ACTA PHYSIOLOGICA, Issue 1-2 2006
F. Schliess
Abstract Insulin- and amino acid-induced signalling by the mammalian target of rapamycin (mTOR) involves hyperphosphorylation of the p70 ribosomal S6 protein kinase (p70S6-kinase) and the eukaryotic initiation factor 4E (eIF4E) binding protein 4E-BP1 and contributes to regulation of protein metabolism. This review considers the impact of cell hydration on mTOR-dependent signalling. Although hypoosmotic hepatocyte swelling in some instances activates p70S6-kinase, the hypoosmolarity-induced proteolysis inhibition in perfused rat liver is insensitive to mTOR inhibition by rapamycin. Likewise, swelling-dependent proteolysis inhibition by insulin and swelling-independent proteolysis inhibition by leucine, a potent activator of p70S6-kinase and 4E-BP1 hyperphosphorylation, in perfused rat liver is insensitive to rapamycin, indicating that at least rapamycin-sensitive mTOR signalling is not involved. Hyperosmotic dehydration in different cell types produces inactivation of signalling components around mTOR, thereby attenuating insulin-induced glucose uptake, glycogen synthesis, and lipogenesis in adipocytes, and MAP-kinase phosphatase MKP-1 expression in hepatoma cells. Direct inactivation of mTOR, stimulation of the AMP-activated protein kinase, and the destabilization of individual proteins may impair mTOR signalling under dehydrating conditions. Further investigation of the crosstalk between the mTOR pathway(s) and hyperosmotic signalling will improve our understanding about the contribution of cell hydration changes in health and disease and will provide further rationale for fluid therapy of insulin-resistant states. [source]

In vivo and in vitro toxicity of decabromodiphenyl ethane, a flame retardant

ENVIRONMENTAL TOXICOLOGY, Issue 4 2010
Tarja Nakari
Abstract Toxicity of a relative new flame retardant, namely decabromodiphenyl ethane (DBDPE), marketed as an alternative to decabromodiphenyl ether (BDE-209) was assessed both in vivo and in vitro using the freshly separated fish hepatocyte assay and standardized water flea and zebrafish egg-larvae tests. The fish hepatocyte assay, based on the synthesis and secretion of vitellogenin from isolated male liver cells produced a clear dose-response curve in the presence of DBDPE. DBDPE induced the induction of hepatic ethoxyresorufin-O-deethylase (EROD) activity at low test concentrations, but started to inhibit the activity at higher concentrations. Also, the induction of the hepatocyte conjugation activity, uridinediphosphoglucuronosyltransferase (UDPGT), was induced with no signs of inhibition even at the highest test concentration. The reduced EROD activity resulted in a drop in the production of vitellogenin by the cells. In vivo tests showed that DBDPE was acutely toxic to water fleas, the 48 h EC-50 value being 19 ,g/L. Moreover, DBDPE reduced the hatching rates of exposed zebra-fish eggs and raised significantly the mortality of hatched larvae. Because there is hardly any information available on the effects of DBDPE on the aquatic environments, it is crucial to obtain more data on the effects and effective concentrations of DBDPE along with its occurrence in the environment. Such data would enable reliable assessments of the risks posed by this flame retardant. © 2009 Wiley Periodicals, Inc. Environ Toxicol 25: 333,338, 2010. [source]

A reversibly immortalized human hepatocyte cell line as a source of hepatocyte-based biological support

ADDICTION BIOLOGY, Issue 4 2001
Naoya Kobayashi
The application of hepatocyte transplantation (HTX) is increasingly envisioned for temporary metabolic support during acute liver failure and provision of specific liver functions in inherited liver-based metabolic diseases. Compared with whole liver transplantation, HTX is a technically simple procedure and hepatocytes can be cryopreserved for future use. A major limitation of this form of therapy in humans is the worldwide shortage of human livers for isolating an adequate number of transplantable human hepatocyes when needed. Furthermore, the numbers of donor livers available for hepatocyte isolation is limited by competition for their use in whole organ transplantation. Considering the cost of hepatocyte isolation and the need for immediate preparation of consistent and functional cells, it is unlikely that human hepatocytes can be obtained on such a scale to treat a large number of patients with falling liver functions. The utilization of xenogenic hepatocytes will result in additional concerns regarding transmission of infectious pathogens and immunological and physiological incompatibilities between animals and humans. An attractive alternative to primary human hepatocytes is the use of tightly regulated human hepatocyte cell lines. Such cell lines can provide the advantages of unlimited availability, sterility and uniformity. We describe here methods for creating transplantable human hepatocyte cell lines using currently available cell cultures and gene transfer technology. [source]

Functional analysis of the rat bile salt export pump gene promoter

FEBS JOURNAL, Issue 14 2002
Regulation by bile acids, drugs, endogenous compounds
The 5, flanking region of the bile salt export pump (Bsep) gene was systematically analysed to provide the basis for understanding the mechanisms which regulate Bsep transcription. In addition substrates and drugs were investigated for their ability to alter Bsep promoter activity. Bsep promoter function was restricted to hepatocyte derived HepG2 cells. The 5, deletional analysis revealed a biphasic shape of reporter gene activities, indicating a suppressive element between nucleotides ,800 and ,512. Two consensus sites for the farnesoid X receptor (FXR) were located at nucleotides ,473 and ,64. The latter was characterized as functionally active in bile acid-mediated feed-back regulation of Bsep transcription. Bsep promoter activity was reduced by rifampin and ,-estradiol. The anti-estrogen tamoxifen stimulated promoter activity. Dexamethasone, hydrocortisone and phenobarbital had no effect on Bsep promoter activity. In conclusion, the data suggest that transcriptional regulation of the Bsep gene can be modulated by a number of endogenous compounds and xenobiotics. FXR was a major regulatory factor, mediating bile acid feed-back stimulation of Bsep transcription. [source]

pH-dependent translocation of ,-tocopherol transfer protein (,-TTP) between hepatic cytosol and late endosomes

GENES TO CELLS, Issue 10 2003
Masakuni Horiguchi
Background:, ,-Tocopherol transfer protein (,-TTP), a member of the Sec14 protein family, plays an important role in transporting ,-tocopherol, a major lipid-soluble anti-oxidant, in the cytosolic compartment of hepatocytes and is known as a product of the causative gene for familial isolated vitamin E deficiency. It has been shown that the secretion of hepatocyte ,-tocopherol taken up with plasma lipoproteins is facilitated by ,-TTP. To explore the mechanism of ,-TTP mediated ,-tocopherol secretion, we investigated drugs which may affect this secretion. Results:, We found that, in a hepatocyte cell culture system, intracellular ,-tocopherol transport is impaired by chloroquine, an agent known for its function of elevating the pH in acidic compartments. Under chloroquine treatment, the diffuse cytosolic distribution of ,-TTP changes to a punctate pattern. Double-staining experiments with endocytosis markers revealed that ,-TTP accumulates transiently on the cytoplasmic surface of late endosomal membranes. This phenomenon is specific for hepatoma cell lines or primarily cultured hepatocytes. Other members of the Sec14 family, such as cellular retinaldehyde-binding protein (CRALBP) and supernatant protein factor (SPF), do not show this accumulation. Furthermore, we elucidate that the obligatory amino acid sequence for this function is located between amino acids 21 and 50, upstream of the N-terminal end of the lipid-binding domain. Conclusion:, We hypothesize that a liver-specific target molecule for ,-TTP exists on the late endosomal membrane surface. This transient binding may explain the mechanism of how ,-tocopherol is transferred from late endosomes to cytosolic ,-TTP. [source]

A Smart Nanoprobe Based On Fluorescence-Quenching PEGylated Nanogels Containing Gold Nanoparticles for Monitoring the Response to Cancer Therapy

ADVANCED FUNCTIONAL MATERIALS, Issue 6 2009
Motoi Oishi
Abstract A biocompatible, caspase-3-responsive, and fluorescence-quenching smart apoptosis nanoprobe based on a PEGylated nanogel that contains gold nanoparticles (GNPs) (fluorescence quenchers) in the cross-linked polyamine gel core and fluorescein isothiocyanate (FITC)-labeled DEVD peptides at the tethered PEG chain ends is prepared for monitoring the cancer response to therapy. FITC,DEVD,nanogel,GNP shows very little fluorescence in the absence of activated caspase-3 (normal cells) through the fluorescence resonance energy transfer (FRET) process between the GNPs and the FITC molecules, while pronounced fluorescence signals are observed in apoptotic cells because of the cleavage of the DEVD peptide by activated caspase-3 present in the cells, which results in the release of FITC molecules. Thus, remarkable quenching and dequenching of fluorescence signals in response to activated caspase-3 is observed. Apoptotic cells are detected in human hepatocyte (HuH-7) multicellular tumor spheroids (MCTSs), a commonly used three-dimensional in vitro model mimicking the in vivo biology of tumors, as early as one day post-treatment with staurosporine, an apoptosis-inducing agent; while growth inhibition (i.e., change in size) of the HuH-7 MCTSs is only observed after a delay of three days (i.e., on day 4). This demonstrates the effectiveness of the FITC,DEVD,nanogel,GNP probe as a smart nanoprobe for real-time monitoring as well as a more rapid assessment of the early response to cancer therapy. [source]

Strain-dependent viral dynamics and virus-cell interactions in a novel in vitro system supporting the life cycle of blood-borne hepatitis C virus,

HEPATOLOGY, Issue 3 2009
Hussein Hassan Aly
We developed an in vitro system that can be used for the study of the life cycle of a wide variety of blood-borne hepatitis C viruses (HCV) from various patients using a three-dimensional hollow fiber culture system and an immortalized primary human hepatocyte (HuS-E/2) cell line. Unlike the conventional two-dimensional culture, this system not only enhanced the infectivity of blood-borne HCV but also supported its long-term proliferation and the production of infectious virus particles. Both sucrose gradient fractionation and electron microscopy examination showed that the produced virus-like particles are within a similar fraction and size range to those previously reported. Infection with different HCV strains showed strain-dependent different patterns of HCV proliferation and particle production. Fluctuation of virus proliferation and particle production was found during prolonged culture and was found to be associated with change in the major replicating virus strain. Induction of cellular apoptosis was only found when strains of HCV-2a genotype were used for infection. Interferon-alpha stimulation also varied among different strains of HCV-1b genotypes tested in this study. Conclusion: These results suggest that this in vitro infection system can reproduce strain-dependent events reflecting viral dynamics and virus-cell interactions at the early phase of blood-borne HCV infection, and that this system can allow the development of new anti-HCV strategies specific to various HCV strains. (HEPATOLOGY 2009.) [source]

Human skin fibroblasts: From mesodermal to hepatocyte-like differentiation,

HEPATOLOGY, Issue 5 2007
Philippe A. Lysy
The phenotypic homology of fibroblasts and mesenchymal stem cells (MSCs) has been recently described. Our study investigated the in vitro potential of human skin fibroblasts to differentiate into mesodermal (osteocyte and adipocyte) and endodermal (hepatocyte) cell lineages by comparison with human bone marrow (hBM) MSCs. The endodermal potential of fibroblasts was then explored in vivo in a mouse model of liver injury. Fibroblasts were able to acquire osteocyte and adipocyte phenotypes as assessed by cytochemistry and gene expression analyses. After exposure to a specific differentiation cocktail, these cells presented hepatocyte-like morphology and acquired liver-specific markers on protein and gene expression levels. Furthermore, these fibroblast-derived hepatocyte-like cells (FDHLCs) displayed the ability to store glycogen and synthesize small amounts of urea. By gene expression analysis, we observed that fibroblasts remained in a mesenchymal-epithelial transition state after hepatocyte differentiation. Moreover, FDHLCs lost their hepatocyte-like phenotype after dedifferentiation. In vivo, human fibroblasts infused directly into the liver of hepatectomized severe combined immunodeficient (SCID) mice engrafted in situ and expressed hepatocyte markers (albumin, alpha-fetoprotein, and cytokeratin 18) together with the mesodermal marker fibronectin. Despite lower liver-specific marker expression, the in vitro and in vivo differentiation profile of fibroblasts was comparable to that of mesenchymal-derived hepatocyte-like cells (MDHLCs). In conclusion, our work demonstrates that human skin fibroblasts are able to display mesodermal and endodermal differentiation capacities and provides arguments that these cells share MSCs features both on the phenotypic and functional levels. (HEPATOLOGY 2007;46:1574,1585.) [source]

An inhibitor of cyclin-dependent kinase, stress-induced p21Waf-1/Cip-1, mediates hepatocyte mito-inhibition during the evolution of cirrhosis,

HEPATOLOGY, Issue 6 2005
John G. Lunz III
During the evolution of cirrhosis, there is a relative decrease in volume percentage of hepatocytes and a relative increase in biliary epithelial cells and myofibroblasts. This is recognized histopathologically as a ductular reaction and leads to gradual distortion of the normal hepatic architecture. The final or decompensated stage of cirrhosis is characterized by a further decline in hepatocyte proliferation and loss of functional liver mass that manifests clinically as ascites, encephalopathy, and other signs of liver failure. In this report, we tested the hypothesis that p21-mediated hepatocyte mito-inhibition accelerates the evolution of cirrhosis using an established mouse model of decompensated biliary cirrhosis, p21-deficient mice, and liver tissue from humans awaiting liver replacement. Despite the same insult of long-term (12-week) bile duct ligation, mice prone to decompensation showed significantly more oxidative stress and hepatocyte nuclear p21 expression, which resulted in less hepatocyte proliferation, an exaggerated ductular reaction, and more advanced disease compared with compensation-prone controls. Mice deficient in p21 were better able than wild-type controls to compensate for long-term bile duct ligation because of significantly greater hepatocyte proliferation, which led to a larger liver mass and less architectural distortion. Mito-inhibitory hepatocyte nuclear p21 expression in humans awaiting liver replacement directly correlated with pathological disease stage and model of end-stage liver disease scoring. In conclusion, stress-induced upregulation of hepatocyte p21 inhibits hepatocyte proliferation during the evolution of cirrhosis. These findings have implications for understanding the evolution of cirrhosis and associated carcinogenesis. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2005.) [source]

Altered gene expression in acute systemic inflammation detected by complete coverage of the human liver transcriptome

HEPATOLOGY, Issue 2 2004
Cédric Coulouarn
The goal of the current study was to provide complete coverage of the liver transcriptome with human probes corresponding to every gene expressed in embryonic, adult, and/or cancerous liver. We developed dedicated tools, namely, the Liverpool nylon array of complementary DNA (cDNA) probes for approximately 10,000 nonredundant genes and the LiverTools database. Inflammation-induced transcriptome changes were studied in liver tissue samples from patients with an acute systemic inflammation and from control subjects. One hundred and fifty-four messenger RNAs (mRNA) correlated statistically with the extent of inflammation. Of these, 134 mRNA samples were not associated previously with an acute-phase (AP) response. The hepatocyte origin and proinflammatory cytokine responsiveness of these mRNAs were confirmed by quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) in cytokine-challenged hepatoma cells. The corresponding gene promoters were enriched in potential binding sites for inflammation-driven transcription factors in the liver. Some of the corresponding proteins may provide novel blood markers of clinical relevance. The mRNAs whose level is most correlated with the AP extent (P < .05) were enriched in intracellular signaling molecules, transcription factors, glycosylation enzymes, and up-regulated plasma proteins. In conclusion, the hepatocyte responded to the AP extent by fine tuning some mRNA levels, controlling most, if not all, intracellular events from early signaling to the final secretion of proteins involved in innate immunity. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2004;39:353,364.) [source]

Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats

HEPATOLOGY, Issue 2 2002
Li Yin
Liver progenitor cells (LPCs) cloned from adult rat livers following allyl alcohol injury express hematopoietic stem cell and early hepatic lineage markers when cultured on feeder layers; under these conditions, neither mature hepatocyte nor bile duct, Ito, stellate, Kupffer cell, or macrophage markers are detected. These phenotypes have remained stable without aneuploidy or morphological transformation after more than 100 population doublings. When cultured without feeder layers, the early lineage markers disappear, and mature hepatocyte markers are expressed; mature hepatocytic differentiation and cell size are also augmented by polypeptide and steroidal growth factors. In contrast to hepatocytic potential, duct-like structures and biliary epithelial markers are expressed on Matrigel. Because they were derived without carcinogens or mutagens, these bipotential LPC lines provide novel tools for models of cellular plasticity and hepatocarcinogenesis, as well as lines for use in cellular transplantation, gene therapy, and bioreactor construction. [source]

Interleukin-6 from intrahepatic cells of bone marrow origin is required for normal murine liver regeneration

HEPATOLOGY, Issue 1 2002
Xavier Aldeguer
Interleukin-6 (IL-6) is required for normal liver regeneration, but the specific cellular source of this growth factor is unknown. We investigated whether this signal originates from the resident macrophage, the Kupffer cell. Using a murine model of bone marrow transplantation, we replaced recipient bone marrow,derived cells, including Kupffer cells, with cells of donor genetic phenotype. Recipients deficient in IL-6 (IL-6,/,) were lethally irradiated, then rescued with 107 donor bone marrow cells capable of expressing IL-6 (IL-6+/+). Conversely, IL-6+/+ recipients received IL-6,/, marrow. Successful engraftment was measured by the presence of the Y chromosome SRY locus in the livers of female recipients receiving male marrow, in situ IL-6 expression by Kupffer cells, and up-regulation of IL-6 in splenocytes after activation with lipopolysaccharide (LPS). Kupffer cell isolation in IL-6,/, females receiving IL-6+/+ male marrow clearly showed the presence of the SRY locus and IL-6 disrupted allele, whereas males receiving female marrow demonstrated no SRY or IL-6 signals, confirming the extent of replacement. Replacement of these cells in IL-6,/, mice with IL-6+/+ bone marrow successfully restored the regenerative response after partial hepatectomy (PHx) as indicated by signal transduction and activator of transcription 3 (STAT3) activation and hepatocyte DNA replication. Alternatively, complete replacement of Kupffer cells in IL-6+/+ mice by transplantation with IL-6,/, cells significantly inhibited liver regeneration and was partially restored by administration of IL-6. This investigation demonstrates a paracrine mechanism by which cells of bone marrow origin, most likely Kupffer cells, regulate the regenerative capacity of the hepatocyte through IL-6 expression. [source]

Hepatocyte growth factor promotes cell survival from Fas-mediated cell death in hepatocellular carcinoma cells via Akt activation and Fas-death,inducing signaling complex suppression

HEPATOLOGY, Issue 4 2000
Atsushi Suzuki
The Akt/PI-3 kinase pathway is a system essential for cell survival. In the current study, we showed that hepatocyte growth factor (HGF) activates the Akt/PI-3 kinase pathway to suppress Fas-mediated cell death in human hepatocellular carcinoma (HCC; 3 lines; SK-Hep1, HLE, and Chang Liver cell lines), hepatoblastoma (1 line; HepG2), and embryonic hepatocyte (1 line; WRL). Five tested cell lines showed the resistance to Fas-mediated cell death by the pretreatment of HGF. This HGF-induced cell survival was suppressed by wortmannin (Akt/PI-3 kinase pathway inhibitor), suggesting an involvement of Akt. When cells were pretreated with HGF, Fas-mediated cell death was suppressed, followed by Akt phosphorylation at Ser473. Fas-death,inducing signaling complex (DISC) formation, especially FADD and caspase 8 interaction, was suppressed by HGF and the suppression of the Akt/PI-3 kinase pathway by transient expression of PTEN, resulting in acquisition of Fas-DISC formation and Fas-mediated cell death in HGF-treated cells. We suggest that HGF promotes cell survival in hepatocyte-derived cell lines (HCC, hepatoblastoma, and embryonic hepatocyte) from Fas-mediated cell death via Fas-DISC suppression as a result of Akt activation. [source]

Potentiation of isoniazid-induced liver toxicity by rifampicin in a combinational therapy of antitubercular drugs (rifampicin, isoniazid and pyrazinamide) in Wistar rats: A toxicity profile study

HEPATOLOGY RESEARCH, Issue 10 2007
Sheikh Abdullah Tasduq
Aim:, Biochemical characterization of long-term toxic manifestations of anti-tubercular (anti-TB) drugs , rifampicin (RIF), isoniazid (INH) and pyrazinamide (PZA) , individually and in two combinations: (i) RIF + INH, and (ii) RIF + INH + PZA in Wistar rats. Methods:, Animals received anti-TB drugs , alone or in combination , once daily p.o. for up to 90 days (doses, in mg/kg: RIF, 250; INH, 50; PZA, 100). Assays for alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin (serum) and lipid peroxidation (LPO), glutathione (GSH), glutathione peroxidase (GPx), catalase, Na+K+-ATPase and CYP 2E1 (liver) were performed to assess liver toxicity. Clinical biochemistry was done by commercial kits. Determinations were made at 0, 15, 30 and 90 days of treatment schedule. Results:, Anti-TB drugs-treated animals showed abnormal rises or falls (>1.5,2 fold) in the serum/liver parameters. Mild hyperlipidemia, hypercholesterolemia and hyperuricemia were the other pathologies. Of all the treated groups, INHalone or in combination with other drugs produced a progressive enhancement of toxicity over 15,90 days. The in vivo results were further supported by in vitro results (MTT assay, GSH and LPO) in primary cultures of rat hepatocyte. Results indicated that anti-TB drugs in combination: (i) caused membrane damage resulting in leakage of ALT, ALP and bilirubin; (ii) caused imbalance in endogenous enzymatic oxidant,antioxidant defense via increased lipid peroxidation and in glutathione homeostasis; and (iii) enhanced the CYP 2E1-mediated bioactivation mechanism. Conclusion:, Toxicity manifestations seemed to be heptocytic injury targeted at hepatocytes, bile ducts or sinusoidal cells related to hepatitis and primary biliary cholestasis. [source]

Growth factor attenuation of IFN,-mediated hepatocyte apoptosis requires p21waf,1

INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2006
Christian T. McCullough
Summary Interferon gamma (IFN,) is an important mediator of inflammatory liver damage as part of a complex cytokine network. In vitro, IFN, induces hepatocyte apoptosis. We hypothesized that the hepatocyte response to IFN signalling is context-dependent, and that specific growth factors, via phosphatidylinositol 3 kinase (PI(3)K) and protein kinase B/Akt signalling pathways, confer a cytoprotective effect. We established an in vitro model of IFN,-mediated primary hepatocyte injury. We show that epidermal growth factor (EGF) and hepatocyte growth factor (HGF) attenuate the IFN,-induced hepatocyte apoptosis. IRF-1, but not p53, is required for IFN,-mediated apoptosis. The loss of p21waf,1 not only sensitizes the hepatocyte to IFN,-mediated injury but is required for survival factor mediated cytoprotection. We show that the PI(3)K inhibitor, LY294002, partially inhibits the apoptotic response of the hepatocyte to IFN,. In summary, we present evidence that a component of pro-apoptotic IFN, signalling in the primary hepatocyte occurs via the PI(3)K pathway. We show that the hepatocyte response to IFN, is modulated by external survival factors and that this survival signalling requires p21waf,1. [source]

The effect of IFN, on the hepatocyte: cell cycle and apoptosis

INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 6 2001
Benjamin J. Tura
The inflammatory cytokine interferon gamma (IFN,) can cause cell cycle arrest and apoptosis in the hepatocyte. Primarily these processes are protective but in chronic liver disease oncogenic mutations may prosper. IFN, signalling is discussed showing how p53 is induced to cause cell cycle arrest. While caspases are are known to be responsible for IFN, induced apoptosis, how they are activated is unclear. Potential mechanisms are reviewed. [source]

Conserved regions from Plasmodium falciparum MSP11 specifically interact with host cells and have a potential role during merozoite invasion of red blood cells

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2010
Ana Zuleima Obando-Martinez
Abstract Despite significant global efforts, a completely effective vaccine against Plasmodium falciparum, the species responsible for the most serious form of malaria, has not been yet obtained. One of the most promising approaches consists in combining chemically synthesized minimal subunits of parasite proteins involved in host cell invasion, which has led to the identification of peptides with high binding activity (named HABPs) to hepatocyte and red blood cell (RBC) surface receptors in a large number of sporozoite and merozoite proteins, respectively. Among these proteins is the merozoite surface protein 11 (MSP11), which shares important structural and immunological features with the antimalarial vaccine candidates MSP1, MSP3, and MSP6. In this study, 20-mer-long synthetic peptides spanning the complete sequence of MSP11 were assessed for their ability to bind specifically to RBCs. Two HABPs with high ability to inhibit invasion of RBCs in vitro were identified (namely HABPs 33595 and 33606). HABP-RBC bindings were characterized by means of saturation assays and Hill analysis, finding cooperative interactions of high affinity for both HABPs (nH of 1.5 and 1.2, Kd of 800 and 600,nM for HABPs 33595 and 33606, respectively). The nature of the possible RBC receptors for MSP11 HABPs was studied in binding assays to enzyme-treated RBCs and cross-linking assays, finding that both HABPs use mainly a sialic acid-dependent receptor. An analysis of the immunological, structural and polymorphic characteristics of MSP11 HABPs supports including these peptides in further studies with the aim of designing a fully effective protection-inducing vaccine against malaria. J. Cell. Biochem. 110: 882,892, 2010. © 2010 Wiley-Liss, Inc. [source]

The sequential syntheses of [76Br]FBAU 3,,5,-dibenzoate and [76Br]FBAU

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 13 2001
Chih-Hao K. Kao
Abstract Thymidine analogs labeled with positron emitting radionuclides are potential proliferation markers for positron emission tomography (PET). Bromine-76 (T1/2=16.2 h) is our choice of radionuclide, because it allows for maximal DNA incorporation of the tracer. Following the literature descriptions, 76Br was produced using the 75As (3He, 2n) 76Br reaction. We then recovered 76Br from the target in the form of [76Br]NH4Br with a yield of 60±12% (n=32). Peracetic acid was used as the oxidant for electrophilic bromodestannylation to prepare [76Br]FBAU 3,,5,-dibenzoate (71.2±12.1%, RCY) and a basic hydrolysis of the dibenzoate then yielded [76Br]FBAU. The yield of the hydrolysis reaction was 53.1±9.2% when heated at 100°C for 15 min or quantitative (decay corrected) when left at room temperature overnight. The sequential synthesis of [76Br]FBAU 3,,5,-dibenzoate and [76Br]FBAU allowed us to perform a side-by-side comparison of their metabolic stabilities. While [76Br]FBAU 3,,5,-dibenzoate was hydrolyzed to [76Br]FBAU within 10 minutes by hepatocyte at 37°C, [76Br]FBAU was stable and no [76Br]Br, was released from either radiopharmaceutical. Both compounds are potential proliferation markers for PET. Copyright © 2001 John Wiley & Sons, Ltd. [source]

Titration of hepatitis B virus infectivity in the sera of pre-acute and late acute phases of HBV infection: Transmission experiments to chimeric mice with human liver repopulated hepatocytes

JOURNAL OF MEDICAL VIROLOGY, Issue 12 2008
Ayako Tabuchi
Abstract Studies of hepatitis B virus (HBV) infection in non-human primates such as chimpanzees are no longer possible due to ethical considerations and the endangered status of chimpanzees since April 2007 in Japan. A human hepatocyte transplanted chimeric mouse was used to characterize HBV infectivity in serial stages of acute infection. Chimeric mice were inoculated intravenously with serum samples obtained from an experimentally infected chimpanzee with HBV. Sera from the pre-acute phases (i.e., rump-up viremia prior to anti-HBc) and late acute phases (i.e., declining phase of HBsAg and anti-HBcAb positive) were collected from the chimpanzees 57 and 244 days after inoculation. These sera contained 2.6,×,106 and 2.8,×,106 copies/ml of HBV DNA, respectively. Three chimeric mice inoculated intravenously with 100 µl of pre-acute serum (equivalent to 100 copy of HBV DNA) developed an HBV infection. The three chimeric mice that received 100 µl of pre-acute serum (equivalent to 101 copies of HBV DNA), developed high levels of serum HBV DNA. None of the three chimeric mice inoculated with 100 µl of 1:104 dilution (equivalent to 101 copies of HBV DNA) of late-acute serum was infected, while only one of three chimeric mice inoculated with 100 µl of 1:103 dilution (equivalent to 102 copies of HBV DNA) of late-acute serum developed an HBV infection. Based on these results, chimeric mice can be used as animal models for the study of HBV infectivity, pathogenesis and control. The results show that pre-acute phase HBV serum is about 100-times more infectious than late acute phase serum. J. Med. Virol. 80:2064,2068, 2008. © 2008 Wiley-Liss, Inc. [source]

Alcohol and Hepatitis C Virus,Interactions in Immune Dysfunctions and Liver Damage

ALCOHOLISM, Issue 10 2010
Gyongyi Szabo
Hepatitis C virus infection affects 170 million people worldwide, and the majority of individuals exposed to HCV develop chronic hepatitis leading to progressive liver damage, cirrhosis, and hepatocellular cancer. The natural history of HCV infection is influenced by genetic and environmental factors of which chronic alcohol use is an independent risk factor for cirrhosis in HCV-infected individuals. Both the hepatitis C virus and alcohol damage the liver and result in immune alterations contributing to both decreased viral clearance and liver injury. This review will capture the major components of the interactions between alcohol and HCV infection to provide better understanding for the molecular basis of the dangerous combination of alcohol use and HCV infection. Common targets of HCV and alcohol involve innate immune recognition and dendritic cells, the critical cell type in antigen presentation and antiviral immunity. In addition, both alcohol and HCV affect intracellular processes critical for hepatocyte and immune cell functions including mitochondrial and proteasomal activation. Finally, both chronic alcohol use and hepatitis C virus infection increase the risk of hepatocellular cancer. The common molecular mechanisms underlying the pathological interactions between alcohol and HCV include the modulation of cytokine production, lipopolysaccharide (LPS)-TLR4 signaling, and reactive oxygen species (ROS) production. LPS-induced chronic inflammation is not only a major cause of progressive liver injury and fibrosis, but it can also contribute to modification of the tissue environment and stem cells to promote hepatocellular cancer development. Alteration of these processes by alcohol and HCV produces an environment of impaired antiviral immune response, greater hepatocellular injury, and activation of cell proliferation and dedifferentiation. [source]

Systematic review: caspase-cleaved fragments of cytokeratin 18 , the promises and challenges of a biomarker for chronic liver disease

ALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 11-12 2009
Y. YILMAZ
Summary Background, Biomarkers hold great promise for detecting chronic liver disease without the use of liver biopsy. Aim, To review the usefulness of cytokeratin (CK) 18 fragments, a marker of hepatocyte apoptosis, to predict the presence of chronic liver injury. Methods, Available literature identified from PubMed was reviewed. Results, Levels of CK18 fragments have been shown to be elevated in hepatocellular carcinoma, viral hepatitis, alcoholic hepatitis, nonalcoholic fatty liver disease and cholestatic liver disease. In the setting of nonalcoholic fatty liver disease, CK18 fragments may distinguish nonalcoholic steatohepatitis from simple fatty liver. Conclusions, Undoubtedly, the most promising application of CK18 fragments is currently in nonalcoholic fatty liver disease, and especially for distinguishing patients with nonalcoholic steatohepatitis vs. those with simple steatosis. Further investigations and technical improvements are required to cross the boundary from research to the clinical application of CK18 fragments as a marker of chronic liver disease. [source]

Hypoxia potentiates transforming growth factor-, expression of hepatocyte during the cirrhotic condition in rat liver

LIVER INTERNATIONAL, Issue 6 2004
Won-II Jeong
Abstract: Background/Aims: Many studies have reported that hypoxia might be associated with angiogenesis and fibrogenesis, and the level of transforming growth factor-,1 (TGF-,1) was increased in fibrotic liver and maximal at cirrhosis. Therefore, we examined the expression of TGF-,1, phosphorylated-Smad2/3 (p-Smad2/3) of the TGF-, immediate down stream signaling system and hypoxic status during hepatic fibrogenesis. Methods: Fibrosis of rats was induced by carbon tetrachloride. Collagens were detected with Azan stain. Immunohistochemistry and immunoblotting was used. Results: TGF-,1 was mainly produced by hypoxic hepatocytes at cirrhosis although myofibroblasts (MFBs) and macrophages producing TGF-,1 were decreased. Moreover, distribution of p-Smad2/3 in hepatocytes was consistent with those of hypoxic hepatocytes regardless of MFBs. Furthermore, in recovery, most MFBs disappeared, whereas positive reactions of p-Smad2/3 still existed in the hepatocytes of hypoxic areas. Therefore, TGF-,1 expression in hepatocytes might have been associated with hypoxia. Conclusions: We put forward the hypothesis that TGF-,1 is mainly produced by MFBs and macrophages at early and middle stages of fibrotic processes, but it is predominantly released by hypoxic hepatocytes in the last fibrotic stage or cirrhosis. [source]

Role of circulating neurotoxins in the pathogenesis of hepatic encephalopathy: potential for improvement following their removal by liver assist devices

LIVER INTERNATIONAL, Issue 2003
Roger F. Butterworth
Abstract Both acute and chronic liver failure result in impaired cerebral function known as hepatic encephalopathy (HE). Evidence suggests that HE is the consequence of the accumulation in brain of neurotoxic and/or neuroactive substance including ammonia, manganese, aromatic amino acids, mercaptans, phenols, short-chain fatty acids, bilirubin and a variety of neuroactive medications prescribed as sedatives to patients with liver failure. Brain ammonia concentrations may attain levels in excess of 2 mm, concentrations which are known to adversely affect both excitatory and inhibitory neurotransmission as well as brain energy metabolism. Manganese exerts toxic effects on dopaminergic neurones. Prevention and treatment of HE continues to rely heavily on the reduction of circulating ammonia either by reduction of gut production using lactulose or antibiotics or by increasing its metabolism using l -ornithine- l -aspartate. No specific therapies have so far been designed to reduce circulating concentrations of other toxins. Liver assist devices offer a potential new approach to the reduction of circulating neurotoxins generated in liver failure. In this regard, the Molecular Absorbents Recirculating System (MARS) appears to offer distinct advantages over hepatocyte-based systems. [source]

High mobility group box 1 protein as a marker of hepatocellular injury in human liver transplantation

LIVER TRANSPLANTATION, Issue 10 2008
Minna Ilmakunnas
High mobility group box 1 protein (HMGB1), a cytokine actively secreted by phagocytes and passively released from necrotic cells, is an inflammatory mediator in experimental hepatic ischemia/reperfusion injury. We characterized its expression in human liver transplantation. In 20 patients, in addition to systemic samples, blood was drawn from portal and hepatic veins during and after reperfusion to assess changes within the graft. Plasma HMGB1, tumor necrosis factor , (TNF-,), and interleukin-6 (IL-6) levels were measured, and HMGB1 immunohistochemistry was performed on biopsies taken before and after reperfusion. Plasma HMGB1 was undetectable before reperfusion, and levels in systemic circulation peaked after graft reperfusion. At portal declamping, HMGB1 levels were substantially higher in the caval effluent [188 (80-371) ng/mL] than in portal venous blood [0 (0-3) ng/mL, P < 0.001]. HMGB1 release from the graft continued thereafter. HMGB1 levels were not related to TNF-, or IL-6 levels. HMGB1 expression was up-regulated in biopsies taken after reperfusion (P = 0.020), with intense hepatocyte and weak neutrophil staining. HMGB1 levels in hepatic venous blood correlated with graft steatosis (r = 0.497, P = 0.03) and peak postoperative alanine aminotransferase levels (r = 0.588, P = 0.008). Our results indicate that HMGB1 originates from the graft and is a marker of hepatocellular injury in human liver transplantation. Liver Transpl 14:1517,1525, 2008. © 2008 AASLD. [source]

Hepatic tissue engineering for adjunct and temporary liver support: Critical technologies

LIVER TRANSPLANTATION, Issue 11 2004
Christina Chan
The severe donor liver shortage, high cost, and complexity of orthotopic liver transplantation have prompted the search for alternative treatment strategies for end-stage liver disease, which would require less donor material, be cheaper, and less invasive. Hepatic tissue engineering encompasses several approaches to develop adjunct internal liver support methods, such as hepatocyte transplantation and implantable hepatocyte-based devices, as well as temporary extracorporeal liver support techniques, such as bioartificial liver assist devices. Many tissue engineered liver support systems have passed the "proof of principle" test in preclinical and clinical studies; however, they have not yet been found sufficiently reliably effective for routine clinical use. In this review we describe, from an engineering perspective, the progress and remaining challenges that must be resolved in order to develop the next generation of implantable and extracorporeal devices for adjunct or temporary liver assist. (Liver Transpl 2004;10:1331,1342.) [source]

Role of hepatocytes and bile duct cells in preservation-reperfusion injury of liver grafts

LIVER TRANSPLANTATION, Issue 5 2001
Marián Kukan
In liver transplantation, it is currently hypothesized that nonparenchymal cell damage and/or activation is the major cause of preservation-related graft injury. Because parenchymal cells (hepatocytes) appear morphologically well preserved even after extended cold preservation, their injury after warm reperfusion is ascribed to the consequences of nonparenchymal cell damage and/or activation. However, accumulating evidence over the past decade indicated that the current hypothesis cannot fully explain preservation-related liver graft injury. We review data obtained in animal and human liver transplantation and isolated perfused animal livers, as well as isolated cell models to highlight growing evidence of the importance of hepatocyte disturbances in the pathogenesis of normal and fatty graft injury. Particular attention is given to preservation time-dependent decreases in high-energy adenine nucleotide levels in liver cells, a circumstance that (1) sensitizes hepatocytes to various stimuli and insults, (2) correlates well with graft function after liver transplantation, and (3) may also underlie the preservation time-dependent increase in endothelial cell damage. We also review damage to bile duct cells, which is increasingly being recognized as important in the long-lasting phase of reperfusion injury. The role of hydrophobic bile salts in that context is particularly assessed. Finally, a number of avenues aimed at preserving hepatocyte and bile duct cell integrity are discussed in the context of liver transplantation therapy as a complement to reducing nonparenchymal cell damage and/or activation. [source]

Differential transcriptome profiling identifies Plasmodium genes encoding pre-erythrocytic stage-specific proteins

MOLECULAR MICROBIOLOGY, Issue 5 2004
Karine Kaiser
Summary Invasive sporozoite and merozoite stages of malaria parasites that infect mammals enter and subsequently reside in hepatocytes and red blood cells respectively. Each invasive stage may exhibit unique adaptations that allow it to interact with and survive in its distinct host cell environment, and these adaptations are likely to be controlled by differential gene expression. We used suppression subtractive hybridization (SSH) of Plasmodium yoelii salivary gland sporozoites versus merozoites to identify stage-specific pre-erythrocytic transcripts. Sequencing of the SSH library and matching the cDNA sequences to the P. yoelii genome yielded 25 redundantly tagged genes including the only two previously characterized sporozoite-specific genes encoding the circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Twelve novel genes encode predicted proteins with signal peptides, indicating that they enter the secretory pathway of the sporozoite. We show that one novel protein bearing a thrombospondin type 1 repeat (TSR) exhibits an expression pattern that suggests localization in the sporozoite secretory rhoptry organelles. In addition, we identified a group of four genes encoding putative low-molecular-mass proteins. Two proteins in this group exhibit an expression pattern similar to TRAP, and thus possibly localize in the sporozoite secretory micronemes. Proteins encoded by the differentially expressed genes identified here probably mediate specific interactions of the sporozoite with the mosquito vector salivary glands or the mammalian host hepatocyte and are not used during merozoite,red blood cell interactions. [source]

IFN-, gene therapy by intrasplenic hepatocyte transplantation: a novel strategy for reversing hepatic fibrosis in Schistosoma japonicum -infected mice

PARASITE IMMUNOLOGY, Issue 1 2001
Lihuang Zhang
Liver-targeted gene therapy using hepatocyte as recipient cells has recently been documented to be effective in treatment of numerous hepatic diseases, such as metabolic diseases and liver carcinoma. IFN-, elicits antipreliferative and antifibrogenic activity in a variety of mesenchymal cells, including hepatic satellite cells. To investigate the antifibrogenic response of liver gene therapy mediated by intrasplenic transplantation of gene-modified hepatocytes, normal mouse liver cell line BNL CL.2 cells were transfected with murine IFN-, gene (BNL.IFN-,) in vitro, and transplanted intrasplenically into Schistosoma japonicum -infected mice. The amounts and distribution of IFN-, (which inhibits collagen synthesis), TGF-, (which stimulates collagen synthesis) and extracellular matrix, including type I and III collagen, were detected. In mice infected with S. japonicum and then treated with BNL.IFN-,, an increase of IFN-, and decrease of TGF-,1 were detected at 20 weeks postinfection compared to untreated S. japonicum -infected mice. Immunohistochemical analysis showed that S. japonicum infection induced a marked increase of type I and III collagen synthesis. Whereas, 4 weeks after treatment with BNL.IFN-,, net synthesis rates of type I and III collagen were markedly decreased in the liver of infected mice. In addition, a decreased expression of TGF-,1 and its receptor TGF-,RII in the liver of BNL.IFN-,-treated mice was also observed. Moreover, the decrease in TGF-,1 and TGF-,RII protein approximately paralleled the decrease in their mRNA expression, which was detected by RNA dot blotting. The data indicate that intrasplenic transplantation of IFN-, gene-modified hepatocyte can be a candidate approach to treat hepatic fibrosis. [source]

Combined hepatocellular carcinoma and cholangiocarcinoma with components of mucinous carcinoma arising in a cirrhotic liver

PATHOLOGY INTERNATIONAL, Issue 4 2006
Daisaku Morita
A rare autopsy case of combined hepatocellular and cholangiocarcinoma, occurring in a 54-year-old man with liver cirrhosis, is presented. Initial laboratory data included CEA 52.1 ng/mL, DUPAN-2 1600 U/mL, AFP 2 ng/mL, and negativity for hepatitis B surface antigen, hepatitis B early antigen and hepatitis B core antibody. Ultrasonography and CT scan showed a large tumor node in the liver with ringed enhancement, swelling of several para-aortic lymph nodes, and ascites. Clinically, it was not possible to determine whether the hepatic tumor was an intrahepatic cholangiocarcinoma or a metastatic carcinoma. Histologically, the primary lesion was composed solely of hepatocellular carcinoma (HCC) with a trabecular pattern, and the intrahepatic metastases consisted of a variable admixture of HCC and cholangiocarcinoma (CC) with excessive mucin production. Interestingly, the tumor cell cluster showing a trabecular growth pattern produced mucin and had immunohistochemical expression of hepatocyte, cytokeratins 7 and 8. It is concluded that these hepatic tumor cells had both HCC and CC characters. [source]

Patterns of hepatic iron distribution in patients with chronically transfused thalassemia and sickle cell disease,

AMERICAN JOURNAL OF HEMATOLOGY, Issue 8 2009
Nilesh R. Ghugre
Patients with sickle cell disease (SCD) appear to be at lower risk of endocrinopathies and cardiac dysfunction than those with thalassemia major (TM). Circulating redox active iron is lower in these patients, possibly due to increased systemic inflammation and circulating cytokines. Hepcidin synthesis is upregulated during chronic inflammation, reducing intestinal iron absorption and promoting retention of iron in the reticuloendothelial cells. Hence, we hypothesized that livers of patients with SCD would exhibit greater iron deposition in sinusoidal spaces relative to hepatocytes and less in portal tracts when compared to patients with TM. To test this hypothesis, iron scoring analysis was performed on 70 clinically indicated liver biopsy specimens from children and young adults with the two syndromes. Sinusoidal scores were lower in around 1 of 4 patients with TM but the relative iron loading in hepatocytes, and portal tracts was identical in both diseases. Sinusoidal iron burdens saturated at low hepatic iron concentration (HIC) while hepatocyte and portal iron depots increased proportionally to HIC. Liver fibrosis was increased in patients with TM regardless of their chronic hepatitis status. Overall, liver iron distribution was relatively insensitive to differences in disease type and to the presence or absence of hepatitis. Am. J. Hematol., 2009. © 2009 Wiley-Liss, Inc. [source]