Surface Of Hepatocytes Research Articles

Structural and functional characterization of FoF1-ATP synthase on the extracellular surface of rat hepatocytes

Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-FoF1-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F1 (α/β and γ) and Fo (FoI-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF1 is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H+ and are inhibited by F1 and Fo-targeting inhibitors. We hypothesise that ecto-FoF1-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.

The hepatic uptake of VLDL in lrp−ldlr−/−vldlr−/− mice is regulated by LPL activity and involves proteoglycans and SR-BI

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp(-)ldlr(-/-)vldlr(-/-) mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp(-)ldlr(-/-)vldlr(-/-) mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.

Reduced expression of low‐density lipoprotein receptor in hepatocellular carcinoma with paraneoplastic hypercholesterolemia

Hepatocellular carcinoma (HCC) is frequently associated with paraneoplastic hypercholesterolemia. In familial hypercholesterolemia, genetic mutation of the low-density lipoprotein (LDL) receptor gene has been recognized as being a pathogenesis of the disease. We investigate the expression of a LDL receptor protein and gene abnormalities of a LDL receptor in HCC cells in cases with paraneoplastic hypercholesterolemia. Eleven patients with HCC associated with paraneoplastic hypercholesterolemia and seven patients with HCC who did not have hypercholesterolemia were studied. Paraffin-embedded tissues were obtained at operative resection, autopsy, or biopsy. Immunohistochemistry was performed using a monoclonal antibody against human LDL receptors. Confocal laser-scanning microscopy was used to observe the FITC-labeled LDL receptor. DNA samples were extracted from paraffin-embedded tissues. Since a LDL receptor gene is located on chromosome 19p13.2, a microsatellite marker, D19S413, was used to analyze the chromosomes. Immunoreactive LDL receptors were observed all over the surface of non-tumorous hepatocytes. However, expression of the LDL receptor was significantly decreased in all HCC cells derived from the 11 patients with hypercholesterolemia. In contrast, the expression was retained in the HCC cells of all patients without hypercholesterolemia. In two patients with hypercholesterolemia, DNA analysis revealed a loss of heterozygosity on chromosome 19p13.2. We demonstrated reduced expression of the LDL receptor in HCC cases with paraneoplastic hypercholesterolemia. LDL receptor genes with genomic disorders may cause decreased expression of the LDL receptor protein, leading to feed-back failure of the cholesterol regulation system, as seen in familial hypercholesterolemia. This is the first report considering the mechanism behind the development of paraneoplastic hypercholesterolemia in HCC.

Cytochrome P450IID6 recognized by LKM1 antibody is not exposed on the surface of hepatocytes.

LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepatocytes with LKM1-positive serum showed slight, if any, cell membrane staining using immunofluorescence, immunoperoxidase and immunoelectron microscopic studies. No staining of the plasma membrane of human hepatocytes was observed when incubations were carried out with immunoaffinity-purified antibody directed against peptide 254-271, the main epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hamster ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by protein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic side of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell membranes did not show the presence of a 50-kD molecule that could have reacted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal sequence of the P450IID6 molecule that could explain a translocation of the molecule to the luminal side of the ER, allowing its expression on the cell surface. These results indicate that, in all likelihood, P450IID6 molecule is not present on the cell surface of normal rat and human hepatocytes. Other mechanisms than antibody-mediated cell lysis directed against membrane P450IID6 antigenic determinants must be found to account for the destruction of hepatocytes observed in this disease.

Antibody-directed complement-mediated cytotoxicity to hepatocytes from patients with chronic hepatitis B.

The susceptibility of hepatocytes from patients with chronic hepatitis B to complement-dependent cytotoxicity mediated by heterologous antibodies to hepatitis B virus core (anti-HBc) and surface (anti-HBs) antigens and to hepatic asialoglycoprotein receptor was examined using a microcytotoxicity assay. The anti-HBc-induced cytotoxicity was found to be markedly enhanced against hepatocytes isolated from patients with chronic active hepatitis (72.6 +/- 9.5% (mean +/- s.e.m.); n = 6) over that against hepatocytes from individuals with chronic persistent hepatitis or inactive liver cirrhosis (40.6 +/- 18.6%; n = 4) (P = 0.019). Overall, values of the anti-HBc-directed cytotoxicity were higher in patients positive for HBcAg in hepatocytes and seropositive for hepatitis B virus e antigen (HBeAg). Hepatocytotoxicity was also exerted by anti-HBs and anti-asialoglycoprotein receptor antibodies in the presence of complement, but it was not seemingly related to disease activity. These results indicate that hepatitis B virus core and surface antigens and asialoglycoprotein receptor at the hepatocyte surface can be recognized by antibodies, and raise the possibility that complement-dependent cytolysis may contribute to the injury of hepatitis B virus-infected hepatocytes. The data also suggest that liver cells of patients with severe chronic hepatitis might be more susceptible to anti-HBc antibody-directed complement-mediated cytotoxicity than those with inactive liver histology.

Hepatitis resulting from liver-specific expression and recognition of self-antigen

Liver-specific immune reactivity in response to aberrant expression of antigen on the surface of hepatocytes is thought to be a major factor in development of autoimmune hepatitis (AIH). Persistent inflammation develops when these antigens are not eliminated and/or responses are not appropriately regulated. We have developed transgenic mice (OVA-HEP), which express chicken ovalbumin on the surface of hepatocytes. These mice are tolerant to ovalbumin, develop normally and have shown no evidence of liver or other disease up to 2 years of age. Adoptive transfer of naïve ovalbumin-specific T cells into OVA-HEP transgenic mice led to liver-specific inflammation in a dose dependent manner. This hepatic necroinflammation was dependent upon CD8 + Vα2 OVA-specific T cells, was limited to the liver, and was augmented by OVA-specific CD4 + T cell help; but did not result from adoptive transfer of ovalbumin-specific CD4 T cells alone. The response was self-limited but persistent inflammation developed after repeated transfer of antigen-specific T cells. This model of T cell recognition of antigen on hepatocytes may be used to understand many liver-specific aspects of the immune response in autoimmune hepatitis.

Ashwell that ends well

A study in Nature Medicine suggests that targeting the Ashwell receptor, a hepatocyte surface protein, or administering bacterial neuraminidase, an enzyme that facilitates the receptor's binding of clotting factors, may be potential treatments for sepsis. The efficacy of these approaches in preventing sepsis-induced systemic clotting, however, must still be explored.

Nutritional regulation of intestine morphology in larval cyprinid fish, silver bream (Vimba vimba)

The present study includes the evaluation of morphological changes in the digestive tract of larval, stomachless fish silver bream (Vimba vimba) fed with various diets – live Artemia nauplii, commercial feed Aglo Norse (NOR) and semi-purified formulated diets: casein–gelatin (CG), dipeptide-protein (50P), dipeptide (100P), no-arginine dipeptide diet (100Pw/oArg) and a free amino acid (FAA) mixture diet. The supranuclear area of enterocytes in the posterior intestine contained enlarged absorptive vacuoles in the FAA, 100P and 100Pw/oArg groups, compared with the remaining groups. Hepatocytes' cytoplasm in fish fed with FAA, 100P and 100Pw/oArg contained mainly glycogen, and no lipid vacuoles were found. Fish fed with 100Pw/oArg showed the lowest hepatocyte surface areas while in those fed with 50P, the largest nuclei diameters were observed. Fish fed with Artemia, NOR and CG diets showed significantly (P<0.05) higher number of proliferating cells compared with the remaining groups. Chromogranin A staining showed endocrine-immunoreactive cells (CgA-IR) in the taste buds in the oral cavity and in the enterocytes' supranuclear areas of the anterior and posterior intestine. We conclude that the growth rate and histological examination of the digestive tract in the 50P group of silver bream showed no nutritional deficiency.

New Insights into Hepatitis B and Hepatitis Delta Virus Entry

Notwithstanding the medical importance of the HBV infection, our understanding of how this pathogen enters hepatocytes is incomplete. This reflects a long-lasting dependence of in vitro infection studies solely on primary human hepatocytes, which are difficult to obtain and maintain in a susceptible state. The establishment of a polarizable HBV-susceptible human hepatoma cell line (HepaRG) and the utilization of Tupaia belangeri hepatocytes (PTHs) resolved this issue. Since then, important insight into viral and cellular determinants participating in HBV binding and infection have been achieved. We now know that the large viral surface protein (L) plays a pivotal role in HBV entry. It mediates diverse functions, commencing binding of virions to heparan sulfate proteoglycans at the hepatocytes surface as a prerequisite for entry. Subsequently, (a) highly specific event(s) involving the myristoylated N-terminal preS1 subdomain of L, as well as the cytosolic and antigenic loops of the S-domain, initiates a series of less well understood steps, resulting in a pH independent, reduction-sensitive fusion of the viral membrane with a cellular membrane. One of these steps is highly sensitive to synthetic N-acylated preS1 lipopeptides and can be blocked in vitro and in vivo at picomolar concentrations. This opens novel therapeutic options addressing virus entry. Future approaches aiming at the elucidation of HBV hepatotropism, the identification of (a) specific receptor(s), the clarification of the endocytic entry pathway and imaging of fluorescently-labeled virions will allow us to decipher more precisely the HBV entry pathway in the near future. Furthermore, clinical efficacy studies with HBV–preS-derived lipopeptides will tell us whether entry inhibition is a passable way to defend acute and chronic HBV and hepatitis delta virus infections.

Atherogenic diet leads to posttranslational down-regulation of murine hepatocyte SR-BI expression

The scavenger receptor class B, type I (SR-BI) mediates selective cholesteryl ester uptake by the liver and thereby exerts a systemic anti-atherogenic effect. It is unknown, however, whether exogenous cholesterol contributes to the regulation of hepatic SR-BI expression. Atherosclerosis resistant SJL and susceptible C57Bl/6 mice were challenged with atherogenic diets that lead to an increase of both plasma and liver cholesterol. The increase of cholesterol was accompanied by a marked posttranslational down-regulation of liver SR-BI expression. On average, we observed a threefold decrease of SR-BI protein while mRNA levels were slightly increased in both strains. Mice with transgenic over-expression of the sterol regulatory element binding proteins (SREBPs) were used as a model that displays endogenously increased liver cholesterol concentrations. In SREBP-1a and -1c transgenic mice on normal chow, SR-BI was transcriptionally down-regulated. When fed the atherogenic diet, the posttranslational SR-BI regulation was still functional on this background. Down-regulation of SR-BI did not alter the hepatocyte surface expression pattern, but was accompanied by a decrease of PDZK1, an adaptor protein recently identified to control SR-BI protein expression levels in the liver and intestine. Taken together, a diet-induced increase of plasma and hepatic cholesterol levels leads to a posttranscriptional down-regulation of SR-BI in mouse liver via a mechanism likely involving PDZK1.

The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer

Fenestrae allow the passage of gene transfer vectors from the sinusoidal lumen to the surface of hepatocytes. We have previously shown that the diameter of fenestrae correlates with species and strain differences of transgene expression following intravenous adenoviral transfer. In the current study, we demonstrate that the diameter of fenestrae in humans without liver pathology is 107+/-1.5 nm. This is similar to the previously reported diameter in New Zealand White (NZW) rabbits (103+/-1.3 nm) and is significantly smaller than in C57BL/6 mice (141+/-5.4 nm) and Sprague-Dawley rats (161+/-2.7 nm). We show that the diameter of fenestrae in one male NZW rabbit and its offspring characterized by a more than 50-fold increase of transgene expression after adenoviral gene transfer is significantly (113+/-1.5 nm; P<0.001) larger than in control NZW rabbits. In vitro filtration experiments using polycarbonate filters with increasing pore sizes demonstrate that a relatively small increment of the diameter of pores potently enhances passage of adenoviral vectors, consistent with in vivo data. In conclusion, the small diameter of fenestrae in humans is likely to be a major obstacle for hepatocyte transduction by adenoviral vectors.

Human LDL Receptor Enhances Sequestration of ApoE4 and VLDL Remnants on the Surface of Hepatocytes but Not Their Internalization in Mice

In humans, apolipoprotein (apo) E4 is associated with elevated plasma cholesterol levels and a high risk of developing atherosclerosis, whereas apoE2 is protective. Here we investigate the mechanism by which mice expressing human apoE isoforms recapitulate this association when they also express high levels of human low-density lipoprotein receptor (LDLR). Primary hepatocytes from apoE4 mice secreted less apoE into the medium than hepatocytes from apoE2 mice. Increased LDLR expression decreased this secretion and increased degradation of apoE4. An apoE4-GFP fusion protein expressed in the liver of apoE-deficient mice accumulated on the hepatocyte surface bordering the space of Disse in an LDLR-dependent manner. Fluorescence-labeled very low-density lipoprotein (VLDL) remnants accumulated on the hepatocyte surface in apoE4 mice with high LDLR, but they were internalized poorly. In contrast, apoE2-GFP did not accumulate on the hepatocyte surface even when the LDLR expression was high, but apoE2 mice with high LDLR internalized the remnants avidly without sequestering them on the hepatocyte surface. The high affinity of apoE4 to the LDLR enhances VLDL sequestration on the hepatocyte surface but delays their internalization. This delay likely increases VLDL conversion to cholesterol-enriched remnants in apoE4 mice with high LDLR, and probably to LDL in humans with apoE4.

Mechanism of Action of Niacin

Nicotinic acid (niacin) has long been used for the treatment of lipid disorders and cardiovascular disease. Niacin favorably affects apolipoprotein (apo) B-containing lipoproteins (eg, very-low-density lipoprotein [VLDL], low-density lipoprotein [LDL], lipoprotein[a]) and increases apo A-I-containing lipoproteins (high-density lipoprotein [HDL]). Recently, new discoveries have enlarged our understanding of the mechanism of action of niacin and challenged older concepts. There are new data on (1) how niacin affects triglycerides (TGs) and apo B-containing lipoprotein metabolism in the liver, (2) how it affects apo A-I and HDL metabolism, (3) how it affects vascular anti-inflammatory events, (4) a specific niacin receptor in adipocytes and immune cells, (5) how niacin causes flushing, and (6) the characterization of a niacin transport system in liver and intestinal cells. New findings indicate that niacin directly and noncompetitively inhibits hepatocyte diacylglycerol acyltransferase-2, a key enzyme for TG synthesis. The inhibition of TG synthesis by niacin results in accelerated intracellular hepatic apo B degradation and the decreased secretion of VLDL and LDL particles. Previous kinetic studies in humans and recent in vitro cell culture findings indicate that niacin retards mainly the hepatic catabolism of apo A-I (vs apo A-II) but not scavenger receptor BI-mediated cholesterol esters. Decreased HDL-apo A-I catabolism by niacin explains the increases in HDL half-life and concentrations of lipoprotein A-I HDL subfractions, which augment reverse cholesterol transport. Initial data suggest that niacin, by inhibiting the hepatocyte surface expression of beta-chain adenosine triphosphate synthase (a recently reported HDL-apo A-I holoparticle receptor), inhibits the removal of HDL-apo A-I. Recent studies indicate that niacin increases vascular endothelial cell redox state, resulting in the inhibition of oxidative stress and vascular inflammatory genes, key cytokines involved in atherosclerosis. The niacin flush results from the stimulation of prostaglandins D(2) and E(2) by subcutaneous Langerhans cells via the G protein-coupled receptor 109A niacin receptor. Although decreased free fatty acid mobilization from adipose tissue via the G protein-coupled receptor 109A niacin receptor has been a widely suggested mechanism of niacin to decrease TGs, physiologically and clinically, this pathway may be only a minor factor in explaining the lipid effects of niacin.

CD58 expression of liver tissue in patients with chronic hepatitis B virus infection

Several kinds of intercellular adhesion molecules closely relate to hepatitis B. The complex of CD(2) and CD58 plays an important role in enhancing the adhesion of T lymphocytes to target cells, hyperplasia and activation of T lymphocytes. In this study, we explored the relationship between the expression of CD58 in liver tissue and chronic hepatitis B infection. We determined the expression of the CD58 molecule on the surface of hepatocytes by using immunohistochemistry and the levels of serum HBV DNA from patients with HBV infection and from normal controls. The biochemical parameters of hepatic function were analyzed as well. CD58 expression in hepatocytes significantly increased with the severity progression of chronic HBV infection. The IOD levels (log10) of CD58 in the control, mild, moderate, and severe chronic HBV infection groups were 0, (7.20+/-4.64) x 10(3), (25.63+/-7.41) x 10(3) and (37.47+/-11.17) x 10(3) respectively (P<0.05 compared with the control group, respectively). CD58 probably increases cell mediated immunity to eliminate hepatitis B virus and leads to damage of hepatocytes.

Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains

Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocytes. The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12- as well as 10-transmembrane domain structures. Which of oatp1a1's four potential N-linked glycosylation sites are actually glycosylated and their influence on transport function have not been investigated in a mammalian system. In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10- from a 12-transmembrane domain model. To map glycosylation sites, the asparagines at the each of the four N-linked glycosylation consensus sites were mutagenized to glutamines. Mutagenized oatp1a1 constructs were expressed in HeLa cells, and effects on protein expression and transport activity were assessed. These studies revealed that oatp1a1 is a 12-transmembrane-domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492, whereas the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain. Constructs in which more than one glycosylation site were eliminated had reduced transport activity but not necessarily reduced transporter expression. This was in accord with the finding that fully unglycosylated oatp1a1 was well expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression.

Human LDL Receptor Enhances Sequestration of ApoE4 and VLDL Remnants on the Surface of Hepatocytes but Not Their Internalization in Mice

Human LDL Receptor Enhances Sequestration of ApoE4 and VLDL Remnants on the Surface of Hepatocytes but Not Their Internalization in Mice

Hepatitis C virus receptor expression in normal and diseased liver tissue

The principal site of hepatitis C virus (HCV) replication is the liver. HCV pseudoparticles infect human liver derived cell lines and this suggests that liver-specific receptors contribute to defining HCV hepatotropism. At least three host cell molecules have been reported to be important for HCV entry: the tetraspanin CD81, scavenger receptor class B member I (SR-BI), and the tight junction (TJ) protein Claudin 1 (CLDN1). Hepatocytes in liver tissue coexpress CD81, SR-BI, and CLDN1, consistent with their ability to support HCV entry. CLDN1 localized at the apical-canalicular TJ region and at basolateral-sinusoidal hepatocyte surfaces in normal tissue and colocalized with CD81 at both sites. In contrast, CLDN1 appeared to colocalize with SR-BI at the basolateral-sinusoidal surface. CLDN1 expression was increased on basolateral hepatocyte membranes in HCV-infected and other chronically inflamed liver tissue compared with normal liver. In contrast, CLDN4 hepatocellular staining was comparable in normal and diseased liver tissue. HCV infection of Huh-7.5 hepatoma cells in vitro significantly increased CLDN1 expression levels, consistent with a direct modulation of CLDN1 by virus infection. In HCV infected livers, immunohistochemical studies revealed focal patterns of CLDN1 staining, suggesting localized areas of increased CLDN1 expression in vivo which may potentiate local viral spread within the liver.

Intracellular Recycling and Iron Donating Ability of Transferrin Receptor 2α Protein Expressed in Transferrin Receptor-Deficient Chinese Hamster Ovary Cells.

[Introduction] Transferrin receptor 2 (TfR2), a homologue of the transferrin receptor 1 (TfR1), is found in two isoforms, α and β. Like the classical TfR1, TfR2α is a type II membrane protein expressing mainly in the hepatocytes as homodimer on their cell surface, but the β form lacks intracellular and transmembrane portions and therefore is likely to be an intracellular protein. Although the main physiological functions of these two isoforms are still not fully understood, TfR2α binds transferrin (Tf), and is therefore thought likely to be involved in cellular iron metabolism. Disruption of TfR2 leads to hemochromatosis, implying that the function of TfR2 might be a regulation of iron metabolism. However, TfR1 have remarkably high binding affinity to diferric Tf and only little expression on the cell surface of hepatocytes so that TfR1 must be fully saturated under physiological conditions. Besides, TfR2 is upregulated in some conditions in that iron accumulation was observed, for instance in the liver of the patients of hepatitis C, indicating that TfR2 might also function as the iron donator for the hepatocytes. The aim of the present study was to investigate the interactions of TfR2α with Tf, and to elucidate whether TfR2α have the ability of iron donation to the cells.[Methods] To investigate the functional properties of TfR2α, we expressed TfR2α protein with FLAG-tagging in transferrin receptor-deficient Chinese hamster ovary (CHO) cells (TRVb). The stably transfected cells expressing TfR2α-FLAG were applied for the 125I -labeled Tf (125I -Tf) binding study at 4 °C. The cells were applied for Tf and iron uptake study using 125I -Tf and 59Fe -loaded at 37 °C. To determine the fate of Tf internalized into the cells, the release of internalized Tf were then investigated. Degradation of released Tf was also investigated by precipitation using trichloroacetic acid/phosphotungustic acid.[Results] The association constant for binding of 125I-Tf to TfR2α was calculated to be 5.6 × 106 M−1 from non-linear least squares curve fitting to a saturable binding isotherm, which is about 50 times lower than that of TfR1. Although CHO cells showed a receptor-independent non-specific association with Tf at 37 °C, we observed cell-associated Tf persisting after acid-washing in TfR2α overexpressing cells, confirming that the existence of Tf internalization via TfR2α. After internalization, efflux of Tf, without substantial degradation, is also confirmed. Thus, Tf internalized by TfR2α, and presumably recycles, like TfR1 pathway. Overexpressed TfR2α protein was also shown to mediate iron uptake although its rate of iron donation is slower than TfR1.[Discussion and Conclusions] TfR2α binds to Tf although its affinity for Tf is low, and TfR2α possesses iron donating ability to the cells when TfR2α is over-expressed in our study. Although TfR2α has been reported to be involved in iron sensing and regulation of hepcidin expression, the involvement of the Tf binding and iron donating properties of TfR2α should be considered when the pathophysiology of iron metabolism is investigated, especially in the conditions that TfR2α is upregulated, for instance in hepatitis C.

Lack of hepatocellular CD10 along bile canaliculi is physiologic in early childhood and persistent in Alagille syndrome

Many tissues, including hepatobiliary cells, express neutral endopeptidase (CD10), encoded by MME. Serum neutral endopeptidase activity (NEA) has been recommended as a marker of cholestasis in adults but not in children with Alagille syndrome (AGS). We investigated ontogenic and disease-related differences in the expression of CD10. CD10 was found on canalicular surfaces of hepatocytes throughout the lobule in 16 adults and in 31 children aged ≥24 months, with and without cholestasis, but not in 39 children aged <24 months, with and without cholestasis. Ten AGS children aged 2 months to 6 years lacked any canalicular CD10 expression. Cholangiocyte apices and/or intrasinusoidal granulocytes marked for CD10 in all subjects. Liver membrane fractions from a child with cholestasis aged <24 months and from 2 AGS patients aged >24 months contained reduced levels of CD10. In contrast, AGS children and all controls expressed CD10 similarly on granulocytes. MME mRNA was found in the liver of children aged <24 months and of adults, all with cholestasis, and of AGS patients. Granulocyte MME mRNA levels were similar among all study subjects; however, liver MME mRNA levels were 6- to 140-fold less than in normal adults in all cholestatic subjects, including AGS children. Methylation of the MME promoter was not detected in the liver of AGS children. In conclusion, hepatocytes in early childhood physiologically lack immunohistochemically detectable CD10. Reduced MME mRNA in AGS is not due to MME promoter methylation. Liver CD10 in childhood appears to undergo reduced synthesis or rapid degradation, which persists in AGS. Absence of CD10 expression thus may limit NEA as a marker of cholestasis in young patients and in AGS.

Liver histopathology and accumulation of melano‐macrophage centres in Hoplias malabaricus after long‐term food deprivation and re‐feeding

In the Neotropical traíra Hoplias malabaricus, hepatocyte surface area declined after 30 days of fasting due to reserve utilization. Changes in normal organization of liver were not confirmed until 180 days of fasting. Severe histopathological changes occurred after 240 days. Pigment accumulation in the hepatocytes and increase in number and size (surface area) of melano‐macrophage centres (MMC) were also verified during long‐term food deprivation. The melano‐macrophages were rich in ferric compounds, probably haemosiderin. This suggests that the activity of hepatic macrophages is related to the intense erythrocyte degradation that occurred in traíra following long‐term food deprivation. After re‐feeding for 30 days, the liver presented a partial restoration, but the large MMC remained. When compared to the respective starved group, the hepatocytes of re‐fed fish increased in size, revealing recovering cell activity and storage of some energy reserves.