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Biomedical subjects

Clare Selden

Publications and source records attributed to Clare Selden.

17 recordsLinked to original sources
PubMed2007

Fat-loaded HepG2 spheroids exhibit enhanced protection from Pro-oxidant and cytokine induced damage.

The mechanisms by which steatosis renders hepatocytes susceptible to damage in non-alcoholic steatohepatitis (NASH) are unclear although fat accumulation is believed to increase hepatocyte susceptibility to inflammatory cytokines and oxidative stress. We therefore investigated the susceptibility of steatotic, hepatocyte-derived cells to TNFalpha and the pro-oxidant, t-butylhydroperoxide (TBH). HepG2 spheroids rendered steatotic by fat-loading with 0.15 mM oleic or palmitic acid for 48 h and treated with TNFalpha or TBH for 18 h exhibited surprisingly lower levels of cytotoxicity, and increased anti-oxidant activity (superoxide dismutase (SOD)) compared with non fat-loaded controls. The protective effect of steatosis was significantly reversed by the inhibition of AMP-activated kinase (AMPK) since spheroids transfected with a kinase-dead AMPKalpha2 subunit, exhibited a significant increase in TBH-induced cytotoxicity when fat-loaded. In conclusion, our findings suggest that fat-loaded hepatocyte-derived cells are surprisingly less susceptible to cytokine and pro-oxidant induced damage via an adaptive mechanism dependent, in part, on AMPK activity.

AMP-Activated Protein Kinases↗
PubMed2007

The level of viral antigen presented by hepatocytes influences CD8 T-cell function.

CD8 T cells exert their antiviral function through cytokines and lysis of infected cells. Because hepatocytes are susceptible to noncytolytic mechanisms of viral clearance, CD8 T-cell antiviral efficiency against hepatotropic viruses has been linked to their capacity to produce gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). On the other hand, intrahepatic cytokine production triggers the recruitment of mononuclear cells, which sustain acute and chronic liver damage. Using virus-specific CD8 T cells and human hepatocytes, we analyzed the modulation of virus-specific CD8 T-cell function after recognition peptide-pulsed or virally infected hepatocytes. We observed that hepatocyte antigen presentation was generally inefficient, and the quantity of viral antigen strongly influenced CD8 T-cell antiviral function. High levels of hepatitis B virus production induced robust IFN-gamma and TNF-alpha production in virus-specific CD8 T cells, while limiting amounts of viral antigen, both in hepatocyte-like cells and naturally infected human hepatocytes, preferentially stimulated CD8 T-cell degranulation. Our data document a mechanism where virus-specific CD8 T-cell function is influenced by the quantity of virus produced within hepatocytes.

Antigen Presentation↗
PubMed2006

Ornithine transcarbamylase and arginase I deficiency are responsible for diminished urea cycle function in the human hepatoblastoma cell line HepG2.

A possible cell source for a bio-artificial liver is the human hepatblastoma-derived cell line HepG2 as it confers many hepatocyte functions, however, the urea cycle is not maintained resulting in the lack of ammonia detoxification via this cycle. We investigated urea cycle activity in HepG2 cells at both a molecular and biochemical level to determine the causes for the lack of urea cycle expression, and subsequently addressed reinstatement of the cycle by gene transfer. Metabolic labelling studies showed that urea production from 15N-ammonium chloride was not detectable in HepG2 conditioned medium, nor could 14C-labelled urea cycle intermediates be detected. Gene expression data from HepG2 cells revealed that although expression of three urea cycle genes Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase was evident, Ornithine Transcarbamylase and Arginase I expression were completely absent. These results were confirmed by Western blot for arginase I, where no protein was detected. Radiolabelled enzyme assays showed that Ornithine Transcarbamylase functional activity was missing but that Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase were functionally expressed at levels comparable to cultured primary human hepatocytes. To restore the urea cycle, HepG2 cells were transfected with full length Ornithine Transcarbamylase and Arginase I cDNA constructs under a CMV promoter. Co-transfected HepG2 cells displayed complete urea cycle activity, producing both labelled urea and urea cycle intermediates. This strategy could provide a cell source capable of urea synthesis, and hence ammonia detoxificatory function, which would be useful in a bio-artificial liver.

Arginase↗
PubMed2005

Hepatocyte progenitors in man and in rodents--multiple pathways, multiple candidates.

In severe injury, liver-cell progenitors may play a role in recovery, proliferating, and subsequently differentiating into mature liver cells. Identifying these progenitors has major therapeutic potential for ex vivo pharmaceutical testing, bioartificial liver support, tissue engineering and gene therapy protocols. Potential liver-cell progenitors have been identified from bone marrow, peripheral blood, cord blood, foetal liver, adult liver and embryonic stem cells. Differences and similarities are found among cells isolated from rodents and humans. This review will discuss identifying markers and differentiation potential in in vitro and in vivo models of these putative progenitors in both humans and rodents.

Animals↗
PubMed2005

Co-transplantation of encapsulated HepG2 and rat Sertoli cells improves outcome in a thioacetamide induced rat model of acute hepatic failure.

Hepatocyte transplantation offers therapeutic opportunities in liver disease. Xenogeneic hepatocytes are a potential resource, but rejection presents a major problem. We combined cell encapsulation with modulation by local generation of an immunosuppressant by co-encapsulating Sertoli cells with HepG2 cells. We assessed in vitro rat leukocyte proliferative responses and HepG2 cell survival after intraperitoneal injection in rats. Empty beads, and beads containing HepG2 cells or HepG2/Sertoli cells were injected intra-peritoneally into rats and survival of implanted cells followed over 4 weeks; in some animals acute hepatic failure (AHF) using thioacetamide (TAA) was also induced. The marked proliferative response of rat leukocytes to HepG2 cells and HepG2-containing beads was reduced by Sertoli cell-conditioned medium and HepG2/Sertoli encapsulates. After intra-peritoneal transplantation, Sertoli cells co-encapsulation protected the HepG2 cells in normal and AHF animals. Combined encapsulation and locally generated immuno-suppression may be a valuable strategy in hepatocyte transplantation.

Animals↗
PubMed2005

Proliferation rates of HepG2 cells encapsulated in alginate are increased in a microgravity environment compared with static cultures.

This study investigates the effect of rotary culture compared with static culture on the proliferation, cell viability, synthetic function and detoxificatory capacity of HepG2 cells encapsulated in 1% alginate. Cell viability and alginate bead morphology were maintained in the rotary culture system at day 10, while cell number showed a 4.5-fold increase compared with static culture. Protein production was increased in rotary cultures with a 4.1-fold increase in total albumin and a 4.4-fold increase in alpha1 antitrypsin levels in rotary compared with static culture at day 10. CYP4501A1/2 activity was maintained between the two culture systems. In conclusion, rotary culture increases proliferation rates leading to improved bead packing and a concomitant increase in total protein synthesis, along with maintenance of detoxificatory capacity. This allows a greater level of hepatic function to be expressed in a given volume, offering clear advantages for the design of liver support systems.

Alginates↗
PubMed2004

Altered mitochondrial function and cholesterol synthesis influences protein synthesis in extended HepG2 spheroid cultures.

Cultures of hepatocytes and HepG2 cells provide useful in vitro models of liver specific function. In this study, we investigated metabolic and biosynthetic function in 3-D HepG2 spheroid cultures, in particular to characterise changes on prolonged culture. We show that HepG2 cells cultured in spheroids demonstrate a reduction in mitochondrial membrane potential and respiration following 10 days of culture. This coincides with a modest reduction in glycolysis but an increase in glucose uptake where increased glycogen synthesis occurs at the expense of the intracellular ATP pool. Lowered biosynthesis coincides with and is linked to mitochondrial functional decline since low glucose-adapted spheroids, which exhibit extended mitochondrial function, have stable biosynthetic activity during extended culture although biosynthetic function is lower. This indicates that glucose is required for biosynthetic output but sustained mitochondrial function is required for the maintenance of biosynthetic function. Furthermore, we show that cholesterol synthesis is markedly increased in spheroids cf. monolayer culture and that inhibition of cholesterol synthesis by lovastatin extends mitochondrial and biosynthetic function. Therefore, increased cholesterol synthesis and/or its derivatives contributes to mitochondrial functional decline in extended HepG2 spheroid cultures.

Albumins↗
PubMed2004

Cellular therapies for liver replacement.

Insufficient donor organs for orthotopic liver transplantation worldwide have urgently increased the requirement for new therapies for acute and chronic liver disease. Whilst none are yet clinically proven there are at least two different approaches for which there is extensive experimental data, some human anecdotal evidence and some data emerging from Phase 1 clinical trials. Both approaches involve bio-engineering. In vivo tissue engineering involves isolated liver cell transplantation into the liver and/or other ectopic sites and in vitro tissue engineering, using an extracorporeal hepatic support system or bioartificial liver. Some questions are common to both these approaches, such as the best cell source and the therapeutic mass required, and are discussed. Others are specific to each approach. For cell transplantation in vivo the initial engraftment and repopulation will make a critical difference to the outcome, and development of markers for transplanted cells has enabled significant advances in understanding, and therefore manipulating, the process. Moreover, the role of immunosuppression is also important and novel approaches to natural immunosuppression are discussed. For use in a bioartificial liver, the ability for hepatocytes to perform ex vivo at in vivo levels is critical. Three dimensional culture improves cell performance over monolayer cultures. Alginate encapsulated cells offer a suitable 3-D environment for a bioartificial liver since they are both easily manipulatable and cryopreservable. The use of cells derived from stem cells or foetal rather than adult liver cells is also emerging as a potential human cell source which may overcome problems associated with xenogeneic cells.

Animals↗
PubMed2004

Stratum corneum acidification in neonatal skin: secretory phospholipase A2 and the sodium/hydrogen antiporter-1 acidify neonatal rat stratum corneum.

At birth, human stratum corneum (SC) displays a near-neutral surface pH, which declines over several days to weeks to months to an acidic pH, comparable to that of adults. Recent studies suggest that an acidic pH is required for normal permeability barrier homeostasis and SC integrity/cohesion. We assessed here the basis for postnatal acidification in the neonatal rat, where SC pH, as measured with a flat surface electrode, declines progressively from near-neutral levels (pH 6.63) on postnatal days 0 to 1 to adult levels (pH 5.9) or even below over the subsequent 7 to 8 d. The postnatal decline in SC pH was paralleled by a progressive activation of a pH-dependent hydrolytic enzyme, beta-glucocerebrosidase. Because SC acidification could not be linked to commonly implicated exogenous factors, such as bacterial colonization, or the deposition of sebaceous gland products. We next assessed whether changes in one or more of three endogenous mechanisms demonstrate postnatal activity changes that contribute to the progressive development of an acidic SC pH. Although the histidine-to-urocanic acid pathway has been implicated in acidification of the adult SC, surface pH is completely normal in histidase-deficient (his/his, Peruvian) mice, ruling out a requirement for this mechanism. In contrast, when sodium/hydrogen antiporter-1 (NHE1), which predominantly acidifies membrane domains at the stratum granulosum-SC interface, is inhibited, postnatal acidification of the SC is partially blocked. Likewise, SC secretory phospholipase A2 (sPLA2) activity, measured with a fluorometric assay, is low at birth, but increases progressively (by 66%) over the first 5 d after birth, and inhibition of sPLA2 between days 0 to 1 and days 5 to 6 delays postnatal SC acidification. Together, these results describe a neonatal model, in which the development of an acidic surface pH can be ascribed, in part, to progressive SC acidification by two endogenous mechanisms, namely, sPLA2 and NHE1, which are known to be important for acidification of adult rodent SC. Conversely, the impaired acidification of neonatal SC, which has important functional and clinical consequences, can be explained by the relatively low activities of one or both of these mechanisms at birth.

Acids↗
PubMed2004

The histopathology of regeneration in massive hepatic necrosis.

Massive hepatic necrosis (MHN) is a condition that offers an opportunity to study the remarkable ability of the liver to become repopulated with hepatocytes. A maximal regenerative stimulus is expected in cases of MHN (Roskams et al. APMIS Suppl 1991;23:32-39). Sequential chronological observations, after a severe degree of liver cell loss, permit study of the human equivalent of the situation in animal models in which circulating and bone marrow-derived stem and liver progenitor cells are recruited to the hepatopoietic process. To date, the bone marrow and circulating precursors have not been identified morphologically in human material. We present data that suggest that the circulating liver progenitor could have a lymphoblastoid morphological appearance. Similar cells are seen among the cellular infiltrate of MHN. We have found that combinations of markers, such as CD117/CD133 positive CD45/tryptase negative are useful to isolate these cells using cell-sorting technology. This may facilitate their expansion in vitro and the development of their use for therapeutic purposes. In MHN, the residual portal tracts and ductular reaction with the associated lymphoid infiltrate (some of which are probably liver cell progenitors derived from the circulation) constitute the fundamental regenerative community unit in which hepatopoiesis takes place. Defining the hepatopoietic process is hindered by the lack of morphological transitional forms in the period between the progenitors within the circulation and when they assume recognizable hepatocytic form as "metaplastic" hepatocytes associated with the ductular reaction. By achieving a better comprehension of these processes of liver cell restoration, we will be better placed to accelerate liver recovery in MHN, for example by the administration of granulocyte colony stimulating factor (GCSF). Thus, more patients will be able to restore their own livers and avoid liver transplantation.

Hepatocytes↗
PubMed2004

Alginate-encapsulated human hepatoblastoma cells in an extracorporeal perfusion system improve some systemic parameters of liver failure in a xenogeneic model.

Previous studies have demonstrated that alginate encapsulation of proliferating hepatocyte-derived cell lines (e.g., HepG2 cells) enhances the expression of differentiated hepatocyte function compared with conventional monolayer culture. Furthermore, such capsules have the advantage of cryopreservability, and can be readily manipulated, e.g., for the charging of extracorporeal devices. We utilize a rabbit model of acute liver failure caused by acetaminophen administration to rabbits pretreated to enhance cytochrome p450 enzyme activity, and demonstrate that encapsulated HepG2 cells, in an extracorporeal chamber, perfused by rabbit plasma separated on-line at a rate of 2-5 mL/min, and perfused over cells at 40-60 mL/min, improve systemic parameters of liver failure (diastolic blood pressure and transjugular venous oxygen saturation). Such encapsulated cells have the potential to be developed for extracorporeal liver support systems for acute liver failure.

Acetaminophen↗
PubMed2003

Triiodothyronine enhances the regenerative capacity of the liver following partial hepatectomy.

This study investigates the effects of administering a primary mitogen, triiodothyronine (T(3)), at the time of 70% partial hepatectomy (PH) in the rat, thus combining the 2 distinct pathways of liver growth: direct hyperplasia and compensatory regeneration. T(3) enhances the proliferative response of hepatocytes within the liver following PH. Flash bromodeoxyuridine (BrdU) labeling showed a cell proliferation index 24 hours after PH alone of 26.5% +/- 2.8%; when T(3) was administered at PH, it increased to 39.5% +/- 5.0% (P <.01 compared with PH alone). Continuous BrdU labeling performed every 6 hours between 15 and 72 hours following surgery showed an index of 84.0% +/- 4.0% when T(3) was administered at PH compared with 71.0% +/- 4.0% with PH alone (P <.01 compared with PH alone). This increase in cell proliferation resulted in a larger liver mass at 4 days in rats receiving T(3) at PH compared with PH alone (P <.05 compared with PH alone). The difference in liver mass was matched with corresponding increases in total DNA and total protein levels as well as cell division, as confirmed by the frequent demonstration of twin daughter cells on histology. In conclusion, this study shows that a single dose of T(3) enhances the regenerative capacity of the liver following PH. The ability to enhance cell proliferation during compensatory hyperplasia following PH could be therapeutically valuable if applicable to humans.

Alanine Transaminase↗
PubMed2003

Epithelial colonies cultured from human explanted liver in subacute hepatic failure exhibit hepatocyte, biliary epithelial, and stem cell phenotypic markers.

The liver in subacute hepatic failure may become enriched for hepatic progenitor cells. Liver tissue from such a patient was collagenase digested and, from the nonparenchymal cell fraction, epithelioid colonies were developed. Albumin and alpha-1-antitrypsin (AAT) were secreted for greater than 120 days from these colonies. Reverse transcription-polymerase chain reaction showed expression of markers of both hepatocyte and biliary epithelial phenotypes (cytokeratins 7, 18, and 19, albumin and AAT, hepatocyte growth factor receptor, transforming growth factor beta receptor type II, gamma-glutamyl transpeptidase, biliary glycoprotein). The cell cycle regulator p21 was also expressed. The POU domain transcription factor octamer-binding protein 4 was present in these cells, but not in RNA or cDNA prepared from adult human liver. These markers were maintained even after 165 days culture. Proliferating epithelial-like cells with combined hepatocyte- and biliary-epithelial-specific functional markers and a stem cell marker can be isolated from the nonparenchymal fraction of liver cells in subacute hepatic failure.

Bile Ducts, Intrahepatic↗
PubMed2003

Characterization of long-term survival of syngeneic hepatocytes in rat peritoneum.

Hepatocyte transplantation is a potential therapy for both acute and chronic hepatic insufficiency and also for treatment of inborn errors of metabolism affecting the liver. The peritoneum is one site for implantation and has several advantages: cells implanted there can be easily identified and observed, and it has a relatively large capacity. Long-term survival using "pure" hepatocytes in the peritoneum have been disappointing. We hypothesized that cotransplantation of hepatocytes with nonparenchymal cells would help maintain differentiated hepatocyte function. Rat liver cells transplanted intraperitoneally into August rats were sacrificed at 7 days, 1, 3, 6, 9, and 12 months and analyzed for presence, basal proliferation, and functionality of hepatocytes. To demonstrate that ectopic hepatocytes remained susceptible to exogenous growth factors affecting cell proliferation, rats 9 and 12 months after transplantation were stimulated with tri-iodothyronine and KGF. Hepatocytes were identified 7 days to >12 months, by H&E and immunohistochemically, as ectopic islands in the omental fat. Functionality was confirmed by glycogen deposition. Basal proliferation in 7-day rats was 28.0 +/- 10/1000 hepatocytes in ectopic islands (cf. 5.70 +/- 2.7/1000 in recipient liver). Proliferation in ectopic islands was greater than host liver. Growth factor-stimulated proliferation in ectopic islands induced a 70-fold increase in DNA synthesis. In conclusion, hepatocytes transplanted with nonparenchymal cells survive, proliferate, and function in the peritoneum of normal rats, and respond to exogenous growth stimuli. Their survival and proliferation in the presence of a normal functioning liver has implications for the potential use of the peritoneal site clinically for supplementation of liver function in metabolic disorders.

Animals↗
PubMed2002

The role of non-parenchymal cells in liver growth.

The main non-parenchymal cells of the liver, Kupffer cells, sinusoidal endothelial cells and stellate cells, participate in liver growth with respect to both their own proliferation, and effects on hepatocyte proliferation. In the well-characterised paradigm of 70% partial hepatectomy, they undergo DNA synthesis and cell division 20-24h later than the hepatocyte population. They exert both positive and negative influences on hepatocyte proliferation, including provision of an extracellular matrix-bound reservoir of hepatocyte growth factor that is activated after damage; priming of hepatocytes for DNA synthesis through rapid generation of TNF-alpha and IL-6; and generation of factors at later time points that curb hepatocyte DNA synthesis (IL-1, TGF-beta) and initiate reconstruction and reformation of matrix proteins.

Animals↗
PubMed2002

Modulation of hepatocyte function in an immortalized human hepatocyte cell line following exposure to liver-failure plasma.

For hepatocytes to function effectively in a bioartificial liver device, maintained function in the milieu of plasma from patients with liver failure will be required. We have investigated the effect of plasma obtained at plasmapheresis from patients with acute liver failure on the performance of the human hepatocyte cell line HHY41 in liver-failure plasma, normal plasma, and culture medium. Cytotoxicity of plasma, DNA synthesis by thymidine incorporation, oxidative status, and cytochrome P450 functions were assayed after a 16 h culture with normal plasma, liver-failure plasma, or culture medium. Some, but not all, samples of liver-failure plasma were deleterious to the performance of the cell line, inducing cytotoxicity and oxidative stress, with diminished DNA synthesis, protein synthesis, and cytochrome P4501A activity. Strategies to minimize the toxic effects of liver-failure plasma may improve the performance of liver cells in extracorporeal liver-support devices.

Biotransformation↗