higher levels of Gal-9 and TGF-β, and upregulate Tim-3 expression and regulatory cytokines TGF-β/IL-10 in co-cultured human CD4+ T cells, driving conventional CD4+ T cells into CD25+Foxp3+ Treg cells. Additionally, recombinant Gal-9 protein can transform TCR-activated CD4+ T cells into Foxp3+ Treg cells in a dose-dependent manner. Importantly, blocking Tim-3/Gal-9 ligations abrogates HCV-mediated Treg-cell induction by HCV-infected hepatocytes, suggesting that Tim-3/Gal-9 interactions may regulate human Foxp3+ Treg-cell development and function during HCV infection.

Permanent repair of the F9 gene is a significant goal to cure Hemophilia B disease. Advanced gene therapy using CRISPR/Cas9 system can increase circulation level of Factor IX proteins to a significant level without the need of demanding infusions of FIX concentrates. Induced pluripotent stem cells represent an ideal cell for gene therapy because patient-derived cells could be reprogrammed into iPSCs, genetically modified, selected, expanded and then induced to differentiate into fully functional hepatocytes in vitro. This study covered a portion of a 5-year project which ultimately aims at establishing therapeutic results in transgenic Hemophilia B mice by injecting genetically corrected iPSC-derived hepatocytes into the liver. The purpose of this thesis is to summarize what has been completed up to now: generation of the proper model of Hemophilia B human iPSCs using CRISPR/Cas9-mediated genome editing and differentiation of healthy and disease specific iPSCs into hepatocytes which will allow disease modelling to look for cell function, viability, homogeneity and drug screening. Further research will be done to effectively knock-in the F9 allele into liver safe harbor site of disease specific iPSCs, which will express FIX at a significant level to show therapeutic effects.

Indiana University-Purdue University Indianapolis (IUPUI) / Liver fibrosis is a common consequence of various chronic liver diseases. Although transforming growth factor β 1 (TGFβ1) expression is known to be associated with liver fibrosis, the reduced clinical efficacy of TGFβ1 inhibition or the inefficiency to completely prevent liver fibrosis in mice with liver-specific knockout of TGF receptor II suggests that other factors can mediate liver fibrogenesis. As a TGFβ superfamily ligand, activin A signaling modulates liver injury by prohibiting hepatocyte proliferation, mediating hepatocyte apoptosis, promoting Kupffer cell activation, and inducing hepatic stellate cell (HSC) activation in vitro. However, the mechanism of action and in vivo functional significance of activin A in liver fibrosis models remain uncertain. Moreover, whether activin B, another ligand structurally related to activin A, is involved in liver fibrogenesis is not yet known. This study aimed to investigate the role of activin A and B in liver fibrosis initiation and progression. The levels of hepatic and circulating activin B and A were analyzed in patients with various chronic liver diseases, including end-stage liver diseases (ESLD), non-alcoholic steatohepatitis (NASH), and alcoholic liver disease (ALD). In addition, their levels were measured in mouse carbon tetrachloride (CCl4), bile duct ligation (BDL), and ALD liver injury models. Mouse primary hepatocytes, RAW264.7 cells, and LX-2 cells were used as in vitro models of hepatocytes, macrophages, and HSCs, respectively. The specificity and potency of anti-activin B monoclonal antibody (mAb) and anti-activin A mAb were evaluated using Smad2/3 luciferase assay. Activin A, activin B, or their combination were immunologically inactivated by the neutralizing mAbs in mice with progressive or established liver fibrosis induced by CCl4 or with developing cholestatic liver fibrosis induced by BDL surgery. In patients with ESLD, NASH, and ALD, increases in hepatic and circulating activin B, but not activin A, were associated with liver fibrosis, irrespective of etiology. In mice with CCl4-, BDL-, or alcohol-induced liver injury, activin B was persistently elevated in the liver and circulation, whereas activin A showed only transient increases. Activin B was expressed and secreted mainly by the hepatocytes and other cells, including cholangiocytes, activated HSCs, and immune cells. Exogenous administration of activin B promoted hepatocyte injury, activated macrophages to release cytokines, and induced a pro-fibrotic expression profile and septa formation in HSCs. Co-treatment of activin A and B interdependently activated the chemokine (C-X-C motif) ligand 1 (CXCL1)/inducible nitric oxide synthase (iNOS) pathway in macrophages and additively upregulated connective tissue growth factor expression in HSCs. Activin B and A had redundant, unique, and interactive effects on the transcripts related to HSC activation. The neutralization of activin B attenuated the development of liver fibrosis and improved liver function in mice with CCl4- or BDL-induced liver fibrosis and largely reversed the already established liver fibrosis in the CCl4 mouse model. These effects were improved by the administration of additional anti-activin A antibody. Combination of both antibodies also inhibited hepatic and circulating inflammatory cytokine production in the BDL mouse model. In conclusion, activin B is a potential circulating biomarker and potent promotor of liver fibrosis. Its levels in the liver and circulation increase significantly in both acute and chronic states of liver injury. Activin B might additively or interdependently cooperate with activin A, which directly acts on multiple liver cell populations during liver injury and fibrosis, as the combination of both proteins increases pro-inflammatory and pro-fibrotic responses in vitro. In addition, the neutralization of both activin A and activin B in vivo enhances the preventive and reversible effects of liver injury and fibrosis compared to that when activin B alone is neutralized. Our data reveal a novel target of liver fibrosis and the mechanism of activin B-mediated initiation of this process by damaging hepatocytes and activating macrophages and HSCs. Our findings show that activin B promotes hepatic fibrogenesis, and that targeting of activin B has anti-inflammatory and anti-fibrotic effects, which ameliorate liver injury by preventing or regressing liver fibrosis. Antagonizing either activin B alone or in combination with activin A prevents and regresses liver fibrosis in multiple animal studies, paving way for future clinical studies.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17896号 / 農博第2019号 / 新制||農||1017(附属図書館) / 学位論文||H25||N4792(農学部図書室) / 30716 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 菅原 達也, 教授 左子 芳彦, 教授 澤山 茂樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

<p> Gene-targeted studies in mice have shown that the murine scavenger receptor class B type I (mSR-BI) is atheroprotective and plays a key role in the clearance of high density lipoprotein (HDL) cholesterol by the liver. We focused on the analysis of human SR-BI (hSR-BI) and the role of its C-terminal cytoplasmic tail on its localization, lipid uptake activity, and regulation in hepatocytes both in vitro and in vivo. Full length hSRBI and hSR-BI lacking its C-terminal cytoplasmic tail (hSR-BI-DM) localized to vesiclelike structures in the cytoplasm, to juxtanuclear regions and to the cell surface in HepG2 cells. Similar cytoplasmic punctate distribution was observed in transfected human and mouse aortic endothelial cells. </p> <p> In HepG2 cells both hSR-BI and hSR-BI-DM mediated HDL-lipid uptake; however, the truncation mutant displayed only half ofthe activity, suggesting that removal ofthe C-terminal cytoplasmic tail reduced but did not eliminate SR-BI's activity. In HepG2 cells treated with the PKC inhibitor, calphostin C, hSR-BI or hSR-BI-DM mediated HDL-lipid uptake was decreased by 40 and 50%, respectively, indicating that this activity is regulated by PKC. </p> <p> In order to determine the effects of hSR-BI and hSR-BI-DM in vivo, we set out to generate transgenic mice with hepatic overexpression ofeach protein using a bipartite expression system requiring driver and responder transgenes. Mice expressing the responder transgenes, PTREhSR-BI and PTREhSR-BI-DM, as well as a reporter transgene (PTRdacZ), driven by the same bi-directional promoter, were generated and mated to mice with a liver-specific driver trans gene, PMuptTA. The mice were analyzed and showed the presence of a reporter protein, ~-galactosidase, in their livers, but not in other tissues tested. Total and HDL cholesterol levels were not altered in PMuPtTA I PrREhSRBI or PMuptTA I PrREhSR-BI-DM transgenic mice. Further characterization ofthe double transgenic mice revealed that hSR-BI m.RNA transcripts were detected in the livers of PMuPtTA I PrREhSR-BI mice, but not in those ofPMuPtTA I PrREhSR-BI-DM mice. However, neither PMuptTA I PrREhSR-BI nor PMuptTA I PrREhSR-BI-DM mice showed increased expression of SR-BI in their livers. </p> / Thesis / Master of Science (MSc)