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Biomedical subjects

John K Olynyk

Publications and source records attributed to John K Olynyk.

At least 19 recordsLinked to original sources
PubMed2006

Evaluation of the "Cellscreen" system for proliferation studies on liver progenitor cells.

Proliferation studies on mammalian cells have been disadvantaged by the limited availability of non-invasive assays as the majority of approaches are based on chemical treatment, sampling or staining of cells removed from culture. In this study, we utilised the Cellscreen system (Innovatis AG, Bielefeld, Germany), a non-invasive automated technique for measuring proliferation of adherent and suspension cells over time. We have evaluated the ability of the Cellscreen system to monitor and quantify growth of adherent liver progenitor cells over time and tested several applications, (i) serum reduction or (ii) treatment with a cytokine. Our results demonstrate that the Cellscreen system reproducibly documents pro- and anti-proliferative effects of cytokines and growth factors and quantifies changes by providing cell-doubling times for control and test cultures. However, we found that for the conversion of cell density values into absolute cell numbers different conversion factors, which better suit the individual growth phases, need to be established. Collectively, these findings reveal that the Cellscreen system is applicable for the determination of cell proliferation of adherent and suspension cells in response to a variety of (growth) factors. It minimises operator participation and thus enables more rapid and larger screens and, being non-invasive, permits multiple assays on the same culture of cells. Hence, this technique proves superior to the common proliferation assays opening up new dimensions of proliferation studies in cell biology.

Animals↗
PubMed2006

Attenuated liver progenitor (oval) cell and fibrogenic responses to the choline deficient, ethionine supplemented diet in the BALB/c inbred strain of mice.

BACKGROUND/AIMS: Liver regeneration following chronic injury is associated with inflammation, the proliferation of liver progenitor (oval) cells and fibrosis. Previous studies identified interferon-gamma as a key mediator of oval cell proliferation. Interferon-gamma is known to regulate Th1 cell activities during immune challenge. Therefore, we hypothesised that progenitor cell-mediated regeneration is associated with a Th1 immune response. METHODS: C57Bl/6 (normal Th1 response) and BALB/c mice (deficient in Th1 signalling) were placed on a carcinogenic diet to induce liver injury, progenitor cell proliferation and fibrosis. RESULTS: Serum transaminases and mortality were elevated in BALB/c mice fed the diet. Proliferation of liver progenitor cells was significantly attenuated in BALB/c animals. The pattern of cytokine expression and inflammation differed between strains. Liver fibrosis and hepatic stellate cell activation were significantly inhibited in BALB/c mice compared to C57Bl/6. In addition, interferon-gamma knockout mice also showed reduced fibrosis compared to wild type. These findings are in contrast to published results, in which interferon-gamma is shown to be anti-fibrogenic. CONCLUSIONS: Our data demonstrate that the hepatic progenitor cell response to a CDE diet is inhibited in mice lacking Th1 immune signalling and further show that this inhibition is associated with reduced liver fibrosis.

Animals↗
PubMed2006

Hepatic oval cell response to the choline-deficient, ethionine supplemented model of murine liver injury is attenuated by the administration of a cyclo-oxygenase 2 inhibitor.

Oval cell proliferation precedes neoplasia in many rodent models of hepatocellular carcinoma and prevention of this proliferative response can reduce the risk of subsequent carcinoma. This study aimed to determine whether a selective cyclo-oxygenase-2 (COX-2) inhibitor, SC-236, affects (i) the oval cell response to liver injury in a mouse model of hepatocarcinogenesis and (ii) an oval cell line. Four-week-old mice were fed either normal chow or a choline deficient, ethionine supplemented (CDE) diet in the presence or absence of SC-236. Liver histology and oval cell numbers were determined after 2, 4, 12 and 52 weeks of treatment. Oval cells were scored using morphological criteria and positive immuno-staining for the M(2)-isozyme of pyruvate kinase (M2PK) or A6. An immortalized oval cell line (PIL-2) was used to study the in vitro effects of SC-236 on oval cell proliferation, apoptosis and Akt phosphorylation. The percentage of M2PK-positive oval cells and COX-2-positive cells was reduced by 80% and 45%, respectively, in CDE-fed mice receiving SC-236 compared with CDE-fed animals not receiving SC-236. Some M2PK-positive oval cells were also COX-2 positive. The percentage of A6-positive cells was not affected by SC-236 administration to CDE-fed mice. Administration of SC-236 increased apoptosis as evidenced by a 73% increase in the number of TUNEL-positive cells at 2 weeks in CDE-fed mice. Primary oval cells and PIL-2 cells expressed COX-2. In vitro treatment of PIL-2 cells with SC-236 resulted in a dose-dependent preferential death of A6-negative cells. Administration of 25 and 50 microM Prostaglandin E(2) partially attenuated SC-236 induced cell death by 25%. In vitro oval cell death was associated with apoptosis and a 70% reduction in Akt phosphorylation. These results suggest that the SC-236 induced reduction of M2PK-positive oval cell numbers may be due to COX-2 dependent inhibition of Akt phosphorylation and induction of apoptosis.

Animals↗
PubMed2006

Antiproliferative effects of interferon alpha on hepatic progenitor cells in vitro and in vivo.

Hepatic progenitor cells (called oval cells in rodents) proliferate during chronic liver injury. They have been suggested as targets of malignant transformation in chronic liver diseases, including chronic hepatitis C. Interferon alpha therapy reduces the risk of hepatocellular carcinoma (HCC) in chronic hepatitis C regardless of viral clearance. The aim of this study was to determine whether interferon alpha could reduce the risk of HCC by modifying preneoplastic events in the hepatic progenitor cell population. Pre- and post-treatment liver biopsies were evaluated for changes in t he hepaticprogenitor cell population in 16 patients with non-responding chronic hepatitis C Interferon alpha-based treatment significantly reduced the numbers of c-kit-positive hepatic progenitor cells by 50%. To determine the mechanism of cell number reduction, the effects of interferon alpha on murinehepatic progenitor cells were studied in vitro. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation assay and proliferating cell nuclear antigen staining showed that interferon alpha had a dose-dependent, anti-proliferative effect Interferon alpha stimulated hepatocytic and biliary differentiation of the oval cell lines reflected by increased expression of albumin and cytokeratin19 accompanied by decreased expression of alphafetoprotein and Thy-1. To validatethese results in vivo, mice were placed on the choline-deficient, ethionine-supplemented diet to induce liver injury and oval cell proliferation and treated with pegylated interferon alpha 2b for 2 weeks. This resulted in a significant four-fold reduction in the number of oval cells (P < .05). In conclusion, interferon alpha-based treatment reduced the number of hepatic progenitor cells in chronic liver injury by modulating apoptosis, proliferation, and differentiation. Supplementay material for this article can

Animals↗
PubMed2006

Population-based study of the relationship between mutations in the hemochromatosis (HFE) gene and arthritis.

BACKGROUND AND AIM: Mutations in the hemochromatosis (HFE) gene are carried by one in three individuals of British Isles descent and may result in increased iron stores. These increased iron stores could potentially induce or exacerbate diseases, such as arthritis, in which iron has a role in pathogenesis. Although arthritis is a well-known association of clinically overt hereditary hemochromatosis, controversy surrounds the role of mutations in the HFE gene as risk factors for arthritis. The aim of the present study was to determine whether mutations in the HFE gene are associated with an increased prevalence of arthritis. METHODS: A population-based study was conducted in Busselton, Western Australia, of the prevalence of arthritis in 1372 individuals of British Isles descent. Participants completed a questionnaire and general physical examination. Analysis for C282Y and H63D HFE mutations was undertaken. Unadjusted and adjusted odds ratios (OR) were calculated for the relationship between HFE mutations and the prevalence of self-reported, doctor-diagnosed arthritis. RESULTS: There was no association between the presence of HFE mutations and the prevalence of self-reported, doctor-diagnosed arthritis (C282Y/wild type (WT) adjusted OR = 1.041 (95% confidence interval (CI) 0.68-1.61), H63D/WT OR = 0.76 (95% CI 0.53-1.08), C282Y/C282Y OR = 0.39 (95% CI 0.04-3.63), C282Y/H 63D OR = 0.808 (95% CI 0.27-2.42), H63D/H63D OR = 0.419 (95% CI 0.13-1.36)). Overall adjusted OR for arthritis in participants with one or more HFE mutations was 0.81 (95% CI 0.61-1.09). CONCLUSIONS: Mutations of the HFE gene are not risk factors for arthritis in populations of British Isles descent.

Adult↗
PubMed2006

Differential findings for CD14-positive hepatic monocytes/macrophages in primary biliary cirrhosis, chronic hepatitis C and nonalcoholic steatohepatitis.

BACKGROUND AND AIMS: Endotoxin-responsive monocytes/macrophages (CD14-positive) are potential sources of profibrogenic factors. The aims of this study were to determine (1) whether hepatic CD14-positive cells are present in various forms of chronic liver disease, and (2) the relationship between CD14-positive cells, myofibroblasts, and fibrosis in these diseases. METHODS: Liver specimens from control subjects (n = 12) and those with primary biliary cirrhosis (n = 18), chronic hepatitis C (n = 13), or nonalcoholic steatohepatitis (n = 13) were immunostained for CD14, CD68, and alpha-smooth muscle actin (SMA) and the number of cells expressing these antigens was determined. Fibrosis and inflammation were also assessed. RESULTS: The total number of hepatic CD68-positive cells was similar in diseased and control livers. The number of CD14-positive cells was increased in advanced fibrosis in primary biliary cirrhosis and hepatitis C but not in nonalcoholic steatohepatitis. The number of CD14-positive cells was also increased in hepatitis C specimens with high inflammatory activity. CD14-positive cells were often associated with alpha-SMA-positive myofibroblasts in fibrous septa. CONCLUSIONS: The number of hepatic CD14-positive cells is increased in advanced fibrosis in subjects with primary biliary cirrhosis and hepatitis C but not in nonalcoholic steatohepatitis. In primary biliary cirrhosis and hepatitis C, CD14-positive macrophages are found in close proximity to fibrous septa and myofibroblasts. In hepatitis C, an increased number of CD14-positive cells are associated with high inflammatory activity.

Biopsy↗
PubMed2006

Normal iron metabolism and the pathophysiology of iron overload disorders.

Iron overload disorders represent a heterogenous group of conditions resulting from inherited and acquired causes. If undiagnosed they can be progressive and fatal. Early detection and phlebotomy prior to the onset of cirrhosis can reduce morbidity and normalise life expectancy. We now have greater insight into the complex mechanisms of normal and disordered iron homeostasis following the discovery of new proteins and genetic defects. Here we review the normal mechanisms and regulation of gastrointestinal iron absorption and liver iron transport and their dysregulation in iron overload states. Advances in the understanding of the natural history of iron overload disorders and new methods for clinical detection and management of hereditary haemochromatosis are also reviewed. The current screening strategies target high-risk groups such as first-degree relatives of affected individuals and those with clinical features suggestive of iron loading. Potential ethical, legal and psychosocial issues arising through application of genetic screening programs need to be resolved prior to implementation of general population screening programs.

Journal Article↗
PubMed2005

Limited iron export by hepatocytes contributes to hepatic iron-loading in the Hfe knockout mouse.

BACKGROUND/AIMS: In hereditary hemochromatosis, iron-loading of hepatocytes is associated with increased iron uptake while little is known about iron release. This study aims to characterise iron release and ferroportin expression by Hfe knockout hepatocytes to determine if they contribute to iron overload in haemochromatosis. METHODS: Iron release by hepatocytes from Hfe knockout, non-iron-loaded and iron-loaded wild-type mice was measured after incubation with nontransferrin-bound iron as iron-citrate. RESULTS: Iron release and ferroportin expression by hepatocytes from Hfe knockout, non-iron-loaded and in vivo iron-loaded wild-type mice were similar although, nontransferrin-bound iron uptake was significantly increased in Hfe knockout hepatocytes and decreased in iron-loaded wild-type hepatocytes compared with non-iron-loaded wild-type cells. When expressed as a percentage of total iron uptake, iron release was decreased in Hfe knockout hepatocytes (4.6+/-0.7 versus 13.7+/-1.2%, P<0.0001) and increased in iron-loaded wild-type hepatocytes (29.5+/-0.5 versus 13.5+/-0.7%; P<0.0001) compared with wild-type hepatocytes. In contrast, in vitro iron-loading increased iron release and ferroportin expression by both Hfe knockout and wild-type hepatocytes. CONCLUSIONS: Hfe knockout hepatocytes accumulate iron as a result of limited iron export and enhanced iron uptake. The correlation between iron release and ferroportin expression suggests that iron export in hepatocytes is mediated by ferroportin.

Animals↗
PubMed2005

Inhibition of adult liver progenitor (oval) cell growth and viability by an agonist of the peroxisome proliferator activated receptor (PPAR) family member gamma, but not alpha or delta.

Multifaceted evidence links the development of liver tumours to the activation and proliferation of adult liver progenitor (oval) cells during the early stages of chronic liver injury. The aim of this study was to examine the role of the peroxisome proliferator activated receptors (PPARs): PPARalpha, delta and gamma, in mediating the behaviour of liver progenitor cells during pre-neoplastic disease and to investigate their potential as therapeutic targets for the treatment of chronic liver injury. We observed increased liver expression of PPARalpha and gamma in concert with expanding oval cell numbers during the first 21 days following commencement of the choline deficient, ethionine supplemented (CDE) dietary model of carcinogenic liver injury in mice. Both primary and immortalized liver progenitor cells were found to express PPARalpha, delta and gamma, but not gamma2, the alternate splice form of PPARgamma. WY14643 (PPARalpha agonist), GW501516 (PPARdelta agonist) and ciglitazone (PPARgamma agonist) were tested for their ability to modulate the behaviour of p53-immortalized liver (PIL) progenitor cell lines in vitro. Both PPARdelta and gamma agonists induced dose-dependent growth inhibition and apoptosis of PIL cells. In contrast, the PPARalpha agonist had no effect on PIL cell growth. None of the drugs affected the maturation of PIL cells along either the hepatocytic or biliary lineages, as judged by their patterns of hepatic gene expression prior to and following treatment. Administration of the PPARgamma agonist ciglitazone to mice fed with the CDE diet for 14 days resulted in a significantly diminished oval cell response and decreased fibrosis compared with those receiving placebo. In contrast, GW501516 did not affect oval cell numbers or liver fibrosis, but inhibited CDE-induced hepatic steatosis. In summary, PPARgamma agonists reduce oval cell proliferation and fibrosis during chronic liver injury and may be useful in the prevention of hepatocellular carcinoma.

Animals↗
PubMed2005

Oncostatin M induces an acute phase response but does not modulate the growth or maturation-status of liver progenitor (oval) cells in culture.

Following acute injury, the liver regenerates through hepatocyte division. If this pathway is impaired, liver repair depends on the recruitment of adult liver progenitor (oval) cells. Mice fed a choline deficient, ethionine supplemented (CDE) diet possess substantial numbers of oval cells, which can be isolated, or examined in vivo. Oncostatin M (OSM) has been shown to induce maturation of murine fetal hepatoblasts into hepatocytes. We recently confirmed this in human fetal liver cultures. Here, we show that liver OSM expression increases in mice fed a CDE diet and CDE-derived oval cell isolates express OSM and its receptor (OSMR). Oval cell lines (PIL cells), as well as primary oval cell cultures, displayed STAT-3 phosphorylation following OSM stimulation. OSM had no effect on the growth of primary oval cells, but it was pro-apoptotic to PIL cells, suggesting that the two cell models are not directly comparable. Expression of PCNA and cyclin D1 was not affected by OSM treatment. No evidence was obtained to suggest an effect on oval cell maturation with OSM treatment. However, decreased albumin production, accompanied by increased expression of haptoglobin and fibrinogen, suggests that OSM induced an acute phase reaction in cultured oval cells.

Acute-Phase Reaction↗
PubMed2005

Jekyll and Hyde: evolving perspectives on the function and potential of the adult liver progenitor (oval) cell.

The liver progenitor cell (LPC) has enormous potential for use in cell therapy to treat liver disease. Since liver regenerates readily from pre-existing hepatocytes, a role for LPCs and, indeed, their existence have been questioned. Research during the last decade has established that LPCs are an important alternative source of cells for liver regeneration. Their utility for cell therapy lies in their ability to generate both hepatocytes and cholangiocytes. However, they are observed in liver diseases that often lead to cancer and there is experimental evidence that implicates LPCs as the source of tumours. This article provides a brief history of the studies that established the functional importance of LPCs in liver disease. It focuses on mouse models that have led to the identification of factors that regulate LPC growth and differentiation and discusses LPCs derived from different sources. Recent promising results from both in vitro and vivo studies suggest that LPCs could be useful for cell therapy. In the context of liver disease, LPCs may indeed be the cell of the future and understandably "our favourite cell".

Aging↗
PubMed2005

Iron overload.

Iron overload disorders represent a heterogenous group of conditions resulting from inherited and acquired causes. With the discovery of new proteins and genetic defects we have gained greater insight into their causation at the molecular level and the complex mechanisms of normal and disordered iron homeostasis. Here we review the normal mechanisms and regulation of gastrointestinal iron absorption and liver iron transport and their dysregulation in iron overload states. Advances in the understanding of the natural history of iron overload disorders and new methods for clinical detection and management of hereditary hemochromatosis are also reviewed.

Ceruloplasmin↗
PubMed2005

Screening for HFE and iron overload.

Type 1 hereditary hemochromatosis is a common disorder of iron overload occurring in individuals homozygous for the C282Y HFE gene mutation. It can be a progressive and fatal condition. Early detection and phlebotomy prior to the onset of cirrhosis can reduce morbidity and normalize life expectancy. It is readily identified through biochemical testing for iron overload using serum transferrin saturation and genetic testing for C282Y homozygosity. General population screening has been waived in preference to targeting high-risk groups such as first-degree relatives of affected individuals and those with clinical features suggestive of iron loading. This screening strategy is likely to continue until uncertainties regarding the natural history of the disease, age-related penetrance, and management of asymptomatic individuals are clarified. Potential ethical, legal, and psychosocial issues arising through application of genetic screening programs also must be resolved prior to implementation of general population screening programs.

Genetic Markers↗
PubMed2005

Liver inflammation and cytokine production, but not acute phase protein synthesis, accompany the adult liver progenitor (oval) cell response to chronic liver injury.

Oval cells are facultative liver progenitor cells, which are invoked during chronic liver injury in order to replenish damaged hepatocytes and bile duct cells. Previous studies have observed inflammation and cytokine production in the liver during chronic injury. Further, it has been proposed that inflammatory growth factors may mediate the proliferation of oval cells during disease progression. We have undertaken a detailed examination of inflammation and cytokine production during a time course of liver injury and repair, invoked by feeding mice a choline-deficient, ethionine-supplemented (CDE) diet. We show that immediately following initial liver injury, B220-expressing leucocytes transiently infiltrate the liver. This inflammatory response occurred immediately before oval cell numbers began to expand in the liver, suggesting that the two events may be linked. Two waves of liver cytokine production were observed during the CDE time course. The first occurred shortly following commencement of the diet, suggesting that it may represent a hepatic acute phase response. However, examination of acute phase marker expression in CDE-fed mice did not support this hypothesis. The second wave of cytokine expression correlated with the expansion of oval cell numbers in the liver, suggesting that these factors may mediate oval cell proliferation. No inflammatory signalling was detected following withdrawal of the injury stimulus. In summary, our results document a close correlation between inflammation, cytokine production and the expansion of oval cells in the liver during experimental chronic injury.

Acute-Phase Proteins↗
PubMed2005

Effects of HFE gene mutations and alcohol on iron status, liver biochemistry and morbidity.

BACKGROUND AND AIMS: The aims of the present study were to determine: (i) whether alcohol consumption is greater in individuals with HFE mutations; and (ii) whether common HFE mutations modify the effects of alcohol on serum iron and liver biochemistry or morbidity. METHODS: The residents of the town of Busselton in Western Australia were subject to cross-sectional health surveys between 1966 and 1983. In 1994/1995 all surviving participants of the earlier surveys were invited to take part in a follow-up survey. Logistic, linear and Poisson log-linear regression analyses were performed in 1490 men and 1452 women from the 1994/1995 survey to assess the relationships between HFE mutations, alcohol, iron levels, liver biochemistry and morbidity. RESULTS: Heavy or moderate alcohol consumption was present in 7% or 36% of men and 0.5% or 12% of women, respectively. Alcohol consumption strongly influenced levels of serum ferritin and gamma glutamyl transpeptidase (GGT) and mean cell volume (MCV) in men and women but only alanine aminotransferase (ALT) levels in women. These effects were independent of HFE gene mutations. Hospital admission rates for respiratory disorders were higher in men with the C282Y mutation. CONCLUSIONS: Alcohol consumption strongly influences serum ferritin and GGT levels and MCV in men and women but only ALT levels in women, and these effects are independent of HFE mutations. HFE gene mutations do not predispose to moderate or heavy alcohol consumption. The C282Y mutation is associated with increased respiratory admission rates in men.

Alanine Transaminase↗
PubMed2005

Lymphotoxin-beta production following bile duct ligation: possible role for Kupffer cells.

BACKGROUND AND AIMS: Lymphotoxin-beta (LT-beta) may play a role in the pathogenesis of chronic liver injury. The aim of this study was to determine in an animal model of bile duct ligation liver injury whether LT-beta expression is induced and whether Kupffer cells are an intrahepatic source of LT-beta. METHODS: Sprague-Dawley rats were divided into two groups: one group received a single dose of GdCl (a Kupffer cell-blocking agent, 10 mg/kg i.v.), whereas the other group received saline. One day later, the groups underwent bile duct ligation or a sham operation. Liver tissue was obtained on days 1, 3, 5, and 8 for assessment of Kupffer cell numbers, early fibrogenic events and LT-beta gene expression. Kupffer cells were isolated using pronase/collagenase perfusion and centrifugal elutriation. RESULTS: Hepatic LT-beta mRNA expression increased early following bile duct ligation. Pretreatment of bile duct-ligated animals with GdCl significantly reduced the number of Kupffer cells, delayed the rise in LT-beta expression, but had no effect on fibrogenesis. Recovery of the Kupffer cell population in these animals was accompanied by increased hepatic LT-beta expression. The LT-beta ligand and receptor were expressed by isolated normal Kupffer cells. CONCLUSIONS: Hepatic LT-beta expression is induced early following bile duct ligation. Kupffer cells may be an intrahepatic source of LT-beta.

Animals↗