Periportal Hepatocytes Research Articles

Challenges of experimental gene therapy for urea cycle disorders

Urea cycle disorders (UCDs) are inborn errors of liver metabolism that often result in hyperammonemia and failure of arginine synthesis due to the deficiencies of one of the six enzymes or two mitochondrial transporters involved. Despite current treatment options, including dietary therapy, stimulation of alternative routes of nitrogen disposal and cell therapies (hepatocyte and orthotopic liver transplantation), significant morbidity and mortality still remain. Gene therapy has emerged as an attractive alternative strategy for providing a definitive cure for patients with metabolic diseases. Successful phenotype correction in pre- clinical animal models is encouraging and research effort is increasingly being focused on translation from the laboratory bench to proof-of-concept human therapy. Gene therapy for UCDs must target the periportal hepatocytes, as these are the only liver cells that express all six enzymes required for full ammonia detoxification. This review explores current efforts to develop and translate liver-directed gene therapy approaches to UCDs caused by each of the defects of the six enzymes. The prominent issues that will need to be addressed and overcome for the future development of clinical trials, including alternatives to mouse mutants and to vectors that are not necessarily conditioned to rodents are also discussed.

Regenerating the liver: not so simple after all?

Under normal homeostatic conditions, hepatocyte renewal is a slow process and complete turnover likely takes at least a year. Studies of hepatocyte regeneration after a two-thirds partial hepatectomy (2/3 PH) have strongly suggested that periportal hepatocytes are the driving force behind regenerative re-population, but recent murine studies have brought greater complexity to the issue. Although periportal hepatocytes are still considered pre-eminent in the response to 2/3 PH, new studies suggest that normal homeostatic renewal is driven by pericentral hepatocytes under the control of Wnts, while pericentral injury provokes the clonal expansion of a subpopulation of periportal hepatocytes expressing low levels of biliary duct genes such as Sox9 and osteopontin. Furthermore, some clarity has been given to the debate on the ability of biliary-derived hepatic progenitor cells to generate physiologically meaningful numbers of hepatocytes in injury models, demonstrating that under appropriate circumstances these cells can re-populate the whole liver.

Increased MMP-7 expression in biliary epithelium and serum underpins native liver fibrosis after successful portoenterostomy in biliary atresia.

The molecular mechanisms underlying progressive liver fibrosis following surgical treatment of biliary atresia (BA) remain unclear. Our aim was to address hepatic gene and protein expression and serum levels of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) after successful portoenterostomy (PE), and relate them to histological signs of liver injury, clinical follow‐up data and biochemical markers of hepatic function. LIver biopsies and serum samples were obtained from 25 children after successful PE at median age of 3.3 years. Serum MMP concentrations were determined by enzyme‐linked immune sorbent assay. Hepatic gene expression of MMPs and TIMPs was analyzed using real‐time reverse‐transcription PCR. Liver expression of MMP‐7 and cytokeratin‐7 was studied using immunohistochemistry. Despite effective clearance of biochemical and histological cholestasis following PE, BA patients showed increased hepatic gene expression of MMP‐7 (29‐fold, p < 0.001), MMP‐2 (3.1‐fold, p < 0.001), MMP‐14 (1.7‐fold, p = 0.007), and TIMP‐1 (1.8‐fold, p < 0.001), when compared to controls. Similar to a biliary epithelial marker cytokeratin‐7, expression of MMP‐7 localized in biliary epithelium of bile ducts and ductal proliferations and periportal hepatocytes and was increased (p < 0.001) in relation to controls. BA patients had 6‐fold higher serum levels of MMP‐7 (p < 0.001), which correlated positively with hepatic MMP‐7 gene (r = 0.548, p = 0.007) and protein (r = 0.532, p = 0.007) expression. Patients showed a positive correlation between biliary MMP‐7 expression and Metavir fibrosis stage (r = 0.605, p = 0.001) and portal fibrosis grade (r = 0.606, p = 0.001). Neither similarly increased MMP‐7 expression nor correlation with liver fibrosis was observed in patients with intestinal failure‐associated liver disease and comparable Metavir stage. In conclusion, our findings support an unique role of altered hepatic expression of MMP‐7 in the progression of liver fibrosis after successful PE and introduce a potential therapeutic target to pharmacologically extend native liver survival by inhibiting MMP‐7 hyperactivity. Serum MMP‐7 may be a valuable postoperative prognostic tool in BA.

169. Expression Studies of Ornithine Transcarbamylase in Liver Using Minicircle Vectors for Gene Therapy: Flag-Tagging of a Mitochondrial Enzyme and Stable Expression Under Control of an Endogenous Otc Promoter-Enhancer

Ornithine transcarbamylase deficiency (OTCD) is the most common inherited defect of the urea cycle resulting in severe hyperammonemia and death if left untreated. We are aiming at correcting OTCD using naked-DNA minicircle (MC) vector mediated gene therapy in the spfash mouse model with low residual OTC activity. A critical parameter is delivery to periportal hepatocytes where the urea cycle is located because of metabolic zonation in the liver. To distinguish between MC-born and endogenous OTC enzyme, we generated an expression cassette with an internally Flag-tagged OTC enzyme. Internal epitope tagging is required because of the N-terminal mitochondrial import sequence. A corresponding Flag-tag sequence was introduced C-terminally of the mitochondrial import signal. The tagged protein performed similar to its non-tagged OTC enzyme upon hydrodynamic tail vein injection for liver targeting in spfash mice, indicating that the tag neither interferes with the mitochondrial import nor the formation of a functional OTC trimer. Furthermore we generated an endogenous Otc promoter-enhancer construct, termed PmO1, for potential specific or “natural” OTC transgene expression. According to others, the promoter (672 bp) sequence derived from mouse Otc was not sufficient for liver specificity (Veres et al, J Biol Chem 261: 7588-7591, 1986) and requires a corresponding enhancer sequence (Nishiyori et al, J Biol Chem 269: 1323-1331, 1994). An enhancer sequence (232 bp) originating from rat Otc was used that contains several binding sites for liver-selective transcription factors (Murakami et al, Mol Cell Biol 10: 1180-1191, 1990). The resulting MC-vector expressing OTC from this PmO1 promoter-enhancer construct was delivered to spfash mice via hydrodynamic tail vein injection, resulting in similar enzymatic activity in whole liver extracts compared to wild-type mice. Vectors designed to express Flag-tagged OTC driven from a natural Otc promoter might be useful to study biodistribution and/or long-term stable expression after MC-based gene therapy for OTCD.

PGC-1α Promotes Ureagenesis in Mouse Periportal Hepatocytes through SIRT3 and SIRT5 in Response to Glucagon.

Excess ammonia is produced during fasting when amino acids are used for glucogenesis. Together with ureagenesis, glucogenesis occurs in periportal hepatocytes mediated mainly through the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). In vivo experiments showed that fasting strongly stimulated mice glucagon secretion, hepatic PGC-1α, sirtuin 3 (SIRT3) and sirtuin 5 (SIRT5) expression and ureagenesis enzymatic activity such as carbamoyl phosphate synthetase 1 (CPS1) and ornithine transcarbamoylase (OTC). Interestingly, 15N-labeled urea and 13C-labeled glucose production in wild-type mice were significantly increased compared with PGC-1α null mice by [15N,13C]alanine perfused liver. Glucagon significantly stimulated ureagenesis, expression of SIRT3, SIRT5 and the activities of CPS1 and OCT but did not stimulate PGC-1α silencing hepatocytes in mice periportal hepatocytes. Contrarily, PGC-1α overexpression significantly increased the expression of SIRT3, SIRT5 and the activities of CPS1 and OTC, but induced no significant changes in CPS1 and OTC expression. Morever, SIRT3 directly deacetylates and upregulates the activity of OTC, while SIRT5 deacetylates and stimulates the activity of CPS1. During fasting, PGC-1α facilitates ureagenesis in mouse periportal hepatocytes by deacetylating CPS1 and OTC modulated by mitochondrial deacetylase, SIRT3 and SIRT5. This mechanism may be relevant to ammonia detoxification and metabolic homeostasis in liver during fasting.

Role of the Orphan Receptors RORα and REV‐Erb α/β in Liver Zonal Gene Regulation

Numerous hepatic metabolic pathways, such as glycolysis and gluconeogenesis, cannot occur in the same cell. To overcome this, opposing pathways are compartmentalized within hepatocytes located within different regions of the liver, a phenomenon called metabolic zonation or zonal regulation. The expression of hepatic genes in different zonal regions is orchestrated by multiple transcription factors. Studies by Benhamouche et al. (Dev. Cell, 10:759) revealed that activation of the β‐catenin signaling pathway, through binding of β‐catenin to TCF4, is required for expression of genes that are active solely in hepatocytes surrounding the central vein (pericentral region). In hepatocytes around the portal triad (periportal region), APC inhibits β‐catenin and prevents expression of pericentral genes. A genome wide study of chromatin occupancy of β‐catenin, TCF4, and the liver transcription factor HNF4α revealed that TCF4 is an opportunistic transcription factor that shifts between interacting with HNF4α in periportal hepatocytes and β‐catenin in pericentral hepatocytes.Previous transgenic studies in our lab demonstrated that enhancer E3 of the activity of mouse alpha‐fetoprotein (AFP) was active in the adult liver, even though AFP expression is silenced at birth, Interestingly, E3 activity is highly restricted to a single layer of hepatocytes surrounding the central vein, thus providing a system to investigate the molecular basis of pericentral gene expression (Peyton et al, PNAS, 97:10890). Additional studies indicate that E3 activity in pericentral hepatocytes requires β‐catenin signaling, since E3 is no longer active in b‐catenin deficient hepatocytes. Additional in vitro studies indicate that a highly conserved TCF4/β‐catenin binding site at the 3′ end of E3 is required for responsiveness to b‐catenin (Clinkenbeard et al, Hepatology, 56:1892). We have also identified a site at the 5′ end of E3 that binds the related orphan nuclear receptors RORα, Rev‐Erbα, and Rev‐Erbβ. Additional data in cultured cells and mice suggest that RORα is a positive regulator of E3, whereas Rev‐Erbα/β repress E3 activity.Based on these data, we propose that differential binding of RORa and Rev‐Erbα/β contribute to zonal E3 activity in the adult liver. We proposed that RORa binding is required for E3 activity in pericentral hepatocytes. In contrast, the binding of Rev‐Erbα/β represses E3 activity in periportal regions. To test this, we are using ChIP to evaluate RORα and Rev‐Erbα/β binding to E3 in enriched pericentral and periportal hepatocytes. We will also knock down Rev‐Erbα/β levels in the adult liver and test whether this results in increased expression of E3 transgenes (and other pericentral genes) in periportal regions. In conclusion, we present data that indicates that RORα and Rev‐Erbα/β regulate the zonal expression of AFP through binding to E3.

Hepatic Macrosteatosis Is Partially Converted to Microsteatosis by Melatonin Supplementation in ob/ob Mice Non-Alcoholic Fatty Liver Disease.

BackgroundObesity is a common risk factor for non-alcoholic fatty liver disease (NAFLD). Currently, there are no specific treatments against NAFLD. Thus, examining any molecule with potential benefits against this condition emerged melatonin as a molecule that influences metabolic dysfunctions. The aim of this study was to determine whether melatonin would function against NAFDL, studying morphological, ultrastuctural and metabolic markers that characterize the liver of ob/ob mice.MethodsLean and ob/ob mice were supplemented with melatonin in the drinking water for 8 weeks. Histology and stereology were performed to assess hepatic steatosis and glycogen deposition. Ultrastructural features of mitochondria, endoplasmic reticulum (ER) and their juxtapositions were evaluated in livers of all experimental groups. Furthermore, hepatic distribution and expression of markers of ER and mitochondria (calnexin, ATP sintase β, GRP78 and CHOP) and metabolic dysfunction (RPB4, β-catenin) and cellular longevity (SIRT1) were analyzed.ResultsMelatonin significantly reduced glycemia, identified also by a decrease of hepatic RBP4 expression, reversed macrosteatosis in microsteatosis at the hepatic pericentral zone, enlarged ER-mitochondrial distance and ameliorated the morphology and organization of these organelles in ob/ob mouse liver. Furthermore, in ob/ob mice, calnexin and ATP synthase β were partially restored, GRP78 and CHOP decreased in periportal and midzonal hepatocytes and β-catenin expression was, in part, restored in peripheral membranes of hepatocytes. Melatonin supplementation to ob/ob mice improves hepatic morphological, ultrastructural and metabolic damage that occurs as a result of NAFLD.ConclusionsMelatonin may be a potential adjuvant treatment to limit NAFLD and its progression into irreversible complications.

Impact of SLC6A Transporters in Physiological Taurine Transport at the Blood-Retinal Barrier and in the Liver.

Cumulative studies showed that taurine (2-aminoethanesulfonic acid) contributes to a variety of physiological events. Transport study suggested the cellular taurine transport in an Na+- and Cl--dependent manner, and the several members of SLC6A family have been shown as taurine transporter. At the inner blood-retinal barrier (BRB), taurine transporter (TauT/SLC6A) is involved in the transport of taurine to the retina from the circulating blood. The involvement of TauT is also suggested in γ-aminobutyric acid (GABA) transport at the inner BRB, and its role is assumed in the elimination of GABA from the retinal interstitial fluid. In the retina, taurine is thought to be a major organic osmolyte, and its influx and efflux through TauT and volume-sensitive organic osmolyte and anion channel (VSOAC) in Müller cells regulate the osmolarity in the retinal microenvironment to maintain a healthy retina. In the liver, hepatocytes take up taurine via GABA transporter 2 (GAT2/SLC6A13, the orthologue of mouse GAT3) expressed at the sinusoidal membrane of periportal hepatocytes, contributing to the metabolism of bile acid. Site-directed mutagenesis study suggests amino acid residues that are crucial in the recognition of substrates by GATs and TauT. The evidence suggests the physiological impact of taurine transporters in tissues.

Role of β-catenin in development of bile ducts

Beta-catenin is known to play stage- and cell-specific functions during liver development. However, its role in development of bile ducts has not yet been addressed. Here we used stage-specific in vivo gain- and loss-of-function approaches, as well as lineage tracing experiments in the mouse, to first demonstrate that β-catenin is dispensable for differentiation of liver precursor cells (hepatoblasts) to cholangiocyte precursors. Second, when β-catenin was depleted in the latter, maturation of cholangiocytes, bile duct morphogenesis and differentiation of periportal hepatocytes from cholangiocyte precursors was normal. In contrast, stabilization of β-catenin in cholangiocyte precursors perturbed duct development and cholangiocyte differentiation. We conclude that β-catenin is dispensable for biliary development but that its activity must be kept within tight limits. Our work is expected to significantly impact on in vitro differentiation of stem cells to cholangiocytes for toxicology studies and disease modeling.

Immunohistochemical analyses of cell cycle progression and gene expression of biliary epithelial cells during liver regeneration after partial hepatectomy of the mouse.

The liver has a remarkable regeneration capacity, and, after surgical removal of its mass, the remaining tissue undergoes rapid regeneration through compensatory growth of its constituent cells. Although hepatocytes synchronously proliferate under the control of various signaling molecules from neighboring cells, there have been few detailed analyses on how biliary cells regenerate for their cell population after liver resection. The present study was undertaken to clarify how biliary cells regenerate after partial hepatectomy of mice through extensive analyses of their cell cycle progression and gene expression using immunohistochemical and RT-PCR techniques. When expression of PCNA, Ki67 antigen, topoisomerase IIα and phosphorylated histone H3, which are cell cycle markers, was immunohistochemically examined during liver regeneration, hepatocytes had a peak of the S phase and M phase at 48–72 h after resection. By contrast, biliary epithelial cells had much lower proliferative activity than that of hepatocytes, and their peak of the S phase was delayed. Mitotic figures were rarely detectable in biliary cells. RT-PCR analyses of gene expression of biliary markers such as Spp1 (osteopontin), Epcam and Hnf1b demonstrated that they were upregulated during liver regeneration. Periportal hepatocytes expressed some of biliary markers, including Spp1 mRNA and protein. Some periportal hepatocytes had downregulated expression of HNF4α and HNF1α. Gene expression of Notch signaling molecules responsible for cell fate decision of hepatoblasts to biliary cells during development was upregulated during liver regeneration. Notch signaling may be involved in biliary regeneration.

Diverse routes to liver regeneration.

The liver's ability to regenerate is indisputable; for example, after a two-thirds partial hepatectomy in rats all residual hepatocytes can divide, questioning the need for a specific stem cell population. On the other hand, there is a potential stem cell compartment in the canals of Hering, giving rise to ductular reactions composed of hepatic progenitor cells (HPCs) when the liver's ability to regenerate is hindered by replicative senescence, but the functional relevance of this response has been questioned. Several papers have now clarified regenerative mechanisms operative in the mouse liver, suggesting that the liver is possibly unrivalled in its versatility to replace lost tissue. Under homeostatic conditions a perivenous population of clonogenic hepatocytes operates, whereas during chronic damage a minor population of periportal clonogenic hepatocytes come to the fore, while the ability of HPCs to completely replace the liver parenchyma has now been shown.

Contribution of Mature Hepatocytes to Biliary Regeneration in Rats with Acute and Chronic Biliary Injury.

Whether hepatocytes can convert into biliary epithelial cells (BECs) during biliary injury is much debated. To test this concept, we traced the fate of genetically labeled [dipeptidyl peptidase IV (DPPIV)-positive] hepatocytes in hepatocyte transplantation model following acute hepato-biliary injury induced by 4,4’-methylene-dianiline (DAPM) and D-galactosamine (DAPM+D-gal) and in DPPIV-chimeric liver model subjected to acute (DAPM+D-gal) or chronic biliary injury caused by DAPM and bile duct ligation (DAPM+BDL). In both models before biliary injury, BECs are uniformly DPPIV-deficient and proliferation of DPPIV-deficient hepatocytes is restricted by retrorsine. We found that mature hepatocytes underwent a stepwise conversion into BECs after biliary injury. In the hepatocyte transplantation model, DPPIV-positive hepatocytes entrapped periportally proliferated, and formed two-layered plates along portal veins. Within the two-layered plates, the hepatocytes gradually lost their hepatocytic identity, proceeded through an intermediate state, acquired a biliary phenotype, and subsequently formed bile ducts along the hilum-to-periphery axis. In DPPIV-chimeric liver model, periportal hepatocytes expressing hepatocyte nuclear factor-1β (HNF-1β) were exclusively DPPIV-positive and were in continuity to DPPIV-positives bile ducts. Inhibition of hepatocyte proliferation by additional doses of retrorsine in DPPIV-chimeric livers prevented the appearance of DPPIV-positive BECs after biliary injury. Moreover, enriched DPPIV-positive BEC/hepatic oval cell transplantation produced DPPIV-positive BECs or bile ducts in unexpectedly low frequency and in mid-lobular regions. These results together suggest that mature hepatocytes but not contaminating BECs/hepatic oval cells are the sources of periportal DPPIV-positive BECs. We conclude that mature hepatocytes contribute to biliary regeneration in the environment of acute and chronic biliary injury through a ductal plate configuration without the need of exogenously genetic or epigenetic manipulation.

Assessment of the hepatic and renal effects of sub-chronic administration of sub-lethal doses of amitraz/xylene in albino Wistar rats

The toxicological effects of increasing doses of amitraz on the liver and kidney of animals treated with it need to be assessed as a recent trend in development of resistance by pests may lead to increasing the quantity of amitraz required to control them. Forty adult male rats 170 ± 10 g assigned using simple random sampling method into four groups were used for the study. Groups A, B, and C were treated daily with 10.0, 2.0 and 0.4 mg/kg body weight, bw of amitraz respectively while group D (control group) was treated with 10 ml/kg bw water. The treatment was done daily using the oral route for 84 days. Parameters assessed were alanine aminotransferase (ALT), alkaline phosphatase (AP), total protein (TP), albumin (ALB), total bilirubin (TB), blood urea nitrogen (BUN), creatinine (CR), and histology of the liver and kidney. The mean TB, AP, and ALT of rats in group A was higher (p < 0.05) than that of groups B, C, and D on day 56 of the experiment. The mean TP and ALB of rats in groups C and D were higher (p < 0.05) than that of group A between day 28 and the end of the experiment. Sections of the liver from rats in group A showed moderate vacuolar degeneration of the periportal hepatocytes, while sections of the kidney obtained from rats in group A showed a widespread, mild to moderate tubular degeneration which involved the pars recta, proximal convoluted tubules and collecting ducts. The kidney and liver sections obtained from rats in group B, C, and D did not reveal any visible lesions.

Mouse white adipose tissue-derived mesenchymal stem cells gain pericentral and periportal hepatocyte features after differentiation invitro, which are preserved invivo after hepatic transplantation.

Mesenchymal stem cells may differentiate into hepatocyte-like cells invitro and invivo. Therefore, they are considered a novel cell resource for the treatment of various liver diseases. Here, the aim was to demonstrate that mesenchymal stem cells may adopt both perivenous and periportal hepatocyte-specific functions invitro and invivo. Adipose tissue-derived mesenchymal stem cells were isolated from immunodeficient C57BL/6 (B6.129S6-Rag2(tm1Fwa) Prf1(tm1Clrk) ) mice and differentiated into the hepatocytic phenotype by applying a simple protocol. Their physiological and metabolic functions were analysed invitro and after hepatic transplantation invivo. Mesenchymal stem cells changed their morphology from a fibroblastoid into shapes of osteocytes, chondrocytes, adipocytes and hepatocytes. Typical for mesenchymal stem cells, hematopoietic marker genes were not expressed. CD90, which is not expressed on mature hepatocytes, decreased significantly after hepatocytic differentiation. Markers indicative for liver development like hepatic nuclear factor 4 alpha, or for perivenous hepatocyte specification like cytochrome P450 subtype 3a11, and CD26 were significantly elevated. Periportal hepatocyte-specific markers like carbamoylphosphate synthetase 1, the entry enzyme of the urea cycle, were up-regulated. Consequently, cytochrome P450 enzyme activity and urea synthesis increased significantly to values comparable to cultured primary hepatocytes. Both perivenous and periportal qualities were preserved after hepatic transplantation and integration into the host parenchyma. Adult mesenchymal stem cells from adipose tissue differentiated into hepatocyte-like cells featuring both periportal and perivenous functions. Hence, they are promising candidates for the treatment of region-specific liver cell damage and may support organ regeneration in acute and chronic liver diseases.

Hybrid Periportal Hepatocytes Regenerate the Injured Liver without Giving Rise to Cancer

Compensatory proliferation triggered by hepatocyte loss is required for liver regeneration and maintenance but also promotes development of hepatocellular carcinoma (HCC). Despite extensive investigation, the cells responsible for hepatocyte restoration or HCC development remain poorly characterized. We used genetic lineage tracing to identify cells responsible for hepatocyte replenishment following chronic liver injury and queried their roles in three distinct HCC models. We found that a pre-existing population of periportal hepatocytes, located in the portal triads of healthy livers and expressing low amounts of Sox9 and other bile-duct-enriched genes, undergo extensive proliferation and replenish liver mass after chronic hepatocyte-depleting injuries. Despite their high regenerative potential, these so-called hybrid hepatocytes do not give rise to HCC in chronically injured livers and thus represent a unique way to restore tissue function and avoid tumorigenesis. This specialized set of pre-existing differentiated cells may be highly suitable for cell-based therapy of chronic hepatocyte-depleting disorders.

Partial hepatectomy induces delayed hepatocyte proliferation and normal liver regeneration in ovariectomized mice.

Estrogens play central roles in sexual development, reproduction, and hepatocyte proliferation. The ovaries are one of the main organs for estradiol (E2) production. Ovariectomies (OVXs) were performed on the female mice, and hepatocyte proliferation was analyzed. The ovariectomized mice exhibited delayed hepatocyte proliferation after partial hepatectomy (PH) and also exhibited delayed and reduced E2 induction. Both E2 administration and PH induced the gene expression of estrogen receptor α (ERα). The transcripts of ERα were detected specifically in periportal hepatocytes after E2 administration and PH. Moreover, the E2 concentrations and hepatocyte proliferation rates were highest in the proestrus period of the estrous cycle. Taken together, these findings indicate that E2 accelerated ERα expression in periportal hepatocytes and hepatocyte proliferation in the female mice.

Proliferation-Independent Initiation of Biliary Cysts in Polycystic Liver Diseases.

Biliary cysts in adult patients affected by polycystic liver disease are lined by cholangiocytes that proliferate, suggesting that initiation of cyst formation depends on proliferation. Here, we challenge this view by analyzing cyst-lining cell proliferation and differentiation in Cpk mouse embryos and in livers from human fetuses affected by Autosomal Recessive Polycystic Kidney Disease (ARPKD), at early stages of cyst formation. Proliferation of fetal cholangiocyte precursors, measured by immunostaining in human and mouse livers, was low and did not differ between normal and ARPKD or Cpk livers, excluding excessive proliferation as an initiating cause of liver cysts. Instead, our analyses provide evidence that the polycystic livers exhibit increased and accelerated differentiation of hepatoblasts into cholangiocyte precursors, eventually coalescing into large biliary cysts. Lineage tracing experiments, performed in mouse embryos, indicated that the cholangiocyte precursors in Cpk mice generate cholangiocytes and periportal hepatocytes, like in wild-type animals. Therefore, contrary to current belief, cyst formation in polycystic liver disease does not necessarily depend on overproliferation. Combining our prenatal data with available data from adult livers, we propose that polycystic liver can be initiated by proliferation-independent mechanisms at a fetal stage, followed by postnatal proliferation-dependent cyst expansion.

Roles of Combined Glypican-3 and Glutamine Synthetase in Differential Diagnosis of Hepatocellular Lesions.

Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer and thirdly leading cause of cancer-related death worldwide. The estimated risk of hepatocellular carcinoma is 15 to 20 times as high among persons infected with HCV as it is among those who are not infected, with most of the excess risk limited to those with advanced hepatic fibrosis or cirrhosis. Glypican3 (GPC3) plays a key role in relation to signaling with growth factors, regulating the proliferative activity of cancer cells. Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia in the mammalian liver. GS was suggested as a specific marker for tracing cell lineage relationships during hepatocarcinogenesis. In normal liver, GS expression is seen in pericentral hepatocytes, but not by midzonal or periportal hepatocytes. In HCC, strong and diffuse GS expression in seen in tumor cells. Glypican3 immunopositvity was highly specific and sensitive indicator for hepatocellular carcinoma as well as glutamine synthetase which was found to be a sensitive and specific indicator for development of hepatocellular carcinoma when compared to cirrhosis, liver cell dyspalsia and metastatic carcinomas. Statistical analysis revealed a significant association between GPC3 and GS with tumor size (P=0.003, p=0.006, respectively). Diffuse staining significantly associated with large tumor size while, focal and mixed staining was detected more with small tumor size. Studying the relation with tumor grade also revealed significant association between diffuse GPC3 and GS staining with high tumor grade. Diffuse staining was detected in 91.7% and 100% respectively of poorly differentiated specimens and only in 33.3% and 22.2% of well differentiated specimens. While using GPC3 and GS to screen for premalignant hepatic lesions remains controversial, our data suggest that GPC3 and GS may be a reliable diagnostic immunomarkers to distinguish HCC from benign hepatocellular lesions. However, negative immunostaining should not exclude the diagnosis of HCC.

Pathology of hepatic iron deposition in hemochromatosis

To identify the type of iron deposition and describe its amount, distribution and associated lesions, in order to support an etiologic diagnosis for hemochromatosis. Hematoxylineosin (HE) stain, reticular fiber stain, Masson's stain and Perl's iron stain were used to assess liver biopsies from 31 patients with hemochromatosis. The Ishak scoring system and Deugnier scoring system were used to assess the histological change in liver and to semi-quantify the excess of hepatic iron. Genetic testing results were received from a portion of the patients and used in analysis. One patient had hereditary (-HFE) hemochromatosis complicated with Gilbert's syndrome, for which the pattern of iron deposition was similar to that of the four patients with Gilbert's syndrome. Iron accumulation appeared as fine granules predominating at the biliary pole of cells and was distributed throughout the lobule with a decreasing gradient spanning from the periportal to centrolobular areas. Mild chronic inflammation was found to be commonly associated with low stage fibrosis.One patient had HFE hemochromatosis complicated with hepatitis B virus infection, and the pattern of iron deposition resembled that in the eight patients with viral hepatitis, wherein the deposition was mainly in the sinusoidal cells and/or portal macrophages. Histological grading and fibrosis staging differed among patients. The five patients with blood disordered showed iron accumulation mainly in the periportal hepatocytes, but mesenchymal iron deposits were also present. The grade of inflammation, as well as of fibrosis,was mild. The five patients with alcoholic disease and the five patients with drug-induced hepatitis showed hepatic iron deposition in swollen or ballooned hepatocytes. The two patients with excessive iron supply showed iron deposition localized within the parenchymal and mesenchymal cells. Etiologic diagnosis of hemochromatosis relies on both the type of iron deposition and the nature of associated lesions. Liver biopsy is necessary for both diagnosis and prognosis.

78. An IND-Enabling GLP-Toxicology and Biodistribution Study Assessing Systemic rAAV9-hNAGLU Gene Delivery for Treating MPS IIIB: Genotype- and Sex-Specific Dose-Limiting Acute Liver Toxicity in Male Wild Type C57BL/6 Mice

No treatment is currently available for mucopolysaccharidosis (MPS) IIIB, a neuropathic lysosomal storage disease due to autosomal recessive defect in α-N-acetylglucosaminidase (NAGLU). In preparation for a clinical trial, we performed an IND-enabling GLP-toxicology study to assess systemic rAAV9-CMV-hNAGLU administration in wt C57BL/6 mice (6-8wk-old), at 1e14 vg/kg or 2e14 vg/kg (n=30/group, M:F=1:1). These were 2 and 4-fold higher, respectively, than our proposed clinical high dose. No adverse clinical signs were observed during the entire 6mo study duration. NAGLU activity above wt levels and vector genomes were detected in all tested CNS and somatic tissues in rAAV-treated mice through 6mo. However, acute mortality was observed in 5 male mice at 2e14 vg/kg dose on Days 6 and 8 pi. All 5 animals exhibited liver discoloration correlating with severe hepatic necrosis and vacuolation, without detectable leukocyte infiltration. At 6, 12 and 24wk pi, minimal single cell hepatocyte necrosis and/or vacuolation, and minimal cardiomyopathy were noted in a dose-dependent manner, with slight transient elevation of ALT or AST at 6wk pi. T-cell responses to AAV9 capsid and rNAGLU were detected at 6wk pi, but partially and completely diminished at 12wk pi, respectively. ELISA showed antibody responses to both AAV9 and rNAGLU in treated animals. In response to the acute mortality in the GLP-tox test, we performed a 6-wk non-GLP study mimicking the GLP-tox study conditions in male MPS IIIB and wt littermates, to further assess the approach and obtain data prior to early mortality. We treated the mice with the same batch of rAAV9-CMV-hNAGLU at the high dose, 2e14 vg/kg, and performed necropsy at 5 days and 6wk pi (n=4/group). No mortality or clinical signs were observed in either rAAV9-treated MPS IIIB or wt mice. At Day 5 pi necropsy, of 4 rAAV9-treated male wt mice, 1 exhibited liver discoloration, correlating with multifocal, periportal and midzonal hepatocyte necrosis, and 2 showed minimal single cell hepatocyte necrosis. Minimal liver damage and elevation of ALT/AST in wt mice persisted to 6wk pi. However, no abnormal changes attributed to vector treatment were detected in the liver in MPS IIIB mice. Therefore, we have identified genotype- and sex-specific dose-limiting toxicity of systemic rAAV9-hNAGLU delivery, supporting a safe profile for our proposed approach for treating MPS IIIB in patients.