Effective Hepatocyte Transplantation Using Rat Hepatocytes with Low Asialoglycoprotein Receptor Expression

PubMed Central

Ise, Hirohiko; Nikaido, Toshio; Negishi, Naoki; Sugihara, Nobuhiro; Suzuki, Fumitaka; Akaike, Toshihiro; Ikeda, Uichi

2004-01-01

Development of a reliable method of isolating highly proliferative potential hepatocytes provides information crucial to progress in the field of hepatocyte transplantation. The aim of this study was to develop reliable hepatocyte transplantation using highly proliferative, eg, progenitor-like hepatocytes, based on asialoglycoprotein receptor (ASGPR) expression levels for hepatocyte transplantation. We have previously reported that mouse hepatocytes with low ASGPR expression levels have highly proliferative potential and can be used as progenitor-like hepatocytes. We therefore fractionated F344 male rat hepatocytes expressing low and high levels of ASGPR and determined the liver repopulation capacity of hepatocytes according to low and high ASGPR expression in the liver. Next, 2 × 105 cells of each type were transplanted into female liver regenerative model dipeptidyl peptidase-deficient rats, and we estimated the rate of liver repopulation by the transplanted hepatocytes in the host liver, as determined by recognition of the Sry gene on the Y-chromosome. At 60 days after hepatocyte transplantation, the transplanted hepatocytes occupied ∼76% of the total hepatocyte mass in the case of the transplantation of hepatocytes with low ASGPR expression, but accounted for ∼12% and 17% of the mass in the case of the transplantation of hepatocytes with high ASGPR expression and unfractionated hepatocytes, respectively. In conclusion, these findings suggest that hepatocytes with low ASGPR expression can result in normal liver function and a high repopulation capacity in vivo. These results provide insight into development of a strategy for effective liver repopulation using transplanted hepatocytes. PMID:15277224

Effective hepatocyte transplantation using rat hepatocytes with low asialoglycoprotein receptor expression.

PubMed

Ise, Hirohiko; Nikaido, Toshio; Negishi, Naoki; Sugihara, Nobuhiro; Suzuki, Fumitaka; Akaike, Toshihiro; Ikeda, Uichi

2004-08-01

Development of a reliable method of isolating highly proliferative potential hepatocytes provides information crucial to progress in the field of hepatocyte transplantation. The aim of this study was to develop reliable hepatocyte transplantation using highly proliferative, eg, progenitor-like hepatocytes, based on asialoglycoprotein receptor (ASGPR) expression levels for hepatocyte transplantation. We have previously reported that mouse hepatocytes with low ASGPR expression levels have highly proliferative potential and can be used as progenitor-like hepatocytes. We therefore fractionated F344 male rat hepatocytes expressing low and high levels of ASGPR and determined the liver repopulation capacity of hepatocytes according to low and high ASGPR expression in the liver. Next, 2 x 10(5) cells of each type were transplanted into female liver regenerative model dipeptidyl peptidase-deficient rats, and we estimated the rate of liver repopulation by the transplanted hepatocytes in the host liver, as determined by recognition of the Sry gene on the Y-chromosome. At 60 days after hepatocyte transplantation, the transplanted hepatocytes occupied approximately 76% of the total hepatocyte mass in the case of the transplantation of hepatocytes with low ASGPR expression, but accounted for approximately 12% and 17% of the mass in the case of the transplantation of hepatocytes with high ASGPR expression and unfractionated hepatocytes, respectively. In conclusion, these findings suggest that hepatocytes with low ASGPR expression can result in normal liver function and a high repopulation capacity in vivo. These results provide insight into development of a strategy for effective liver repopulation using transplanted hepatocytes.

Kupffer cells induce Notch-mediated hepatocyte conversion in a common mouse model of intrahepatic cholangiocarcinoma

PubMed Central

Terada, Maiko; Horisawa, Kenichi; Miura, Shizuka; Takashima, Yasuo; Ohkawa, Yasuyuki; Sekiya, Sayaka; Matsuda-Ito, Kanae; Suzuki, Atsushi

2016-01-01

Intrahepatic cholangiocarcinoma (ICC) is a malignant epithelial neoplasm composed of cells resembling cholangiocytes that line the intrahepatic bile ducts in portal areas of the hepatic lobule. Although ICC has been defined as a tumor arising from cholangiocyte transformation, recent evidence from genetic lineage-tracing experiments has indicated that hepatocytes can be a cellular origin of ICC by directly changing their fate to that of biliary lineage cells. Notch signaling has been identified as an essential factor for hepatocyte conversion into biliary lineage cells at the onset of ICC. However, the mechanisms underlying Notch signal activation in hepatocytes remain unclear. Here, using a mouse model of ICC, we found that hepatic macrophages called Kupffer cells transiently congregate around the central veins in the liver and express the Notch ligand Jagged-1 coincident with Notch activation in pericentral hepatocytes. Depletion of Kupffer cells prevents the Notch-mediated cell-fate conversion of hepatocytes to biliary lineage cells, inducing hepatocyte apoptosis and increasing mortality in mice. These findings will be useful for uncovering the pathogenic mechanism of ICC and developing prevenient and therapeutic strategies for this refractory disease. PMID:27698452

Role of YAP activation in nuclear receptor CAR-mediated proliferation of mouse hepatocytes.

PubMed

Abe, Taiki; Amaike, Yuto; Shizu, Ryota; Takahashi, Miki; Kano, Makoto; Hosaka, Takuomi; Sasaki, Takamitsu; Kodama, Susumu; Matsuzawa, Atsushi; Yoshinari, Kouichi

2018-06-08

Constitutive androstane receptor (CAR) is a xenobiotic-responsive nuclear receptor that is highly expressed in the liver. CAR activation induces hepatocyte proliferation and hepatocarcinogenesis in rodents, but the mechanisms remain unclear. In this study, we investigated the association of CAR-dependent cell proliferation with Yes-associated protein (YAP), which is a transcriptional cofactor controlling organ size and cell growth through the interaction with various transcriptional factors including TEAD. In mouse livers, TCPOBOP (a mouse CAR activator) treatment increased the nuclear YAP accumulation and mRNA levels of YAP target genes as well as cell-cycle related genes along with liver hypertrophy and verteporfin (an inhibitor of YAP/TEAD interaction) cotreatment tended to attenuate them. Furthermore, in cell-based reporter gene assays, CAR activation enhanced the YAP/TEAD-dependent transcription. To investigate the role of YAP/TEAD activation in the CAR-dependent hepatocyte proliferation, we sought to establish an in vitro system completely reproducing CAR-dependent cell proliferation. Since CAR was only slightly expressed in cultured mouse primary hepatocytes compared to mouse livers and no proliferation was observed after treatment with TCPOBOP, we overexpressed CAR using mouse CAR expressing adenovirus (Ad-mCAR-V5) in mouse primary hepatocytes. Ad-mCAR-V5 infection and TCPOBOP treatment induced hepatocyte proliferation. Similar results were obtained with immortalized normal mouse hepatocytes as well. In the established in vitro system, CAR-dependent proliferation was strongly inhibited by Yap knockdown and completely abolished by verteporfin treatment. Our present results obtained in in vivo and in vitro experiments suggest that YAP/TEAD activation plays key roles in CAR-dependent proliferation of murine hepatocytes.

Hanging Drop, A Best Three-Dimensional (3D) Culture Method for Primary Buffalo and Sheep Hepatocytes.

PubMed

Shri, Meena; Agrawal, Himanshu; Rani, Payal; Singh, Dheer; Onteru, Suneel Kumar

2017-04-26

Livestock, having close resemblance to humans, could be a better source of primary hepatocytes than rodents. Herein, we successfully developed three-dimensional (3D) culturing system for primary sheep and buffalo hepatocytes. The 3D-structures of sheep hepatocytes were formed on the fifth-day and maintained until the tenth-day on polyHEMA-coated plates and in hanging drops with William's E media (HDW). Between the cultured and fresh cells, we observed a similar expression of GAPDH, HNF4α, ALB, CYP1A1, CK8 and CK18. Interestingly, a statistically significant increase was noted in the TAT, CPS, AFP, AAT, GSP and PCNA expression. In buffalo hepatocytes culture, 3D-like structures were formed on the third-day and maintained until the sixth-day on polyHEMA and HDW. The expression of HNF4α, GSP, CPS, AFP, AAT, PCNA and CK18 was similar between cultured and fresh cells. Further, a statistically significant increase in the TAT and CK8 expression, and a decrease in the GAPDH, CYP1A1 and ALB expression were noted. Among the culture systems, HDW maintained the liver transcript markers more or less similar to the fresh hepatocytes of the sheep and buffalo for ten and six days, respectively. Taken together, hanging drop is an efficient method for 3D culturing of primary sheep and buffalo hepatocytes.

Hepatocyte-specific deletion of hepatocyte nuclear factor-4α in adult mice results in increased hepatocyte proliferation.

PubMed

Walesky, Chad; Gunewardena, Sumedha; Terwilliger, Ernest F; Edwards, Genea; Borude, Prachi; Apte, Udayan

2013-01-01

Hepatocyte nuclear factor-4α (HNF4α) is known as the master regulator of hepatocyte differentiation. Recent studies indicate that HNF4α may inhibit hepatocyte proliferation via mechanisms that have yet to be identified. Using a HNF4α knockdown mouse model based on delivery of inducible Cre recombinase via an adeno-associated virus 8 viral vector, we investigated the role of HNF4α in the regulation of hepatocyte proliferation. Hepatocyte-specific deletion of HNF4α resulted in increased hepatocyte proliferation. Global gene expression analysis showed that a majority of the downregulated genes were previously known HNF4α target genes involved in hepatic differentiation. Interestingly, ≥500 upregulated genes were associated with cell proliferation and cancer. Furthermore, we identified potential negative target genes of HNF4α, many of which are involved in the stimulation of proliferation. Using chromatin immunoprecipitation analysis, we confirmed binding of HNF4α at three of these genes. Furthermore, overexpression of HNF4α in mouse hepatocellular carcinoma cells resulted in a decrease in promitogenic gene expression and cell cycle arrest. Taken together, these data indicate that, apart from its role in hepatocyte differentiation, HNF4α actively inhibits hepatocyte proliferation by repression of specific promitogenic genes.

Increased reprogramming of human fetal hepatocytes compared with adult hepatocytes in feeder-free conditions.

PubMed

Hansel, Marc C; Gramignoli, Roberto; Blake, William; Davila, Julio; Skvorak, Kristen; Dorko, Kenneth; Tahan, Veysel; Lee, Brian R; Tafaleng, Edgar; Guzman-Lepe, Jorge; Soto-Gutierrez, Alejandro; Fox, Ira J; Strom, Stephen C

2014-01-01

Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes, and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome, type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors, while fetal hepatocytes could be reprogrammed with three (OCT4, SOX2, NANOG) or four factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation.

Comparison of the effects of sodium phenobarbital in wild type and humanized constitutive androstane receptor (CAR)/pregnane X receptor (PXR) mice and in cultured mouse, rat and human hepatocytes.

PubMed

Haines, Corinne; Elcombe, Barbara M; Chatham, Lynsey R; Vardy, Audrey; Higgins, Larry G; Elcombe, Clifford R; Lake, Brian G

2018-03-01

Phenobarbital (PB), a constitutive androstane receptor (CAR) activator, produces liver tumours in rodents by a mitogenic mode of action involving CAR activation. In this study, the hepatic effects of sodium phenobarbital (NaPB) were compared in male C57BL/6J wild type (WT) mice and in humanized mice, where both the mouse CAR and pregnane X receptor (PXR) have been replaced by their human counterparts (hCAR/hPXR mice). Investigations were also performed in cultured male C57BL/6J and CD-1 mouse, male Sprague-Dawley rat and male and female human hepatocytes. The treatment of WT and hCAR/hPXR mice with 186-984 ppm NaPB in the diet for 7 days resulted in increased relative liver weight, hypertrophy and induction of cytochrome P450 (CYP) enzyme activities. Treatment with NaPB also produced dose-dependent increases in hepatocyte replicative DNA synthesis (RDS), with the effect being more marked in WT than in hCAR/hPXR mice. While the treatment of cultured C57BL/6J and CD-1 mouse, Sprague-Dawley rat and human hepatocytes with 100 and/or 1000 μM NaPB for 4 days induced CYP enzyme activities, increased RDS was only observed in mouse and rat hepatocytes. However, as a positive control, epidermal growth factor increased RDS in hepatocytes from all three species. In summary, although human hepatocytes are refractory to the mitogenic effects of NaPB, treatment with NaPB induced RDS in vivo in hCAR/hPXR mice, which is presumably due to the human CAR and PXR receptors operating in a mouse hepatocyte regulatory environment. As the response of the hCAR/hPXR mouse to the CAR activator NaPB differs markedly from that of human hepatocytes, the hCAR/hPXR mouse is thus not a suitable animal model for studies on the hepatic effects of nongenotoxic rodent CAR activators. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of peroxisome proliferator-activiated receptor alpha (PPARalpha)-independent effects of PPARalpha activators in the rodent liver: Di(2-ethylehexyl) phthalate activates the constitutive activated receptor

EPA Science Inventory

Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). Recent studies indicate that the plasticizer di-2-ethylhexyl ph...

[Proliferation of hepatocytes after delivery of exogenous hepatocyte growth factor gene].

PubMed

Lin, Yong; Xie, Wei fen; Chen, Wei-zhong; Zhang, Xin; Zeng, Xin; Chen, Yue-xiang; Yang, Xiu-jiang; Zhang, Zhong-bing

2003-06-01

To explore the proliferation of primary cultured rats hepatocytes after delivery of exogenous hepatocyte growth factor (HGF) gene which was inserted into the genome of replication-deficient recombinant adenovirus vector. The recombinant adenovirus-AdHGF which could express HGF was generated by homologous recombination. After the HGF gene was delivered into the hepatocytes, the expression of both HGF and c-met/HGF receptor mRNA in the cells was detected by RT-PCR and the level of HGF in the culture supernatant was also assayed by ELISA. On the other hand, cell proliferation was compared between before and after delivery of the HGF gene by MTS assay and the percentages of cell cycles were analyzed by flow cytometry. In addition, the expression of proliferating cell nuclear antigen (PCNA) was determined by immunocytofluorescent stain. 4 x 10(10) efu/ml titer of AdHGF was obtained after recombination, RT-PCR indicated that the expression of HGF mRNA in hepatocytes increased on the third day after infected by the viruses and c-met/HGF receptor mRNA was also up-regulated. The HGF level in the culture supernatant assayed by ELISA was (5,939.0+/-414.39) pg/ml, which was much higher than that in the control (208.1pg/ml+/-37.20pg/ml, F=13.661, P<0.01). In addition, the proliferation of hepatocytes infected with AdHGF increased significantly according to MTS method (F>or=15.158, P<0.01) and more hepatocytes in G0/G1 stages changed into S stage (chi2=41.616, P<0.01), accordingly, PCNA index increased from 6.42+/- 1.88 to 14.56+/-2.85 (F=42.122, P<0.01). show that HGF gene delivered into hepatocytes by AdHGF can be expressed with high efficiency in the cells, which can stimulate hepatocytes proliferation. It may be an effective tool for hepatocyte transplantation by gene modified donor hepatocytes.

Hepatocyte Polarity

PubMed Central

Treyer, Aleksandr; Müsch, Anne

2013-01-01

Hepatocytes, like other epithelia, are situated at the interface between the organism’s exterior and the underlying internal milieu and organize the vectorial exchange of macromolecules between these two spaces. To mediate this function, epithelial cells, including hepatocytes, are polarized with distinct luminal domains that are separated by tight junctions from lateral domains engaged in cell-cell adhesion and from basal domains that interact with the underlying extracellular matrix. Despite these universal principles, hepatocytes distinguish themselves from other nonstriated epithelia by their multipolar organization. Each hepatocyte participates in multiple, narrow lumina, the bile canaliculi, and has multiple basal surfaces that face the endothelial lining. Hepatocytes also differ in the mechanism of luminal protein trafficking from other epithelia studied. They lack polarized protein secretion to the luminal domain and target single-spanning and glycosylphosphatidylinositol-anchored bile canalicular membrane proteins via transcytosis from the basolateral domain. We compare this unique hepatic polarity phenotype with that of the more common columnar epithelial organization and review our current knowledge of the signaling mechanisms and the organization of polarized protein trafficking that govern the establishment and maintenance of hepatic polarity. The serine/threonine kinase LKB1, which is activated by the bile acid taurocholate and, in turn, activates adenosine monophosphate kinase-related kinases including AMPK1/2 and Par1 paralogues has emerged as a key determinant of hepatic polarity. We propose that the absence of a hepatocyte basal lamina and differences in cell-cell adhesion signaling that determine the positioning of tight junctions are two crucial determinants for the distinct hepatic and columnar polarity phenotypes. PMID:23720287

Characterization of Peroxisome Proliferator-Activated Receptor a (PPARa) -Independent Effects of PPARa Activators in the Rodent Liver: Di-(2-ethylhexyl) phthalate Also Activates the Constitutive Activated Receptor

EPA Science Inventory

Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferatoractivated receptor alpha (PPARa). Recent studies indicate that one such PPC, the plasticizer di2- et...

Extensive conversion of hepatic biliary epithelial cells to hepatocytes after near total loss of hepatocytes in zebrafish.

PubMed

Choi, Tae-Young; Ninov, Nikolay; Stainier, Didier Y R; Shin, Donghun

2014-03-01

Biliary epithelial cells (BECs) are considered to be a source of regenerating hepatocytes when hepatocyte proliferation is compromised. However, there is still controversy about the extent to which BECs can contribute to the regenerating hepatocyte population, and thereby to liver recovery. To investigate this issue, we established a zebrafish model of liver regeneration in which the extent of hepatocyte ablation can be controlled. Hepatocytes were depleted by administration of metronidazole to Tg(fabp10a:CFP-NTR) animals. We traced the origin of regenerating hepatocytes using short-term lineage-tracing experiments, as well as the inducible Cre/loxP system; specifically, we utilized both a BEC tracer line Tg(Tp1:CreER(T2)) and a hepatocyte tracer line Tg(fabp10a:CreER(T2)). We also examined BEC and hepatocyte proliferation and liver marker gene expression during liver regeneration. BECs gave rise to most of the regenerating hepatocytes in larval and adult zebrafish after severe hepatocyte depletion. After hepatocyte loss, BECs proliferated as they dedifferentiated into hepatoblast-like cells; they subsequently differentiated into highly proliferative hepatocytes that restored the liver mass. This process was impaired in zebrafish wnt2bb mutants; in these animals, hepatocytes regenerated but their proliferation was greatly reduced. BECs contribute to regenerating hepatocytes after substantial hepatocyte depletion in zebrafish, thereby leading to recovery from severe liver damage. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Fibrinogen-like protein 1, a hepatocyte derived protein is an acute phase reactant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Zhilin; Ukomadu, Chinweike

2008-01-25

Fibrinogen-like protein 1 (FGL1) is a hepatocyte derived protein that is upregulated in regenerating rodent livers following partial hepatectomy. It has been implicated as a mitogen for liver cell proliferation. In this study, we show that recombinant human IL-6 induces FGL1 expression in Hep G2 cells in a pattern similar to those of acute phase reactants. Following induction of acute inflammation in rats by subcutaneous injection of turpentine oil, serum FGL1 levels are also enhanced. Although, a recent report suggests that FGL1 associates almost exclusively with the fibrin matrix, we report here that approximately 20% of the total plasma FGL1more » remains free. The enhancement of FGL1 levels in vitro by IL-6 and its induction after turpentine oil injection suggest that it is an acute phase reactant. Its presence in bound and free forms in the blood also implies biological roles that extend beyond the proposed autocrine effect it has on hepatocytes during regeneration.« less

The rodent liver undergoes weaning-induced involution and supports breast cancer metastasis

PubMed Central

Goddard, Erica T.; Hill, Ryan C.; Nemkov, Travis; D’Alessandro, Angelo; Hansen, Kirk C.; Maller, Ori; Mongoue-Tchokote, Solange; Mori, Motomi; Partridge, Ann H.; Borges, Virginia F.; Schedin, Pepper

2017-01-01

Postpartum breast cancer patients are at increased risk for metastasis compared to age-matched nulliparous or pregnant patients. Here, we address whether circulating tumor cells have a metastatic advantage in the postpartum host and find the post-lactation rodent liver preferentially supports metastasis. Upon weaning, we observed liver weight loss, hepatocyte apoptosis, ECM remodeling including deposition of collagen and tenascin-C, and myeloid cell influx, data consistent with weaning-induced liver involution and establishment of a pro-metastatic microenvironment. Using intracardiac and intraportal metastasis models, we observed increased liver metastasis in post-weaning Balb/c mice compared to nulliparous controls. Human relevance is suggested by a ~3-fold increase in liver metastasis in postpartum breast cancer patients (n=564) and by liver-specific tropism (n=117). In sum, our data reveal a previously unknown biology of the rodent liver, weaning-induced liver involution, which may provide insight into the increased liver metastasis and poor prognosis of women diagnosed with postpartum breast cancer. PMID:27974414

The molecular functions of hepatocyte nuclear factors - In and beyond the liver.

PubMed

Lau, Hwee Hui; Ng, Natasha Hui Jin; Loo, Larry Sai Weng; Jasmen, Joanita Binte; Teo, Adrian Kee Keong

2018-05-01

The hepatocyte nuclear factors (HNFs) namely HNF1α/β, FOXA1/2/3, HNF4α/γ and ONECUT1/2 are expressed in a variety of tissues and organs, including the liver, pancreas and kidney. The spatial and temporal manner of HNF expression regulates embryonic development and subsequently the development of multiple tissues during adulthood. Though the HNFs were initially identified individually based on their roles in the liver, numerous studies have now revealed that the HNFs cross-regulate one another and exhibit synergistic relationships in the regulation of tissue development and function. The complex HNF transcriptional regulatory networks have largely been elucidated in rodent models, but less so in human biological systems. Several heterozygous mutations in these HNFs were found to cause diseases in humans but not in rodents, suggesting clear species-specific differences in mutational mechanisms that remain to be uncovered. In this review, we compare and contrast the expression patterns of the HNFs, the HNF cross-regulatory networks and how these liver-enriched transcription factors serve multiple functions in the liver and beyond, extending our focus to the pancreas and kidney. We also summarise the insights gained from both human and rodent studies of mutations in several HNFs that are known to lead to different disease conditions. Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Hepatocyte-specific PPARA expression exclusively promotes agonist-induced cell proliferation without influence from nonparenchymal cells

PubMed Central

Brocker, Chad N.; Yue, Jiang; Kim, Donghwan; Qu, Aijuan; Bonzo, Jessica A.

2017-01-01

Peroxisome proliferator-activated receptor-α (PPARA) is a nuclear transcription factor and key mediator of systemic lipid metabolism. Prolonged activation in rodents causes hepatocyte proliferation and hepatocellular carcinoma. Little is known about the contribution of nonparenchymal cells (NPCs) to PPARA-mediated cell proliferation. NPC contribution to PPARA agonist-induced hepatomegaly was assessed in hepatocyte (Ppara△Hep)- and macrophage (Ppara△Mac)-specific Ppara null mice. Mice were treated with the agonist Wy-14643 for 14 days, and response of conditional null mice was compared with conventional knockout mice (Ppara−/−). Wy-14643 treatment caused weight loss and severe hepatomegaly in wild-type and Ppara△Mac mice, and histological analysis revealed characteristic hepatocyte swelling; Ppara△Hep and Ppara−/− mice were protected from these effects. Ppara△Mac serum chemistries, as well as aspartate aminotransferase and alanine aminotransferase levels, matched wild-type mice. Agonist-treated Ppara△Hep mice had elevated serum cholesterol, phospholipids, and triglycerides when compared with Ppara−/− mice, indicating a possible role for extrahepatic PPARA in regulating circulating lipid levels. BrdU labeling confirmed increased cell proliferation only in wild-type and Ppara△Mac mice. Macrophage PPARA disruption did not impact agonist-induced upregulation of lipid metabolism, cell proliferation, or DNA damage and repair-related gene expression, whereas gene expression was repressed in Ppara△Hep mice. Interestingly, downregulation of inflammatory cytokines IL-15 and IL-18 was dependent on macrophage PPARA. Cell type-specific regulation of target genes was confirmed in primary hepatocytes and Kupffer cells. These studies conclusively show that cell proliferation is mediated exclusively by PPARA activation in hepatocytes and that Kupffer cell PPARA has an important role in mediating the anti-inflammatory effects of PPARA agonists. PMID

Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes.

PubMed

Runge, D M; Runge, D; Dorko, K; Pisarov, L A; Leckel, K; Kostrubsky, V E; Thomas, D; Strom, S C; Michalopoulos, G K

1999-02-01

Serum-free primary cultures of hepatocytes are a useful tool to study factors triggering hepatocyte proliferation and regeneration. We have developed a chemically defined serum-free system that allows human hepatocyte proliferation in the presence of epidermal growth factor and hepatocyte growth factor. DNA synthesis and accumulation were determined by [3H]thymidine incorporation and fluorometry, respectively. Western blot analyses and co-immunoprecipitations were used to investigate the association of proteins involved in epidermal growth factor and hepatocyte growth factor activation and signaling: epidermal growth factor receptor, hepatocyte growth factor receptor (MET), urokinase-type plasminogen activator and its receptor, and a member of the signal transducer and activator of transcription family, STAT-3. Primary human hepatocytes proliferated under serum-free conditions in a chemically defined medium for up to 12 days. Epidermal growth factor-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to varying degrees. Proliferation of human hepatocytes can improve by modification of a chemically defined medium originally used for rat hepatocyte cultures. In these long-term cultures of human hepatocytes, hepatocyte growth factor and epidermal growth factor can stimulate growth and differentiation by interacting with their receptors and initiating downstream signaling. This involves complex formation of the receptors with other plasma membrane components for MET (urokinase-type plasminogen activator in context of its receptor) and activation of STAT-3 for both receptors.

Strategies for immortalization of primary hepatocytes

PubMed Central

Eva, Ramboer; Bram, De Craene; Joery, De Kock; Tamara, Vanhaecke; Geert, Berx; Vera, Rogiers; Mathieu, Vinken

2014-01-01

The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications. PMID:24911463

A metabolic screening study of trichostatin A (TSA) and TSA-like histone deacetylase inhibitors in rat and human primary hepatocyte cultures.

PubMed

Elaut, G; Laus, G; Alexandre, E; Richert, L; Bachellier, P; Tourwé, D; Rogiers, V; Vanhaecke, T

2007-04-01

Hydroxamic acid (HA)-based histone deacetylase (HDAC) inhibitors, with trichostatin A (TSA) as the reference compound, are potential antitumoral drugs and show promise in the creation of long-term primary cell cultures. However, their metabolic properties have barely been investigated. TSA is rapidly inactivated in rodents both in vitro and in vivo. We previously found that 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxyamide or 4-Me2N-BAVAH (compound 1) is metabolically more stable upon incubation with rat hepatocyte suspensions. In this study, we show that human hepatocytes also metabolize TSA more rapidly than compound 1 and that similar pathways are involved. Furthermore, structural analogs of compound 1 (compounds 2-9) are reported to have the same favorable metabolic properties. Removal of the dimethylamino substituent of compound 1 creates a very stable but 50% less potent inhibitor. Chain lengthening (4 to 5 carbon spacer) slightly improves both potency and metabolic stability, favoring HA reduction to hydrolysis. On the other hand, Calpha-unsaturation and spacer methylation not only reduce HDAC inhibition but also increase the rate of metabolic inactivation approximately 2-fold, mainly through HA reduction. However, in rat hepatocyte monolayer cultures, compound 1 is shown to be extensively metabolized by phase II conjugation. In conclusion, this study suggests that simple structural modifications of amide-linked TSA analogs can improve their phase I metabolic stability in both rat and human hepatocyte suspensions. Phase II glucuronidation, however, can compensate for their lower phase I metabolism in rat hepatocyte monolayers and could play a yet unidentified role in the determination of their in vivo clearance.

Repolarization of hepatocytes in culture.

PubMed

Talamini, M A; Kappus, B; Hubbard, A

1997-01-01

We have evaluated the biochemical, morphological, and functional redevelopment of polarity in freshly isolated hepatocytes cultured using a double layer collagen gel sandwich technique. Western blot analysis showed increased cellular levels of the cell adhesion protein uvomorulin as cultured hepatocytes repolarized. Immunofluorescence studies using antibodies against domain-specific membrane proteins showed polarity as early as 48 hours, although the pattern of the polymeric Immunoglobulin-A receptor (pIgA-R) differed from in vivo liver. Electron microscopy showed developing bile canaliculi at 1 day. However, the functional presence of tight junctions was absent at 1 day, but present at 5 days. We further showed functional polarity to be present at 4 days by documenting the ability of cultured hepatocytes to metabolize and excrete fluorescein diacetate into visible bile canaliculi. We conclude that hepatocytes cultured appropriately develop morphological and functional polarity. Hepatocyte culture is therefore a useful tool for the study of mechanisms responsible for the development of polarized function.

Physiological Ranges of Matrix Rigidity Modulate Primary Mouse Hepatocyte Function In Part Through Hepatocyte Nuclear Factor 4 Alpha

PubMed Central

Desai, Seema S.; Tung, Jason C.; Zhou, Vivian X.; Grenert, James P.; Malato, Yann; Rezvani, Milad; Español-Suñer, Regina; Willenbring, Holger; Weaver, Valerie M.; Chang, Tammy T.

2016-01-01

Matrix rigidity has important effects on cell behavior and is increased during liver fibrosis; however, its effect on primary hepatocyte function is unknown. We hypothesized that increased matrix rigidity in fibrotic livers would activate mechanotransduction in hepatocytes and lead to inhibition of hepatic-specific functions. To determine the physiologically relevant ranges of matrix stiffness at the cellular level, we performed detailed atomic force microscopy analysis across liver lobules from normal and fibrotic livers. We determined that normal liver matrix stiffness was around 150Pa and increased to 1–6kPa in areas near fibrillar collagen deposition in fibrotic livers. In vitro culture of primary hepatocytes on collagen matrix of tunable rigidity demonstrated that fibrotic levels of matrix stiffness had profound effects on cytoskeletal tension and significantly inhibited hepatocyte-specific functions. Normal liver stiffness maintained functional gene regulation by hepatocyte nuclear factor 4 alpha (HNF4α) whereas fibrotic matrix stiffness inhibited the HNF4α transcriptional network. Fibrotic levels of matrix stiffness activated mechanotransduction in primary hepatocytes through focal adhesion kinase (FAK). In addition, blockade of the Rho/Rho-associated protein kinase (ROCK) pathway rescued HNF4α expression from hepatocytes cultured on stiff matrix. Conclusion Fibrotic levels of matrix stiffness significantly inhibit hepatocyte-specific functions in part by inhibiting the HNF4α transcriptional network mediated through the Rho/ROCK pathway. Increased appreciation of the role of matrix rigidity in modulating hepatocyte function will advance our understanding of the mechanisms of hepatocyte dysfunction in liver cirrhosis and spur development of novel treatments for chronic liver disease. PMID:26755329

Liver-enriched transcription factors are critical for the expression of hepatocyte marker genes in mES-derived hepatocyte-lineage cells.

PubMed

Kheolamai, Pakpoom; Dickson, Alan J

2009-04-23

Induction of stem cell differentiation toward functional hepatocytes is hampered by lack of knowledge of the hepatocyte differentiation processes. The overall objective of this project is to characterize key stages in the hepatocyte differentiation process. We established a mouse embryonic stem (mES) cell culture system which exhibited changes in gene expression profiles similar to those observed in the development of endodermal and hepatocyte-lineage cells previously described in the normal mouse embryo. Transgenic mES cells were established that permitted isolation of enriched hepatocyte-lineage populations. This approach has isolated mES-derived hepatocyte-lineage cells that express several markers of mature hepatocytes including albumin, glucose-6-phosphatase, tyrosine aminotransferase, cytochrome P450-3a, phosphoenolpyruvate carboxykinase and tryptophan 2,3-dioxygenase. In addition, our results show that the up-regulation of the expression levels of hepatocyte nuclear factor-3alpha, -4alpha, -6, and CCAAT-enhancer binding protein-beta might be critical for passage into late-stage differentiation towards functional hepatocytes. These data present important steps for definition of regulatory phenomena that direct specific cell fate determination. The mES cell culture system generated in this study provides a model for studying transition between stages of the hepatocyte development and has significant potential value for studying the molecular basis of hepatocyte differentiation in vitro.

Three Dimensional Primary Hepatocyte Culture

NASA Technical Reports Server (NTRS)

Yoffe, Boris

1998-01-01

Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

Isolated hepatocytes--past, present and future.

PubMed

Berry, M N; Grivell, A R; Grivell, M B; Phillips, J W

1997-07-01

The first technique for large-scale preparation of isolated hepatocytes was described in 1953 and involved perfusion of rat liver under pressure with a Ca(2+)-free solution containing a chelating agent. Various modifications of this technique were in use over the next ten years, until it was demonstrated that cells prepared in this manner were grossly damaged, losing most of their cytoplasmic enzymes during the preparative procedure. The successful preparation of intact isolated hepatocytes by collagenase-treatment of liver was achieved in 1967, and the widespread use of intact hepatocyte suspensions was accelerated by the development soon after of high-yield preparative techniques involving perfusion of the liver with a medium containing collagenase. The introduction of the isolated hepatocyte preparation has enabled experimental studies that otherwise would not be feasible. Important advances have been the use of cultured hepatocytes, frequently of human origin, for the investigation of the metabolism and toxicology of potential therapeutic agents. Success in this field has been achieved through the steady improvement in techniques for the maintenance in culture of differentiated hepatocytes, and in particular their cytochrome P450 complexes. Another area showing considerable promise is the employment of hepatocytes, generally from a porcine source, in temporary support systems for patients with acute liver failure. Our own studies have concentrated on the demonstration of long-range interactions between hepatocyte compartments which suggest that energy transfer between cell compartments can take place without ATP turnover.

Physiological ranges of matrix rigidity modulate primary mouse hepatocyte function in part through hepatocyte nuclear factor 4 alpha.

PubMed

Desai, Seema S; Tung, Jason C; Zhou, Vivian X; Grenert, James P; Malato, Yann; Rezvani, Milad; Español-Suñer, Regina; Willenbring, Holger; Weaver, Valerie M; Chang, Tammy T

2016-07-01

Matrix rigidity has important effects on cell behavior and is increased during liver fibrosis; however, its effect on primary hepatocyte function is unknown. We hypothesized that increased matrix rigidity in fibrotic livers would activate mechanotransduction in hepatocytes and lead to inhibition of liver-specific functions. To determine the physiologically relevant ranges of matrix stiffness at the cellular level, we performed detailed atomic force microscopy analysis across liver lobules from normal and fibrotic livers. We determined that normal liver matrix stiffness was around 150 Pa and increased to 1-6 kPa in areas near fibrillar collagen deposition in fibrotic livers. In vitro culture of primary hepatocytes on collagen matrix of tunable rigidity demonstrated that fibrotic levels of matrix stiffness had profound effects on cytoskeletal tension and significantly inhibited hepatocyte-specific functions. Normal liver stiffness maintained functional gene regulation by hepatocyte nuclear factor 4 alpha (HNF4α), whereas fibrotic matrix stiffness inhibited the HNF4α transcriptional network. Fibrotic levels of matrix stiffness activated mechanotransduction in primary hepatocytes through focal adhesion kinase. In addition, blockade of the Rho/Rho-associated protein kinase pathway rescued HNF4α expression from hepatocytes cultured on stiff matrix. Fibrotic levels of matrix stiffness significantly inhibit hepatocyte-specific functions in part by inhibiting the HNF4α transcriptional network mediated through the Rho/Rho-associated protein kinase pathway. Increased appreciation of the role of matrix rigidity in modulating hepatocyte function will advance our understanding of the mechanisms of hepatocyte dysfunction in liver cirrhosis and spur development of novel treatments for chronic liver disease. (Hepatology 2016;64:261-275). © 2016 by the American Association for the Study of Liver Diseases.

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes

PubMed Central

Baxter, Melissa; Withey, Sarah; Harrison, Sean; Segeritz, Charis-Patricia; Zhang, Fang; Atkinson-Dell, Rebecca; Rowe, Cliff; Gerrard, Dave T.; Sison-Young, Rowena; Jenkins, Roz; Henry, Joanne; Berry, Andrew A.; Mohamet, Lisa; Best, Marie; Fenwick, Stephen W.; Malik, Hassan; Kitteringham, Neil R.; Goldring, Chris E.; Piper Hanley, Karen; Vallier, Ludovic; Hanley, Neil A.

2015-01-01

Background & Aims Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Methods Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. Results HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. Conclusions HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes. PMID:25457200

Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival.

PubMed

Hang, Hua-Lian; Liu, Xin-Yu; Wang, Hai-Tian; Xu, Ning; Bian, Jian-Min; Zhang, Jian-Jun; Xia, Lei; Xia, Qiang

2017-11-15

Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of β-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote β-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF. Copyright © 2017 Elsevier Inc. All rights reserved.

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes.

PubMed

Baxter, Melissa; Withey, Sarah; Harrison, Sean; Segeritz, Charis-Patricia; Zhang, Fang; Atkinson-Dell, Rebecca; Rowe, Cliff; Gerrard, Dave T; Sison-Young, Rowena; Jenkins, Roz; Henry, Joanne; Berry, Andrew A; Mohamet, Lisa; Best, Marie; Fenwick, Stephen W; Malik, Hassan; Kitteringham, Neil R; Goldring, Chris E; Piper Hanley, Karen; Vallier, Ludovic; Hanley, Neil A

2015-03-01

Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

PubMed Central

Jitraruch, Suttiruk; Hughes, Robin D.; Filippi, Celine; Lehec, Sharon C.; Glover, Leanne; Mitry, Ragai R.

2017-01-01

Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimized protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300 mM) glucose and CryoStor CS10 showed better postthawing cell viability, attachment, and hepatocyte functions than with histidine–tryptophan–ketoglutarate/10% DMSO/5% glucose and Bambanker. The 2 freezing solutions that gave better results were studied with human and rat hepatocytes microbeads. Similar effects on cryopreserved microbead morphology (external and ultrastructural), viability, and hepatocyte-functions post thawing were observed over 7 d in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone [ZVAD]), an antioxidant (desferoxamine [DFO]), and a buffering and mechanical protectant (human serum albumin [HSA]) on RMBs. ZVAD (60 µM) had a beneficial effect on cell viability that was greater than with DFO (1 mM), HSA (2%), and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes and a lower degree of cell apoptosis were observed with all 3 cytoprotectants, with ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the 3 cytoprotectant groups than with fresh microbeads. In conclusion, developing an optimized cryopreservation protocol by adding

Failure of hepatocyte marker-expressing hematopoietic progenitor cells to efficiently convert into hepatocytes in vitro.

PubMed

Lian, Gewei; Wang, Chengyan; Teng, Chunbo; Zhang, Cong; Du, Liying; Zhong, Qian; Miao, Chenglin; Ding, Mingxiao; Deng, Hongkui

2006-03-01

Whether bone marrow (BM) hematopoietic stem/progenitor cells can directly differentiate into nonhematopoietic cells remains controversial. The aim of this study is to further investigate the potentiality of BM hematopoietic progenitor cells to convert into hepatocytes in vitro. Different subsets of BM cells from C57/BL6 mice were isolated using markers of hematopoietic stem cells by magnetic cell sorting and by flow cytometry. These cells were induced to transdifferentiate to hepatocytes in vitro in the presence of various cytokines or of hepatocytes (or tissue) from damaged liver, which have been reported to stimulate the conversion. Hepatic gene markers in freshly isolated or cultured BM cells were determined by reverse transcriptase polymerase chain reaction and immunofluorescence. Freshly isolated hematopoietic progenitor cells (HPC) expressed a low level of messenger RNAs of hepatic cell-specific markers including albumin and alpha-fetoprotein (AFP), but did not significantly upregulate expression of these markers, even in the presence of cytokines or cocultured hepatocytes (or tissue). HPCs induced in vitro did not express the message of alpha-anti-trypsin-a mature hepatocyte marker. At protein level, the specific staining of AFP was not detected in the HPCs, either freshly isolated or in vitro induced. Albumin protein was detected in freshly isolated albumin mRNA-positive and -negative BM cell subpopulations. Albumin-stained BM cells disappeared after being induced for 5 days, but restained if mouse serum was supplemented in medium for a 24-hour extended culture, suggesting that albumin was absorbed by BM cells instead of de novo expression. HPCs expressed mRNAs of hepatic cell markers, but could not efficiently convert into hepatocytes in vitro under our experimental conditions. Our observation raises a cautionary note in determining whether in vitro transdifferentiation of BM cells to hepatocytes can actually take place.

Hepatocyte transplantation for liver-based metabolic disorders.

PubMed

Dhawan, Anil; Mitry, Ragai R; Hughes, Robin D

2006-01-01

Hepatocyte transplantation is being investigated as an alternative to orthotopic liver transplantation in patients with liver-based metabolic disorders. The progress made in this field to date is reviewed. Protocols have been developed using collagenase perfusion to isolate human hepatocytes from unused donor liver tissue. Hepatocytes with a high viability can often be obtained and can be cryopreserved for later use, though with loss of function on thawing. For clinical use, hepatocytes must be prepared in clean GMP conditions with cells meeting criteria of function and lack of microbial contamination before patient use. Hepatocytes are infused intraportally into the patient's liver, where a proportion of cells will engraft and replace the deficient metabolic function without the need for major surgery. Twenty patients have now received hepatocyte transplantation, including eight children at King's College Hospital. There was a range of aetiologies of liver disease: familial hypercholesterolaemia, Crigler-Najjar syndrome type 1, urea cycle defects, infantile Refsum disease, glycogen storage disease type Ia, inherited factor VII deficiency and progressive familial intrahepatic cholestasis type 2. Clinical improvement and partial correction of the metabolic abnormality was observed in most cases. Considerable progress has been made in developing the technique, but hepatocyte transplantation is limited by the available supply of liver tissue. Hepatocytes derived from stem cells could provide alternative sources of cells in the future.

Hepatocyte nuclear factor-4alpha induces transdifferentiation of hematopoietic cells into hepatocytes.

PubMed

Khurana, Satish; Jaiswal, Amit K; Mukhopadhyay, Asok

2010-02-12

Hematopoietic stem cells can directly transdifferentiate into hepatocytes because of cellular plasticity, but the molecular basis of transdifferentiation is not known. Here, we show the molecular basis using lineage-depleted oncostatin M receptor beta-expressing (Lin(-)OSMRbeta(+)) mouse bone marrow cells in a hepatic differentiation culture system. Differentiation of the cells was marked by the expression of albumin. Hepatocyte nuclear factor (HNF)-4alpha was expressed and translocated into the nuclei of the differentiating cells. Suppression of its activation in OSM-neutralized culture medium inhibited cellular differentiation. Ectopic expression of full-length HNF4alpha in 32D myeloid cells resulted in decreased myeloid colony-forming potential and increased expression of hepatocyte-specific genes and proteins. Nevertheless, the neohepatocytes produced in culture expressed active P450 enzyme. The obligatory role of HNF4alpha in hepatic differentiation was confirmed by transfecting Lin(-)OSMRbeta(+) cells with dominant negative HNF4alpha in the differentiation culture because its expression inhibited the transcription of the albumin and tyrosine aminotransferase genes. The loss and gain of functional activities strongly suggested that HNF4alpha plays a central role in the transdifferentiation process. For the first time, this report demonstrates the mechanism of transdifferentiation of hematopoietic cells into hepatocytes, in which HNF4alpha serves as a molecular switch.

Activation of the Nrf2-ARE Pathway in Hepatocytes Protects Against Steatosis in Nutritionally Induced Non-alcoholic Steatohepatitis in Mice

PubMed Central

Lee, Lung-Yi; Köhler, Ulrike A.; Zhang, Li; Roenneburg, Drew; Werner, Sabine; Johnson, Jeffrey A.; Foley, David P.

2014-01-01

Oxidative stress is implicated in the development of non-alcoholic steatohepatitis (NASH). The Nrf2-antioxidant response element pathway protects cells from oxidative stress. Studies have shown that global Nrf2 deficiency hastens the progression of NASH. The purpose of this study was to determine whether long-term hepatocyte-specific activation of Nrf2 mitigates NASH progression. Transgenic mice expressing a constitutively active Nrf2 construct in hepatocytes (AlbCre+/caNrf2+) and littermate controls were generated. These mice were fed standard or methionine-choline-deficient (MCD) diet, a diet used to induce NASH development in rodents. After 28 days of MCD dietary feeding, mice developed significant increases in steatosis, inflammation, oxidative stress, and HSC activation compared with those mice on standard diet. AlbCre+/caNrf2+ animals had significantly decreased serum transaminases and reduced steatosis when compared with the AlbCre+/caNrf2− animals. This significant reduction in steatosis was associated with increased expression of genes involved in triglyceride export (MTTP) and β-oxidation (CPT2). However, there were no differences in the increased oxidative stress, inflammation, and HSC activation from MCD diet administration between the AlbCre+/caNrf2− and AlbCre+/caNrf2+ animals. We conclude that hepatocyte-specific activation of Nrf2-mediated gene expression decreased hepatocellular damage and steatosis in a dietary model of NASH. However, hepatocyte-specific induction of Nrf2-mediated gene expression alone is insufficient to mitigate inflammation, oxidative stress, and HSC activation in this nutritional NASH model. PMID:25294219

Oxidative stress triggers cytokinesis failure in hepatocytes upon isolation.

PubMed

Tormos, A M; Taléns-Visconti, R; Bonora-Centelles, A; Pérez, S; Sastre, J

2015-01-01

Primary hepatocytes are highly differentiated cells and proliferatively quiescent. However, the stress produced during liver digestion seems to activate cell cycle entry by proliferative/dedifferentiation programs that still remain unclear. The aim of this work was to assess whether the oxidative stress associated with hepatocyte isolation affects cell cycle and particularly cytokinesis, the final step of mitosis. Hepatocytes were isolated from C57BL/6 mice by collagenase perfusion in the absence and presence of N-acetyl cysteine (NAC). Polyploidy, cell cycle, and reactive oxygen species (ROS) were studied by flow cytometry (DNA, phospho-histone 3, and CellROX(®) Deep Red) and Western blotting (cyclins B1 and D1, and proliferating cell nuclear antigen). mRNA expression of cyclins A1, B1, B2, D1, and F by reverse transcription (RT)-PCR was also assessed. Glutathione levels were measured by mass spectrometry. Here we show that hepatocyte isolation enhanced cell cycle entry, increased hepatocyte binucleation, and caused marked glutathione oxidation. Addition of 5 mM NAC to the hepatocyte isolation media prevented glutathione depletion, partially blocked ROS production and cell cycle entry of hepatocytes, and avoided the blockade of mitosis progression, abrogating defective cytokinesis and diminishing the formation of binucleated hepatocytes during isolation. Therefore, addition of NAC to the isolation media decreased the generation of polyploid hepatocytes confirming that oxidative stress occurs during hepatocyte isolation and it is responsible, at least in part, for cytokinesis failure and hepatocyte binucleation.

Lipopolysaccharide potentiates the effect of hepatocyte growth factor on hepatocyte replication in rats by augmenting AP-1 activity.

PubMed

Gao, C; Jokerst, R; Gondipalli, P; Cai, S R; Kennedy, S; Flye, M W; Ponder, K P

1999-12-01

The liver regenerates by replication of differentiated hepatocytes after damage or removal of part of the liver. Although several growth factors and signaling pathways are activated during regeneration, it is unclear as to which of these are essential for hepatocyte replication. We show here that low- (1 mg/kg) and high- (10 mg/kg) dose hepatocyte growth factor (HGF) induced replication of 2.1% and 11.1% of hepatocytes in rats, respectively. Lipopolysaccharide (LPS), an inducer of the acute phase response, augmented hepatocyte replication in response to low- and high-dose HGF by 4- and 2-fold, respectively. HGF alone induced moderate levels of c-Jun-N-terminal kinase (JNK) and p44/p42 mitogen-activated protein kinase (MAPK), resulting in moderate levels of AP-1-DNA binding activity. The combination of LPS + HGF increased JNK and AP-1-DNA binding activity more than levels seen with LPS or HGF alone. The activation of Stat3 that was observed after administration of LPS + HGF, but not HGF alone, could contribute to increased transcription of AP-1 components. Because phosphorylation of the c-Jun component of AP-1 by JNK increases its ability to activate transcription, the AP-1 in hepatocytes from animals treated with LPS + HGF may be more active than in rats treated with LPS or HGF alone. LPS may contribute to hepatocyte replication by potentiating the effect of HGF on the activation of both AP-1-DNA binding and transcriptional activity.

Breaking through the false coincidence barrier in electron–ion coincidence experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osborn, David L.; Hayden, Carl C.; Hemberger, Patrick

Photoelectron Photoion Coincidence (PEPICO) spectroscopy holds the promise of a universal, isomer-selective, and sensitive analytical technique for time-resolved quantitative analysis of bimolecular chemical reactions. Unfortunately, its low dynamic range of ~10 3 has largely precluded its use for this purpose, where a dynamic range of at least 10 5 is generally required. This limitation is due to the false coincidence background common to all coincidence experiments, especially at high count rates. Electron/ion pairs emanating from separate ionization events but arriving within the ion time of flight (TOF) range of interest constitute the false coincidence background. Although this background has uniformmore » intensity at every m/z value, the Poisson scatter in the false coincidence background obscures small signals. In this paper, temporal ion deflection coupled with a position-sensitive ion detector enables suppression of the false coincidence background, increasing the dynamic range in the PEPICO TOF mass spectrum by 2–3 orders of magnitude. The ions experience a time-dependent electric deflection field at a well-defined fraction of their time of flight. This deflection defines an m/z- and ionization-time dependent ion impact position for true coincidences, whereas false coincidences appear randomly outside this region and can be efficiently suppressed. When cold argon clusters are ionized, false coincidence suppression allows us to observe species up to Ar 9 +, whereas Ar 4 + is the largest observable cluster under traditional operation. As a result, this advance provides mass-selected photoelectron spectra for fast, high sensitivity quantitative analysis of reacting systems.« less

Breaking through the false coincidence barrier in electron–ion coincidence experiments

DOE PAGES

Osborn, David L.; Hayden, Carl C.; Hemberger, Patrick; ...

2016-10-31

Photoelectron Photoion Coincidence (PEPICO) spectroscopy holds the promise of a universal, isomer-selective, and sensitive analytical technique for time-resolved quantitative analysis of bimolecular chemical reactions. Unfortunately, its low dynamic range of ~10 3 has largely precluded its use for this purpose, where a dynamic range of at least 10 5 is generally required. This limitation is due to the false coincidence background common to all coincidence experiments, especially at high count rates. Electron/ion pairs emanating from separate ionization events but arriving within the ion time of flight (TOF) range of interest constitute the false coincidence background. Although this background has uniformmore » intensity at every m/z value, the Poisson scatter in the false coincidence background obscures small signals. In this paper, temporal ion deflection coupled with a position-sensitive ion detector enables suppression of the false coincidence background, increasing the dynamic range in the PEPICO TOF mass spectrum by 2–3 orders of magnitude. The ions experience a time-dependent electric deflection field at a well-defined fraction of their time of flight. This deflection defines an m/z- and ionization-time dependent ion impact position for true coincidences, whereas false coincidences appear randomly outside this region and can be efficiently suppressed. When cold argon clusters are ionized, false coincidence suppression allows us to observe species up to Ar 9 +, whereas Ar 4 + is the largest observable cluster under traditional operation. As a result, this advance provides mass-selected photoelectron spectra for fast, high sensitivity quantitative analysis of reacting systems.« less

Generation of functional hepatocytes from human spermatogonial stem cells.

PubMed

Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

2016-02-23

To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration.

Generation of functional hepatocytes from human spermatogonial stem cells

PubMed Central

Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

2016-01-01

To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration. PMID:26840458

Reversal of hepatocyte senescence after continuous in vivo cell proliferation.

PubMed

Wang, Min-Jun; Chen, Fei; Li, Jian-Xiu; Liu, Chang-Cheng; Zhang, Hai-Bin; Xia, Yong; Yu, Bing; You, Pu; Xiang, Dao; Lu, Lian; Yao, Hao; Borjigin, Uyunbilig; Yang, Guang-Shun; Wangensteen, Kirk J; He, Zhi-Ying; Wang, Xin; Hu, Yi-Ping

2014-07-01

A better understanding of hepatocyte senescence could be used to treat age-dependent disease processes of the liver. Whether continuously proliferating hepatocytes could avoid or reverse senescence has not yet been fully elucidated. We confirmed that the livers of aged mice accumulated senescent and polyploid hepatocytes, which is associated with accumulation of DNA damage and activation of p53-p21 and p16(ink4a)-pRB pathways. Induction of multiple rounds continuous cell division is hard to apply in any animal model. Taking advantage of serial hepatocyte transplantation assays in the fumarylacetoacetate hydrolase-deficient (Fah(-/-)) mouse, we studied the senescence of hepatocytes that had undergone continuous cell proliferation over a long time period, up to 12 rounds of serial transplantations. We demonstrated that the continuously proliferating hepatocytes avoided senescence and always maintained a youthful state. The reactivation of telomerase in hepatocytes after serial transplantation correlated with reversal of senescence. Moreover, senescent hepatocytes harvested from aged mice became rejuvenated upon serial transplantation, with full restoration of proliferative capacity. The same findings were also true for human hepatocytes. After serial transplantation, the high initial proportion of octoploid hepatocytes decreased to match the low level of youthful liver. These findings suggest that the hepatocyte "ploidy conveyer" is regulated differently during aging and regeneration. The findings of reversal of hepatocyte senescence could enable future studies on liver aging and cell therapy. © 2014 by the American Association for the Study of Liver Diseases.

Cryopreservation and gel collagen culture of porcine hepatocytes

PubMed Central

Liu, Hong-Ling; Wang, Ying-Jie; Guo, Hai-Tao; Wang, Yu-Ming; Liu, Jun; Yu, Yue-Cheng

2004-01-01

AIM: To study the method of cryopreserving porcine hepatocytes and gel collagen culture measure after its cryopreservation. METHODS: Hepatocytes, isolated from Chinese experimental suckling mini-pigs by two-step perfusion with collagenase using an extra corporeal perfusion apparatus, were cryopreserved with 50 mL/L to 200 mL/L DMSO in liquid nitrogen for 4 mo, then thawed and seeded in 1 or between 2 layers of gel collagen. The expression of porcine albumin message RNA, cellular morphology and content of aspartate aminotransferase (AST) and urea nitrogen (UN) were examined during culture in gel. RESULTS: Viability of 150 mL/L DMSO group thawed hepatocytes was (83 ± 4)%, but after purification, its viability was (90 ± 5)%, attachment efficiency was (86 ± 7)%, the viability of thawed hepatocytes was near to fresh cells. When the thawed hepatocytes were cultivated in gel collagen with culture medium adding epidermal growth factor, the hepatocytes grew in various administrative levels in mixed collagen gel, and bunchy in the sandwich configuration cultures. For up to 10 days’ culture, the typical cellular morphological characteristics of cultivated hepatocytes could be observed. The leakage of AST was lower during culture in gel than that in common culture. At the same time, the UN synthesized by cells cultivated in mixed gel collagen was higher than that in other groups. CONCLUSION: Storage in liquid nitrogen can long keep hepatocytes’ activities, the concentration of 150 mL/L DMSO is fit for porcine hepatocytes’ cryopreservation. Thawed hepatocytes can be cultivated with collagenous matrix, which provides an environment that more closely resembles that in vivo and maintain the expression of certain liver-specific function of hepatocytes. PMID:15052684

Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species

PubMed Central

Semple, Bridgette D.; Blomgren, Klas; Gimlin, Kayleen; Ferriero, Donna M.; Noble-Haeusslein, Linda J.

2013-01-01

Hypoxic-ischemic and traumatic brain injuries are leading causes of long-term mortality and disability in infants and children. Although several preclinical models using rodents of different ages have been developed, species differences in the timing of key brain maturation events can render comparisons of vulnerability and regenerative capacities difficult to interpret. Traditional models of developmental brain injury have utilized rodents at postnatal day 7–10 as being roughly equivalent to a term human infant, based historically on the measurement of post-mortem brain weights during the 1970s. Here we will examine fundamental brain development processes that occur in both rodents and humans, to delineate a comparable time course of postnatal brain development across species. We consider the timing of neurogenesis, synaptogenesis, gliogenesis, oligodendrocyte maturation and age-dependent behaviors that coincide with developmentally regulated molecular and biochemical changes. In general, while the time scale is considerably different, the sequence of key events in brain maturation is largely consistent between humans and rodents. Further, there are distinct parallels in regional vulnerability as well as functional consequences in response to brain injuries. With a focus on developmental hypoxicischemic encephalopathy and traumatic brain injury, this review offers guidelines for researchers when considering the most appropriate rodent age for the developmental stage or process of interest to approximate human brain development. PMID:23583307

Selective insulin resistance in hepatocyte senescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aravinthan, Aloysious; Challis, Benjamin; Shannon, Nicholas

Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied inmore » three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance.« less

Urban resident attitudes toward rodents, rodent control products, and environmental effects

EPA Science Inventory

Rodent control in urban areas can result in the inadvertent mortality of non-target species (e.g., bobcats). However, there is little detailed information about rodent control practices of urban residents. Our objective was to evaluate urban rodent control behaviors in two area...

Hepatocyte polyploidization and its association with pathophysiological processes.

PubMed

Wang, Min-Jun; Chen, Fei; Lau, Joseph T Y; Hu, Yi-Ping

2017-05-18

A characteristic cellular feature of the mammalian liver is the progressive polyploidization of the hepatocytes, where individual cells acquire more than two sets of chromosomes. Polyploidization results from cytokinesis failure that takes place progressively during the course of postnatal development. The proportion of polyploidy also increases with the aging process or with cellular stress such as surgical resection, toxic stimulation, metabolic overload, or oxidative damage, to involve as much as 90% of the hepatocytes in mice and 40% in humans. Hepatocyte polyploidization is generally considered an indicator of terminal differentiation and cellular senescence, and related to the dysfunction of insulin and p53/p21 signaling pathways. Interestingly, the high prevalence of hepatocyte polyploidization in the aged mouse liver can be reversed when the senescent hepatocytes are serially transplanted into young mouse livers. Here we review the current knowledge on the mechanism of hepatocytes polyploidization during postnatal growth, aging, and liver diseases. The biologic significance of polyploidization in senescent reversal, within the context of new ways to think of liver aging and liver diseases is considered.

Hepatocyte polyploidization and its association with pathophysiological processes

PubMed Central

Wang, Min-Jun; Chen, Fei; Lau, Joseph T Y; Hu, Yi-Ping

2017-01-01

A characteristic cellular feature of the mammalian liver is the progressive polyploidization of the hepatocytes, where individual cells acquire more than two sets of chromosomes. Polyploidization results from cytokinesis failure that takes place progressively during the course of postnatal development. The proportion of polyploidy also increases with the aging process or with cellular stress such as surgical resection, toxic stimulation, metabolic overload, or oxidative damage, to involve as much as 90% of the hepatocytes in mice and 40% in humans. Hepatocyte polyploidization is generally considered an indicator of terminal differentiation and cellular senescence, and related to the dysfunction of insulin and p53/p21 signaling pathways. Interestingly, the high prevalence of hepatocyte polyploidization in the aged mouse liver can be reversed when the senescent hepatocytes are serially transplanted into young mouse livers. Here we review the current knowledge on the mechanism of hepatocytes polyploidization during postnatal growth, aging, and liver diseases. The biologic significance of polyploidization in senescent reversal, within the context of new ways to think of liver aging and liver diseases is considered. PMID:28518148

Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments

PubMed Central

1985-01-01

The endocytic compartments of the asialoglycoprotein (ASGP) pathway in rat hepatocytes were studied using a combined morphological and biochemical approach in the isolated perfused liver. Use of electron microscopic tracers and a temperature-shift protocol to synchronize ligand entry confirmed the route of ASGP internalization observed in our previous in vivo studies (1) and established conditions under which we could label the contents of successive compartments in the pathway for subcellular fractionation studies. Three endosomal compartments were demonstrated in which ASGPs appear after they enter the cell via coated pits and vesicles but before they reach their site of degradation in lysosomes. These three compartments could be distinguished by their location within the hepatocyte, by their morphological appearance in situ, and by their density in sucrose gradients. The distributions of ASGP receptors, both accessible and latent (revealed by detergent permeabilization), were also examined and compared with that of ligand during subcellular fractionation. Most accessible ASGP receptors co-distributed with conventional plasma membrane markers. However, hepatocytes contain a substantial intracellular pool of latent ASGP binding sites that exceeds the number of cell surface receptors and whose presence is not dependent on ASGP exposure. The distribution of these latent ASGP receptors on sucrose gradients (detected either immunologically or by binding assays) was coincident with that of ligand sequestered within the early endosome compartments. In addition, both early endosomes and the membrane vesicles containing latent ASGP receptors had high cholesterol content, because both shifted markedly in density upon exposure to digitonin. PMID:2866191

Interactions between macrophage/Kupffer cells and hepatocytes in surgical sepsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

West, M.A.

Experiments were performed to investigate the role of Kupffer cell/macrophage interactions with hepatocytes in modulating liver function during infections using direct in vitro cocultivation of rat macrophages or Kupffer cells with rat hepatocytes. Protein synthesis was assayed as a sensitive indicator of integrated hepatocellular function by measuring {sup 3}H-leucine incorporation into hepatocyte protein. Septic stimuli such as lipoploysaccharide and killed bacteria were added to cocultures of hepatocytes and macrophages or Kupffer cells and the responses compared to hepatocytes alone. Information about the types of proteins synthesized by hepatocytes under various culture conditions was determined using polyacrylamide gel electrophoresis and autoradiography.more » These experiments showed that septic stimuli alter the amount and type of protein synthesized by hepatocytes and had no direct effect on hepatocytes in the absence of macrophages or Kupffer cells. The mediator(s) appears to be a heat labile, soluble monokine(s) which is distinct from interleukin-1 or tumor necrosis factor. The important role of Kupffer cells/macrophages in mediating alterations in hepatocellular function in sepsis may ultimately improve patient care.« less

The potential of induced pluripotent stem cell derived hepatocytes.

PubMed

Hannoun, Zara; Steichen, Clara; Dianat, Noushin; Weber, Anne; Dubart-Kupperschmitt, Anne

2016-07-01

Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

A non-human primate model of human radiation-induced venocclusive liver disease and hepatocyte injury

PubMed Central

Yamamoto, Toshiyuki; Ito, Ryotaro; Brooks, Jenna M.; Guzman-Lepe, Jorge; Galambos, Csaba; Fong, Jason V.; Deutsch, Melvin; Quader, Mubina A.; Yamanouchi, Kosho; Kabarriti, Rafi; Mehta, Keyur; Soto-Gutierrez, Alejandro; Roy-Chowdhury, Jayanta; Locker, Joseph; Abe, Michio; Enke, Charles A.; Baranowska-Kortylewicz, Janina; Solberg, Timothy D.; Guha, Chandan; Fox, Ira J.

2014-01-01

Background Human liver has an unusual sensitivity to radiation that limits its use in cancer therapy or in preconditioning for hepatocyte transplantation. Since the characteristic venocclusive lesions of radiation-induced liver disease do not occur in rodents, there has been no experimental model to investigate the limits of safe radiation therapy or explore the pathogenesis of hepatic venocclusive disease. Methods We performed a dose escalation study in a primate, the cynomolgus monkey, using hypofractionated stereotactic body radiotherapy in 13 animals. Results At doses ≥40Gy, animals developed a systemic syndrome resembling human radiation-induced liver disease, consisting of decreased albumin, elevated alkaline phosphatase, loss of appetite, ascites, and normal bilirubin. Higher radiation doses were lethal, causing severe disease that required euthanasia approximately 10 weeks after radiation. Even at lower doses where radiation-induced liver disease was mild or non-existent, latent and significant injury to hepatocytes was demonstrated by asialoglycoprotein-mediated functional imaging. These monkeys developed hepatic failure with encephalopathy when they received parenteral nutrition containing high concentrations of glucose. Histologically, livers showed central obstruction via an unusual intimal swelling that progressed to central fibrosis. Conclusions The cynomolgus monkey, as the first animal model of human venocclusive radiation-induced liver disease, provides a resource for characterizing the early changes and pathogenesis of venocclusion, for establishing nonlethal therapeutic dosages, and for examining experimental therapies to minimize radiation injury. PMID:24315566

Reactive oxygen species mediate human hepatocyte injury during hypoxia/reoxygenation.

PubMed

Bhogal, Ricky Harminder; Curbishley, Stuart M; Weston, Christopher J; Adams, David H; Afford, Simon C

2010-11-01

Increasing evidence shows that reactive oxygen species (ROS) may be critical mediators of liver damage during the relative hypoxia of ischemia/reperfusion injury (IRI) associated with transplant surgery or of the tissue microenvironment created as a result of chronic hepatic inflammation or infection. Much work has been focused on Kupffer cells or liver resident macrophages with respect to the generation of ROS during IRI. However, little is known about the contribution of endogenous hepatocyte ROS production or its potential impact on the parenchymal cell death associated with IRI and chronic hepatic inflammation. For the first time, we show that human hepatocytes isolated from nondiseased liver tissue and human hepatocytes isolated from diseased liver tissue exhibit marked differences in ROS production in response to hypoxia/reoxygenation (H-R). Furthermore, several different antioxidants are able to abrogate hepatocyte ROS-induced cell death during hypoxia and H-R. These data provide clear evidence that endogenous ROS production by mitochondria and nicotinamide adenine dinucleotide phosphate oxidase drives human hepatocyte apoptosis and necrosis during hypoxia and H-R and may therefore play an important role in any hepatic diseases characterized by a relatively hypoxic liver microenvironment. In conclusion, these data strongly suggest that hepatocytes and hepatocyte-derived ROS are active participants driving hepatic inflammation. These novel findings highlight important functional/metabolic differences between hepatocytes isolated from normal donor livers, hepatocytes isolated from normal resected tissue obtained during surgery for malignant neoplasms, and hepatocytes isolated from livers with end-stage disease. Furthermore, the targeting of hepatocyte ROS generation with antioxidants may offer therapeutic potential for the adjunctive treatment of IRI and chronic inflammatory liver diseases. © 2010 AASLD.

Metabolism of para-aminophenol by rat hepatocytes.

PubMed

Yan, Z; Nikelly, J G; Killmer, L; Tarloff, J B

2000-08-01

Autoxidation of para-aminophenol (PAP) has been proposed to account for the selective nephrotoxicity of this compound. However, other studies suggest that hepatic metabolites of PAP rather than the parent compound may be responsible for renal damage. These studies were designed to investigate PAP metabolism in isolated hepatocytes. We synthesized several proposed metabolites for analysis by HPLC/mass spectrometry and compared those results with HPLC/mass spectrometric analyses of metabolites found after incubating hepatocytes with PAP. Hepatocytes prepared from male Sprague-Dawley rats were incubated in Krebs-Henseleit buffer at 37 degrees C for 5 h with 2.3 mM PAP under an atmosphere of 5% CO2/95% O2. Aliquots were withdrawn at 0.1 h of incubation and then hourly through 5 h of incubation. Reactions were terminated by the addition of acetonitrile. Hepatocyte viability was unaltered with PAP present in the incubation medium. We found that hepatocytes converted PAP to two major metabolites (PAP-GSH conjugates and PAP-N-acetylcysteine conjugates) and several minor metabolites [PAP-O-glucuronide, acetaminophen (APAP), APAP-O-glucuronide, APAP-GSH conjugates, and 4-hydroxyformanilide]. Preincubating hepatoyctes with 1-aminobenzotriazole, an inhibitor of cytochromes P450, did not alter the pattern of PAP metabolism. In conclusion, we found that PAP was metabolized in hepatocytes predominantly to PAP-GSH conjugates and PAP-N-acetylcysteine conjugates in sufficient quantities to account for the nephrotoxicity of PAP.

CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface fas ligand expression and amplifies fas-mediated hepatocyte death during allograft rejection.

PubMed

Afford, S C; Randhawa, S; Eliopoulos, A G; Hubscher, S G; Young, L S; Adams, D H

1999-01-18

We propose that a novel mechanism of hepatocyte apoptosis, involving a cooperative interaction between CD40 and Fas, is involved in the hepatocyte loss of chronic liver allograft rejection. We detected increased hepatocyte expression of Fas, Fas ligand (FasL), and CD40 associated with dropout of centrilobular (acinar zone 3) hepatocytes in chronic allograft rejection. Expression of CD40 ligand (CD40L) was also increased but was largely restricted to CD68(+) macrophages. A functional role for CD40 and Fas in hepatocyte apoptosis was demonstrated in vitro using primary human hepatocytes and the HepG2 cell line in both of which apoptosis was induced, not only by cross-linking Fas directly but also via CD40 activation. Our data suggest that CD40 activation induces apoptosis via Fas because (a) ligation of CD40 upregulated hepatocyte FasL expression, and (b) apoptosis induced via activation of CD40 was prevented by a neutralizing monoclonal antibody to FasL. Thus, CD40 engagement triggers apoptosis of human hepatocytes and might amplify Fas-dependent hepatocyte apoptosis in chronic rejection and other inflammatory liver diseases in which Fas-mediated apoptosis is involved.

TRANSPLANTATION OF HEPATOCYTES FROM GENETICALLY-ENGINEERED PIGS IN BABOONS

PubMed Central

Iwase, Hayato; Liu, Hong; Schmelzer, Eva; Ezzelarab, Mohamed; Wijkstrom, Martin; Hara, Hidetaka; Lee, Whayoung; Singh, Jagjit; Long, Cassandra; Lagasse, Eric; Gerlach, Jörg C.; Cooper, David K.C.; Gridelli, Bruno

2017-01-01

Background Some patients with acute or acute-on-chronic hepatic failure die before a suitable human liver allograft becomes available. Encouraging results have been achieved in such patients by the transplantation of human hepatocyte progenitor cells from fetal liver tissue. The aim of the study was to explore survival of hepatocytes from genetically-engineered pigs after direct injection into the spleen and other selected sites in immunosuppressed baboons to monitor the immune response and the metabolic function and survival of the transplanted hepatocytes. Methods Baboons (n=3) were recipients of GTKO/hCD46 pig hepatocytes. All three baboons received anti-thymocyte globulin (ATG) induction and tapering methylprednisolone. Baboon 1 received maintenance immunosuppressive therapy with tacrolimus and rapamycin. Baboons 2 and 3 received an anti-CD40mAb/rapamycin-based regimen that prevents sensitization to pig solid organ grafts. The baboons were euthanized 4 or 5 weeks after hepatocyte transplantation. The baboon immune response was monitored by measurement of anti-nonGal IgM and IgG antibodies (by flow cytometry) and CFSE-mixed lymphocyte reaction. Monitoring for hepatocyte survival and function was by (i) real-time PCR detection of porcine DNA, (ii) real-time PCR for porcine gene expression, and (iii) pig serum albumin levels (by ELISA). The sites of hepatocyte injection were examined microscopically. Results Detection of porcine DNA and porcine gene expression was minimal at all sites of hepatocyte injection. Serum levels of porcine albumen were very low – 500–1,000-fold lower than in baboons with orthotopic pig liver grafts, and approximately 5,000-fold lower than in healthy pigs. No hepatocytes or infiltrating immune cells were seen at any of the injection sites. Two baboons (Baboons 1 and 3) demonstrated a significant increase in anti-pig IgM and an even greater increase in IgG, indicating sensitization to pig antigens. Discussion and Conclusions As a

Rodent phylogeny revised: analysis of six nuclear genes from all major rodent clades

PubMed Central

Blanga-Kanfi, Shani; Miranda, Hector; Penn, Osnat; Pupko, Tal; DeBry, Ronald W; Huchon, Dorothée

2009-01-01

Background Rodentia is the most diverse order of placental mammals, with extant rodent species representing about half of all placental diversity. In spite of many morphological and molecular studies, the family-level relationships among rodents and the location of the rodent root are still debated. Although various datasets have already been analyzed to solve rodent phylogeny at the family level, these are difficult to combine because they involve different taxa and genes. Results We present here the largest protein-coding dataset used to study rodent relationships. It comprises six nuclear genes, 41 rodent species, and eight outgroups. Our phylogenetic reconstructions strongly support the division of Rodentia into three clades: (1) a "squirrel-related clade", (2) a "mouse-related clade", and (3) Ctenohystrica. Almost all evolutionary relationships within these clades are also highly supported. The primary remaining uncertainty is the position of the root. The application of various models and techniques aimed to remove non-phylogenetic signal was unable to solve the basal rodent trifurcation. Conclusion Sequencing and analyzing a large sequence dataset enabled us to resolve most of the evolutionary relationships among Rodentia. Our findings suggest that the uncertainty regarding the position of the rodent root reflects the rapid rodent radiation that occurred in the Paleocene rather than the presence of conflicting phylogenetic and non-phylogenetic signals in the dataset. PMID:19341461

[State of hepatocyte transplantation: a risk or a chance?].

PubMed

Leckel, K; Blaheta, R A; Markus, B H

2003-04-01

Over the past few years, hepatocyte transplantation has been considered as an alternative method for orthotopic liver transplantation for the treatment of various liver diseases. Beside curative approach for genetic metabolic deficiencies (familial hypercholesterolemia, hemophilia, etc.), it could be a useful tool for bridging the waiting period until an appropriate donor organ is obtained. In preclinical animal studies, hepatocytes injected intraperitoneally, intraportally or into the spleen settle down in the diseased liver. This enables genetic modification to correct inborn metabolic deficiencies and improves survival in acute liver failure. In 1992, the first clinical transplantation of isolated hepatocytes in 10 patients was performed. In 1998, Fox and coworkers described the successful transplantation of allogeneic liver cells in a child with Crigler-Najjar syndrome. Accomplished studies of Strom et al. resp. Bilir et al. of the same year proved the effectiveness of liver cell transplantation for transient treatment of acute liver failure. Prerequisite of this cell-based therapeutic strategy is a sufficient amount of highly differentiated hepatocytes, hence, a well established in-vitro cell-culture technique is necessary to yield a reproducible number of proliferating hepatocytes and to preserve the physiological cell function. This review discusses the different experimental approaches regarding the cultivation of human hepatocytes and also the use of alternative cell sources (like animal hepatocytes, immortalized cells of human origin, progenitor cells from fetal human liver/liver stem cells) for hepatocyte transplantation.

Low asialoglycoprotein receptor expression as markers for highly proliferative potential hepatocytes.

PubMed

Ise, H; Sugihara, N; Negishi, N; Nikaido, T; Akaike, T

2001-07-13

Development of a reliable method to isolate highly proliferative potential hepatocytes will provide insight into the molecular mechanisms of liver regeneration, as well as proving crucial for the development of a biohybrid artificial liver. The aim of this study is to isolate highly proliferative, e.g., progenitor-like, hepatocytes. To this end, we fractionated hepatocytes expressing low and high levels of the asialoglycoprotein receptor (ASGP-R) based on the difference in their adhesion to poly[N-p-vinylbenzyl-O-beta-d-galactopyranosyl-(1-->4)-d-gluconamide] (PVLA), and examined the proliferative activity and gene expression of these fractionated hepatocytes. The results showed that approximately 0.5 to 1% of the total number of hepatocytes, which showed low adhesion to PVLA, expressed low levels of the ASGP-R, while the rest of hepatocyte population with high adhesion to PVLA expressed high levels of the ASGP-R. Interestingly hepatocytes with low ASGP-R expression levels had much higher DNA synthesizing activity (i.e., are much more proliferative) than those with high ASGP-R expression levels. Moreover, hepatocytes with low ASGP-R expression levels expressed higher levels of epidermal growth factor receptor (EGF-R), CD29 (beta1 integrin) and CD49f (alpha6 integrin) and lower levels of glutamine synthetase than those with high ASGP-R expression. These findings suggested that hepatocytes with low adhesion to PVLA due to their low ASGP-R expression could be potential candidates for progenitor-like hepatocytes due to their high proliferative capacity; hence, the low expression of the ASGP-R could be a unique marker for progenitor hepatocytes. The isolation of hepatocytes with different functional phenotypes using PVLA may provide a new research tool for a better understanding of the biology of hepatocytes and the mechanisms regulating their proliferation and differentiation in health and disease. Copyright 2001 Academic Press.

Mfsd2a+ hepatocytes repopulate the liver during injury and regeneration

PubMed Central

Pu, Wenjuan; Zhang, Hui; Huang, Xiuzhen; Tian, Xueying; He, Lingjuan; Wang, Yue; Zhang, Libo; Liu, Qiaozhen; Li, Yan; Li, Yi; Zhao, Huan; Liu, Kuo; Lu, Jie; Zhou, Yingqun; Huang, Pengyu; Nie, Yu; Yan, Yan; Hui, Lijian; Lui, Kathy O.; Zhou, Bin

2016-01-01

Hepatocytes are functionally heterogeneous and are divided into two distinct populations based on their metabolic zonation: the periportal and pericentral hepatocytes. During liver injury and regeneration, the cellular dynamics of these two distinct populations remain largely elusive. Here we show that major facilitator super family domain containing 2a (Mfsd2a), previously known to maintain blood–brain barrier function, is a periportal zonation marker. By genetic lineage tracing of Mfsd2a+ periportal hepatocytes, we show that Mfsd2a+ population decreases during liver homeostasis. Nevertheless, liver regeneration induced by partial hepatectomy significantly stimulates expansion of the Mfsd2a+ periportal hepatocytes. Similarly, during chronic liver injury, the Mfsd2a+ hepatocyte population expands and completely replaces the pericentral hepatocyte population throughout the whole liver. After injury recovery, the adult liver re-establishes the metabolic zonation by reprogramming the Mfsd2a+-derived hepatocytes into pericentral hepatocytes. The evidence of entire zonation replacement during injury increases our understanding of liver biology and disease. PMID:27857132

Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes

PubMed Central

Tarlow, Branden D.; Pelz, Carl; Naugler, Willscott E.; Wakefield, Leslie; Wilson, Elizabeth M.; Finegold, Milton J.; Grompe, Markus

2014-01-01

Summary Adult liver progenitor cells are biliary-like epithelial cells that emerge only under injury conditions in the periportal region of the liver. They exhibit phenotypes of both hepatocytes and bile ducts. However, their origin and their significance to injury repair remain unclear. Here, we used a chimeric lineage tracing system to demonstrate that hepatocytes contribute to the progenitor pool. RNA-sequencing, ultrastructural analysis, and in vitro progenitor assays revealed that hepatocyte-derived progenitors were distinct from their biliary-derived counterparts. In vivo lineage tracing and serial transplantation assays showed that hepatocyte-derived proliferative ducts retained a memory of their origin and differentiated back into hepatocytes upon cessation of injury. Similarly, human hepatocytes in chimeric mice also gave rise to biliary progenitors in vivo. We conclude that human and mouse hepatocytes can undergo reversible ductal metaplasia in response to injury, expand as ducts and subsequently contribute to restoration of the hepatocyte mass. PMID:25312494

Hepatocyte-induced CD4+ T cell alloresponse is associated with major histocompatibility complex class II up-regulation on hepatocytes and suppressible by regulatory T cells.

PubMed

DeTemple, Daphne E; Oldhafer, Felix; Falk, Christine S; Chen-Wacker, Chen; Figueiredo, Constanca; Kleine, Moritz; Ramackers, Wolf; Timrott, Kai; Lehner, Frank; Klempnauer, Juergen; Bock, Michael; Vondran, Florian W R

2018-03-01

Hepatocyte transplantation is a promising therapeutic approach for various liver diseases. Despite the liver's tolerogenic potential, early immune-mediated loss of transplanted cells is observed, and longterm acceptance has not been achieved yet. Patients deemed tolerant after liver transplantation presented an increased frequency of regulatory T cells (Tregs), which therefore also might enable reduction of posttransplant cell loss and enhance longterm allograft acceptance. We hence characterized hepatocyte-induced immune reactions and evaluated the immunomodulatory potential of Tregs applying mixed lymphocyte cultures and mixed lymphocyte hepatocyte cultures. These were set up using peripheral blood mononuclear cells and primary human hepatocytes, respectively. Polyclonally expanded CD4 + CD25 high CD127 low Tregs were added to cocultures in single-/trans-well setups with/without supplementation of anti-interferon γ (IFNγ) antibodies. Hepatocyte-induced alloresponses were then analyzed by multicolor flow cytometry. Measurements indicated that T cell response upon stimulation was associated with IFNγ-induced major histocompatibility complex (MHC) class II up-regulation on hepatocytes and mediated by CD4 + T cells. An indirect route of antigen presentation could be ruled out by use of fragmented hepatocytes and culture supernatants of hepatocytes. Allospecific proliferation was accompanied by inflammatory cytokine secretion. CD8 + T cells showed early up-regulation of CD69 despite lack of cell proliferation in the course of coculture. Supplementation of Tregs effectively abrogated hepatocyte-induced alloresponses and was primarily cell contact dependent. In conclusion, human hepatocytes induce a CD4 + T cell alloresponse in vitro, which is associated with MHC class II up-regulation on hepatocytes and is susceptible to suppression by Tregs. Liver Transplantation 24 407-419 2018 AASLD. © 2018 The Authors. Liver Transplantation published by Wiley Periodicals, Inc

A Nonhuman Primate Model of Human Radiation-Induced Venocclusive Liver Disease and Hepatocyte Injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yannam, Govardhana Rao; Han, Bing; Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi

Background: Human liver has an unusual sensitivity to radiation that limits its use in cancer therapy or in preconditioning for hepatocyte transplantation. Because the characteristic veno-occlusive lesions of radiation-induced liver disease do not occur in rodents, there has been no experimental model to investigate the limits of safe radiation therapy or explore the pathogenesis of hepatic veno-occlusive disease. Methods and Materials: We performed a dose-escalation study in a primate, the cynomolgus monkey, using hypofractionated stereotactic body radiotherapy in 13 animals. Results: At doses ≥40 Gy, animals developed a systemic syndrome resembling human radiation-induced liver disease, consisting of decreased albumin, elevatedmore » alkaline phosphatase, loss of appetite, ascites, and normal bilirubin. Higher radiation doses were lethal, causing severe disease that required euthanasia approximately 10 weeks after radiation. Even at lower doses in which radiation-induced liver disease was mild or nonexistent, latent and significant injury to hepatocytes was demonstrated by asialoglycoprotein-mediated functional imaging. These monkeys developed hepatic failure with encephalopathy when they received parenteral nutrition containing high concentrations of glucose. Histologically, livers showed central obstruction via an unusual intimal swelling that progressed to central fibrosis. Conclusions: The cynomolgus monkey, as the first animal model of human veno-occlusive radiation-induced liver disease, provides a resource for characterizing the early changes and pathogenesis of venocclusion, for establishing nonlethal therapeutic dosages, and for examining experimental therapies to minimize radiation injury.« less

Exposure of small rodents to plague during epizootics in black-tailed prairie dogs.

PubMed

Stapp, Paul; Salkeld, Daniel J; Eisen, Rebecca J; Pappert, Ryan; Young, John; Carter, Leon G; Gage, Kenneth L; Tripp, Daniel W; Antolin, Michael F

2008-07-01

Plague, caused by the bacterium Yersinia pestis, causes die-offs of colonies of prairie dogs (Cynomys ludovicianus). It has been argued that other small rodents are reservoirs for plague, spreading disease during epizootics and maintaining the pathogen in the absence of prairie dogs; yet there is little empirical support for distinct enzootic and epizootic cycles. Between 2004 and 2006, we collected blood from small rodents captured in colonies in northern Colorado before, during, and for up to 2 yr after prairie dog epizootics. We screened 1,603 blood samples for antibodies to Y. pestis, using passive hemagglutination and inhibition tests, and for a subset of samples we cultured blood for the bacterium itself. Of the four species of rodents that were common in colonies, the northern grasshopper mouse (Onychomys leucogaster) was the only species with consistent evidence of plague infection during epizootics, with 11.1-23.1% of mice seropositive for antibody to Y. pestis during these events. Seropositive grasshopper mice, thirteen-lined ground squirrels (Spermophilus tridecemlineatus), and deer mice (Peromyscus maniculatus) were captured the year following epizootics. The appearance of antibodies to Y. pestis in grasshopper mice coincided with periods of high prairie dog mortality; subsequently, antibody prevalence rates declined, with no seropositive individuals captured 2 yr after epizootics. We did not detect plague in any rodents off of colonies, or on colonies prior to epizootics, and found no evidence of persistent Y. pestis infection in blood cultures. Our results suggest that grasshopper mice could be involved in epizootic spread of Y. pestis, and possibly, serve as a short-term reservoir for plague, but provide no evidence that the grasshopper mouse or any small rodent acts as a long-term, enzootic host for Y. pestis in prairie dog colonies.

Transplantation of hepatocytes from genetically engineered pigs into baboons.

PubMed

Iwase, Hayato; Liu, Hong; Schmelzer, Eva; Ezzelarab, Mohamed; Wijkstrom, Martin; Hara, Hidetaka; Lee, Whayoung; Singh, Jagjit; Long, Cassandra; Lagasse, Eric; Gerlach, Jörg C; Cooper, David K C; Gridelli, Bruno

2017-03-01

Some patients with acute or acute-on-chronic hepatic failure die before a suitable human liver allograft becomes available. Encouraging results have been achieved in such patients by the transplantation of human hepatocyte progenitor cells from fetal liver tissue. The aim of the study was to explore survival of hepatocytes from genetically engineered pigs after direct injection into the spleen and other selected sites in immunosuppressed baboons to monitor the immune response and the metabolic function and survival of the transplanted hepatocytes. Baboons (n=3) were recipients of GTKO/hCD46 pig hepatocytes. All three baboons received anti-thymocyte globulin (ATG) induction and tapering methylprednisolone. Baboon 1 received maintenance immunosuppressive therapy with tacrolimus and rapamycin. Baboons 2 and 3 received an anti-CD40mAb/rapamycin-based regimen that prevents sensitization to pig solid organ grafts. The baboons were euthanized 4 or 5 weeks after hepatocyte transplantation. The baboon immune response was monitored by the measurement of anti-non-Gal IgM and IgG antibodies (by flow cytometry) and CFSE-mixed lymphocyte reaction. Monitoring for hepatocyte survival and function was by (i) real-time PCR detection of porcine DNA, (ii) real-time PCR for porcine gene expression, and (iii) pig serum albumin levels (by ELISA). The sites of hepatocyte injection were examined microscopically. Detection of porcine DNA and porcine gene expression was minimal at all sites of hepatocyte injection. Serum levels of porcine albumen were very low-500-1000-fold lower than in baboons with orthotopic pig liver grafts, and approximately 5000-fold lower than in healthy pigs. No hepatocytes or infiltrating immune cells were seen at any of the injection sites. Two baboons (Baboons 1 and 3) demonstrated a significant increase in anti-pig IgM and an even greater increase in IgG, indicating sensitization to pig antigens. As a result of this disappointing experience, the following points

Disruption of Redox Homeostasis in Tumor Necrosis Factor-Induced Apoptosis in a Murine Hepatocyte Cell Line

PubMed Central

Pierce, Robert H.; Campbell, Jean S.; Stephenson, Alyssa B.; Franklin, Christopher C.; Chaisson, Michelle; Poot, Martin; Kavanagh, Terrance J.; Rabinovitch, Peter S.; Fausto, Nelson

2000-01-01

Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytes in vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours ∼50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFκB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant α-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses. PMID

Zoonoses of rabbits and rodents.

PubMed

Hill, William Allen; Brown, Julie Paige

2011-09-01

Millions of households in the US own rabbits or rodents, including hamsters, guinea pigs, and gerbils. Activities such as hunting and camping also involve human interactions with wild rabbits and rodents. In many environments, feral rabbits and rodents live in close proximity to humans, domesticated animals, and other wildlife. Education of rodent and rabbit owners and individuals with occupational or recreational exposures to these species is paramount to reduce the prevalence of zoonoses associated with rabbit and rodent exposure.

Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation

PubMed Central

Xiang, D; Liu, C-C; Wang, M-J; Li, J-X; Chen, F; Yao, H; Yu, B; Lu, L; Borjigin, U; Chen, Y-X; Zhong, L; Wangensteen, K J; He, Z-Y; Wang, X; Hu, Y-P

2014-01-01

Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)–/– mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation. PMID:24853430

Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Atienzar, Franck A., E-mail: franck.atienzar@ucb.com; Novik, Eric I.; Gerets, Helga H.

Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine modelmore » along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes.« less

Destruction of the hepatocyte junction by intercellular invasion of Leptospira causes jaundice in a hamster model of Weil's disease.

PubMed

Miyahara, Satoshi; Saito, Mitsumasa; Kanemaru, Takaaki; Villanueva, Sharon Y A M; Gloriani, Nina G; Yoshida, Shin-ichi

2014-08-01

Weil's disease, the most severe form of leptospirosis, is characterized by jaundice, haemorrhage and renal failure. The mechanisms of jaundice caused by pathogenic Leptospira remain unclear. We therefore aimed to elucidate the mechanisms by integrating histopathological changes with serum biochemical abnormalities during the development of jaundice in a hamster model of Weil's disease. In this work, we obtained three-dimensional images of infected hamster livers using scanning electron microscope together with freeze-cracking and cross-cutting methods for sample preparation. The images displayed the corkscrew-shaped bacteria, which infiltrated the Disse's space, migrated between hepatocytes, detached the intercellular junctions and disrupted the bile canaliculi. Destruction of bile canaliculi coincided with the elevation of conjugated bilirubin, aspartate transaminase and alkaline phosphatase levels in serum, whereas serum alanine transaminase and γ-glutamyl transpeptidase levels increased slightly, but not significantly. We also found in ex vivo experiments that pathogenic, but not non-pathogenic leptospires, tend to adhere to the perijunctional region of hepatocyte couplets isolated from hamsters and initiate invasion of the intercellular junction within 1 h after co-incubation. Our results suggest that pathogenic leptospires invade the intercellular junctions of host hepatocytes, and this invasion contributes in the disruption of the junction. Subsequently, bile leaks from bile canaliculi and jaundice occurs immediately. Our findings revealed not only a novel pathogenicity of leptospires, but also a novel mechanism of jaundice induced by bacterial infection. © 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.

Destruction of the hepatocyte junction by intercellular invasion of Leptospira causes jaundice in a hamster model of Weil's disease

PubMed Central

Miyahara, Satoshi; Saito, Mitsumasa; Kanemaru, Takaaki; Villanueva, Sharon Y A M; Gloriani, Nina G; Yoshida, Shin-ichi

2014-01-01

Weil's disease, the most severe form of leptospirosis, is characterized by jaundice, haemorrhage and renal failure. The mechanisms of jaundice caused by pathogenic Leptospira remain unclear. We therefore aimed to elucidate the mechanisms by integrating histopathological changes with serum biochemical abnormalities during the development of jaundice in a hamster model of Weil's disease. In this work, we obtained three-dimensional images of infected hamster livers using scanning electron microscope together with freeze-cracking and cross-cutting methods for sample preparation. The images displayed the corkscrew-shaped bacteria, which infiltrated the Disse's space, migrated between hepatocytes, detached the intercellular junctions and disrupted the bile canaliculi. Destruction of bile canaliculi coincided with the elevation of conjugated bilirubin, aspartate transaminase and alkaline phosphatase levels in serum, whereas serum alanine transaminase and γ-glutamyl transpeptidase levels increased slightly, but not significantly. We also found in ex vivo experiments that pathogenic, but not non-pathogenic leptospires, tend to adhere to the perijunctional region of hepatocyte couplets isolated from hamsters and initiate invasion of the intercellular junction within 1 h after co-incubation. Our results suggest that pathogenic leptospires invade the intercellular junctions of host hepatocytes, and this invasion contributes in the disruption of the junction. Subsequently, bile leaks from bile canaliculi and jaundice occurs immediately. Our findings revealed not only a novel pathogenicity of leptospires, but also a novel mechanism of jaundice induced by bacterial infection. PMID:24945433

Targeted transplantation of mitochondria to hepatocytes

PubMed Central

Gupta, Nidhi; Wu, Catherine H; Wu, George Y

2016-01-01

Background Mitochondrial defects in hepatocytes can result in liver dysfunction and death. Hepatocytes have cell-surface asialoglycoprotein receptors (AsGRs) which internalize AsGs within endosomes. The aim of this study was to determine whether mitochondria could be targeted to hepatocytes by AsGR-mediated endocytosis. Materials and methods An AsG, AsOR, was linked to polylysine to create a conjugate, AsOR-PL, and complexed with healthy and functional mitochondria (defined by normal morphology, cytochrome c assays, and oxygen-consumption rates). Huh7 (AsGR+) and SK Hep1 (AsGR−) cells were treated with a mitochondrial toxin to form Huh7-Mito− and SK Hep1-Mito− cells, lacking detectable mitochondrial DNA. An endosomolytic peptide, LLO, was coupled to AsOR to form AsOR-LLO. A lysosomal inhibitor, amantadine, was used in mitochondria-uptake studies as a control for nonspecific endosomal release. Results Coincubation of complexed mitochondria and AsOR-LLO with Huh7-Mito− cells increased mitochondrial DNA to >9,700-fold over control at 7 days (P<0.001), and increased mitochondrial oxygen-consumption rates to >90% of control by 10 days. Conclusion Rescue of mitochondria-damaged hepatocytes can be achieved by targeted uptake of normal mitochondria through receptor-mediated endocytosis. PMID:27942238

GROWTH INHIBITORY ACTIONS OF PROTHROMBIN ON NORMAL HEPATOCYTES

PubMed Central

Carr, Brian I.; Kar, Siddhartha; Wang, Meifang; Wang, Ziqiu

2007-01-01

Most hepatomas have a defect in prothrombin carboxylation, and can secrete under-carboxylated prothrombin or des-γ-carboxy-prothrombin (DCP), the function of which is unknown. We considered that prothrombin-DCP axis might also be involved in growth control. Hepatocytes and hepatoma cells were treated with prothrombin, and DNA synthesis and cytoskeleton were studied. Prothrombin inhibited DNA synthesis in hepatocytes on fibronectin, but not collagen matrix. Hepatoma cell lines were not inhibited. We found that hepatoma cell matrix conferred resistance to hepatocytes. Prothrombin decreased fibronectin but not collagen amounts, but only in the presence of hepatocytes and not hepatoma cells, indicating that it has a differential action on matrix proteins. It also caused changes in cell shape and actin depolymerization. In vivo, there was a decrease in plasma prothrombin activity after a partial hepatectomy (PH) concomitant with a peak of DNA synthesis by the hepatocyte at 24 h after PH. Injection of warfarin at the time of PH, further inhibited PT activity and enhanced this 24 h peak of DNA synthesis. Furthermore, repeated injection of prothrombin lowered the peak DNA synthesis after PH. The data support the hypothesis that prothrombin can act as a hepatocyte growth inhibitor, likely at the level of fibronectin loss and result in cytoskeletal changes. Hepatomas resist this action, possibly due to their different matrix proteins. This represents a novel mechanism for growth regulation and provides a possible biological significance for the tumor marker DCP. PMID:17490900

MicroRNAs control hepatocyte proliferation during liver regeneration.

PubMed

Song, Guisheng; Sharma, Amar Deep; Roll, Garrett R; Ng, Raymond; Lee, Andrew Y; Blelloch, Robert H; Frandsen, Niels M; Willenbring, Holger

2010-05-01

MicroRNAs (miRNAs) constitute a new class of regulators of gene expression. Among other actions, miRNAs have been shown to control cell proliferation in development and cancer. However, whether miRNAs regulate hepatocyte proliferation during liver regeneration is unknown. We addressed this question by performing 2/3 partial hepatectomy (2/3 PH) on mice with hepatocyte-specific inactivation of DiGeorge syndrome critical region gene 8 (DGCR8), an essential component of the miRNA processing pathway. Hepatocytes of these mice were miRNA-deficient and exhibited a delay in cell cycle progression involving the G(1) to S phase transition. Examination of livers of wildtype mice after 2/3 PH revealed differential expression of a subset of miRNAs, notably an induction of miR-21 and repression of miR-378. We further discovered that miR-21 directly inhibits Btg2, a cell cycle inhibitor that prevents activation of forkhead box M1 (FoxM1), which is essential for DNA synthesis in hepatocytes after 2/3 PH. In addition, we found that miR-378 directly inhibits ornithine decarboxylase (Odc1), which is known to promote DNA synthesis in hepatocytes after 2/3 PH. Our results show that miRNAs are critical regulators of hepatocyte proliferation during liver regeneration. Because these miRNAs and target gene interactions are conserved, our findings may also be relevant to human liver regeneration.

[Study on the hepatocytic cell targetability of liposomes].

PubMed

Hou, Xin-pu; Wang, Li; Wang, Xiang-tao; Li, Sha

2003-02-01

To target for hepatocytic cell, liposomes was modified by special ligand. Sterically stabilized liposomes (SSL) was conjugated with asialofeticin (AF), the ligand of asialoglycoprotein receptor (ASGP-R) of hepatocyte. ASGP-R-BLM is the ASGP-R reconstructed on bilayer lipid membrane (BLM). The recognition reaction between AF-SSL and ASGP-R-BLM can be monitored by the varieties of membrane electrical parameters. The targetability of AF-SSL mediated to hepatocyte was detected by radioisotopic labeled in vitro and in vivo. The therapeutic effect of antihepatocarcinoma was observed also. The lifetime of ASGP-R-BLM decreased with the added amount of AF-SSL. It was demonstrated that there was recognition reaction between AF-SSL and ASGP-R-BLM. The combination of AF-SSL with hepatocyte was significantly higher than that of SSL without AF-modified in vitro and in vivo. The survival time of rat for AF-SSL carriered ADM (adriamycin) group was much longer and the toxicities on heart, kidney and lung were lower than those SSL carried ADM group. It is possible to actively target the cell with specific receptor by ligand modified liposomes. The result prvide scientific basis of hepatocyte targeted liposomes.

Experience of microbiological screening of human hepatocytes for clinical transplantation.

PubMed

Lehec, Sharon C; Hughes, Robin D; Mitry, Ragai R; Graver, Michelle A; Verma, Anita; Wade, Jim J; Dhawan, Anil

2009-01-01

Hepatocyte transplantation is being used in patients with liver-based metabolic disorders and acute liver failure. Hepatocytes are isolated from unused donor liver tissue under GMP conditions. Cells must be free of microbiological contamination to be safe for human use. The experience of microbiological screening during 72 hepatocyte isolation procedures at one center is reported. Samples were taken at different stages of the process and tested using a blood culture bottle system and Gram stain. Bacterial contamination was detected in 37.5% of the UW organ preservative solutions used to transport the liver tissue to the Cell Isolation Unit. After tissue processing the contamination was reduced to 7% overall in the final hepatocyte product, irrespective of the presence of initial contamination of the transport solution. The most common organisms recovered were coagulase-negative staphylococci, a skin commensal. A total of 41 preparations of fresh or cryopreserved hepatocytes were used for cell transplantation in children with liver-based metabolic disorders without any evidence of sepsis due to infusion of hepatocytes. In conclusion, the incidence of bacterial contamination of the final product was low, confirming the suitability of the organs used, hepatocyte isolation procedure, and the environmental conditions of the clean room.

Functional assessment of hepatocytes after transplantation into rat spleen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woods, R.J.; Fuller, B.J.; Attenburrow, V.D.

1982-02-01

The retention of structural integrity and metabolic function by isolated hepatocytes after ectopic transplantation has been investigated in autografted rats. Rats were partially hepatectomized and isolated hepatocytes prepared from the excised liver lobes were implanted into their spleens. Histological examination of the spleens 7 or more weeks after implantation revealed aggregates of hepatocytes in the red pulp. Two tests of biochemical function were applied to the hepatocytes after tranplantation. In the first the hepatobiliary imaging agent technetium-99m N-(N'-(2,6-dimethylphenyl)carbamoylmethyl)iminodiacetic acid (/sup 99//sup m/Tc HIDA), which was shown to be avidly taken up by isolated hepatocytes in vitro, was infused into themore » tail veins of autograft and control rats. Radioactivity accumulating in the spleens of autografted rats was markedly greater than that in controls implanted with lethally damaged cells or in nontransplanted rats. In the second the presence of bilirubin metabolites was sought in autograft spleens after intravenous infusion of bilirubin. Both mono- and diglucuronides of bilirubin were recovered from the spleens of autograft rats but no conjugates were recovered from the spleens of unoperated controls. We conclude that after autotransplantation isolated hepatocytes retain their morphology and at least some of their functional activities.« less

Functional assessment of hepatocytes after transplantation into rat spleen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woods, R.J.; Fuller, B.J.; Attenburrow, V.D.

1982-02-01

The retention of structural integrity and metabolic function by isolated hepatocytes after ectopic transplantation has been investigated in autografted rats. Rats were partially hepatectomized and isolated hepatocytes prepared from the excised liver lobes were implanted into their spleens. Histological examination of the spleens 7 or more weeks after implantation revealed aggregates of hepatocytes in the red pulp. Two tests of biochemical function were applied to the hepatocytes after transplantation. In the first the hepatobiliary imaging agent technetium-99m N-(N'-(2, 6-dimethylphenyl)carbamoylmethyl)iminodiacetic acid (99mTc HIDA), which was shown to be avidly taken up by isolated hepatocytes in vitro, was infused into the tailmore » veins of autograft and control rats. Radioactivity accumulating in the spleens of autografted rats was markedly greater than that in controls implanted with lethally damaged cells or in nontransplanted rats. In the second the presence of bilirubin metabolites was sought in autograft spleens after intravenous infusion of bilirubin. Both mono- and diglucuronides of bilirubin were recovered from the spleens of autograft rats but no conjugates were recovered from the spleens of unoperated controls. We conclude that after autotransplantation isolated hepatocytes retain their morphology and at least some of their functional activities.« less

Billion-scale production of hepatocyte-like cells from human induced pluripotent stem cells.

PubMed

Yamashita, Tomoki; Takayama, Kazuo; Sakurai, Fuminori; Mizuguchi, Hiroyuki

2018-02-19

Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells are expected to be utilized in drug screening and regenerative medicine. However, hepatocyte-like cells have not been fully used in such applications because it is difficult to produce such cells on a large scale. In this study, we tried to establish a method to mass produce hepatocyte-like cells using a three-dimensional (3D) cell culture bioreactor called the Rotary Cell Culture System (RCCS). RCCS enabled us to obtain homogenous hepatocyte-like cells on a billion scale (>10 9  cells). The gene expression levels of some hepatocyte markers (alpha-1 antitrypsin, cytochrome (CYP) 1A2, CYP2D6, and hepatocyte nuclear factor 4alpha) were higher in 3D-cultured hepatocyte-like cells than in 2D-cultured hepatocyte-like cells. This result suggests that RCCS could provide more suitable conditions for hepatocyte maturation than the conventional 2D cell culture conditions. In addition, more than 90% of hepatocyte-like cells were positive for albumin and could uptake low-density lipoprotein in the culture medium. We succeeded in the large-scale production of homogenous and functional hepatocyte-like cells from human iPS cells. This technology will be useful in drug screening and regenerative medicine, which require enormous numbers of hepatocyte-like cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Monitoring liver damage using hepatocyte-specific methylation markers in cell-free circulating DNA.

PubMed

Lehmann-Werman, Roni; Magenheim, Judith; Moss, Joshua; Neiman, Daniel; Abraham, Ofri; Piyanzin, Sheina; Zemmour, Hai; Fox, Ilana; Dor, Talya; Grompe, Markus; Landesberg, Giora; Loza, Bao-Li; Shaked, Abraham; Olthoff, Kim; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

2018-06-21

Liver damage is typically inferred from serum measurements of cytoplasmic liver enzymes. DNA molecules released from dying hepatocytes are an alternative biomarker, unexplored so far, potentially allowing for quantitative assessment of liver cell death. Here we describe a method for detecting acute hepatocyte death, based on quantification of circulating, cell-free DNA (cfDNA) fragments carrying hepatocyte-specific methylation patterns. We identified 3 genomic loci that are unmethylated specifically in hepatocytes, and used bisulfite conversion, PCR, and massively parallel sequencing to quantify the concentration of hepatocyte-derived DNA in mixed samples. Healthy donors had, on average, 30 hepatocyte genomes/ml plasma, reflective of basal cell turnover in the liver. We identified elevations of hepatocyte cfDNA in patients shortly after liver transplantation, during acute rejection of an established liver transplant, and also in healthy individuals after partial hepatectomy. Furthermore, patients with sepsis had high levels of hepatocyte cfDNA, which correlated with levels of liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Duchenne muscular dystrophy patients, in which elevated AST and ALT derive from damaged muscle rather than liver, did not have elevated hepatocyte cfDNA. We conclude that measurements of hepatocyte-derived cfDNA can provide specific and sensitive information on hepatocyte death, for monitoring human liver dynamics, disease, and toxicity.

Development of scaffold architectures and heterotypic cell systems for hepatocyte transplantation

NASA Astrophysics Data System (ADS)

Alzebdeh, Dalia Abdelrahim

In vitro assembly of functional liver tissue is needed to enable the transplantation of tissue-engineered livers. In addition, there is an increasing demand for in vitro models that replicate complex events occurring in the liver. However, tissue engineering of sizable implantable liver systems is currently limited by the difficulty of assembling three dimensional hepatocyte cultures of a useful size, while maintaining full cell viability, an issue which is closely related to the high metabolic rate of hepatocytes. In this study, we first compared two designs of highly porous chitosan-heparin scaffolds seeded with hepatocytes in dynamic perfusion bioreactor systems. The aim was to promote cell seeding efficiency by effectively entrapping 100 million hepatocytes at high density. We found that scaffolds with radially tapering pore architecture had highly efficient cell entrapment that maximized donor hepatocyte utilization, compared to alternate pore structures. Hepatocytes showed higher seeding efficiency and metabolic function when seeded as single cell suspensions as opposed to pre-formed, 100microm aggregates. Seeding efficiency was found to increase with flow rate, with single cell and aggregate suspension exhibiting different optimal flow rates. However, metabolic performance results indicated significant shear damage to cells at high efficiency flow rates. To better maintain hepatocyte basement membrane and cell polarity, spheroid co-cultures with mesenchymal stem cells (MSC) were investigated. Hepatocytes and MSCs were seeded in three different architectures in an effort to optimize the spatial arrangement of the two cell types. MSC co-culture greatly enhanced hepatocyte metabolic function in agitated cultures. Interestingly, the effects of diffusion limitations in spheroid culture, coupled with shear damage and subsequent removal of outer hepatocyte layers produced a defined oscillation of urea production rates in certain co-culture arrangements. A

HGF Secreted by Activated Kupffer Cells Induces Apoptosis of Plasmodium-Infected Hepatocytes

PubMed Central

Gonçalves, Lígia Antunes; Rodo, Joana; Rodrigues-Duarte, Lurdes; de Moraes, Luciana Vieira; Penha-Gonçalves, Carlos

2017-01-01

Malaria liver stage infection is an obligatory parasite development step and represents a population bottleneck in Plasmodium infections, providing an advantageous target for blocking parasite cycle progression. Parasite development inside hepatocytes implies a gross cellular insult evoking innate host responses to counteract intra-hepatocytic infection. Using primary hepatocyte cultures, we investigated the role of Kupffer cell-derived hepatocyte growth factor (HGF) in malaria liver stage infection. We found that Kupffer cells from Plasmodium-infected livers produced high levels of HGF, which trigger apoptosis of infected hepatocytes through a mitochondrial-independent apoptosis pathway. HGF action in infected hepatocyte primary cultures results in a potent reduction of parasite yield by specifically sensitizing hepatocytes carrying established parasite exo-erythrocytic forms to undergo apoptosis. This apoptosis mechanism is distinct from cell death that is spontaneously induced in infected cultures and is governed by Fas signaling modulation through a mitochondrial-dependent apoptosis pathway. This work indicates that HGF and Fas signaling pathways are part of an orchestrated host apoptosis response that occurs during malaria liver stage infection, decreasing the success of infection of individual hepatocytes. Our results raise the hypothesis that paracrine signals derived from Kupffer cell activation are implicated in directing death of hepatocytes infected with the malaria parasite. PMID:28220125

HGF Secreted by Activated Kupffer Cells Induces Apoptosis of Plasmodium-Infected Hepatocytes.

PubMed

Gonçalves, Lígia Antunes; Rodo, Joana; Rodrigues-Duarte, Lurdes; de Moraes, Luciana Vieira; Penha-Gonçalves, Carlos

2017-01-01

Malaria liver stage infection is an obligatory parasite development step and represents a population bottleneck in Plasmodium infections, providing an advantageous target for blocking parasite cycle progression. Parasite development inside hepatocytes implies a gross cellular insult evoking innate host responses to counteract intra-hepatocytic infection. Using primary hepatocyte cultures, we investigated the role of Kupffer cell-derived hepatocyte growth factor (HGF) in malaria liver stage infection. We found that Kupffer cells from Plasmodium -infected livers produced high levels of HGF, which trigger apoptosis of infected hepatocytes through a mitochondrial-independent apoptosis pathway. HGF action in infected hepatocyte primary cultures results in a potent reduction of parasite yield by specifically sensitizing hepatocytes carrying established parasite exo-erythrocytic forms to undergo apoptosis. This apoptosis mechanism is distinct from cell death that is spontaneously induced in infected cultures and is governed by Fas signaling modulation through a mitochondrial-dependent apoptosis pathway. This work indicates that HGF and Fas signaling pathways are part of an orchestrated host apoptosis response that occurs during malaria liver stage infection, decreasing the success of infection of individual hepatocytes. Our results raise the hypothesis that paracrine signals derived from Kupffer cell activation are implicated in directing death of hepatocytes infected with the malaria parasite.

Rat hepatocytes transport water mainly via a non-channel-mediated pathway.

PubMed

Yano, M; Marinelli, R A; Roberts, S K; Balan, V; Pham, L; Tarara, J E; de Groen, P C; LaRusso, N F

1996-03-22

During bile formation by the liver, large volumes of water are transported across two epithelial barriers consisting of hepatocytes and cholangiocytes (i.e. intrahepatic bile duct epithelial cells). We recently reported that a water channel, aquaporin-channel-forming integral protein of 28 kDa, is present in cholangiocytes and suggested that it plays a major role in water transport by these cells. Since the mechanisms of water transport across hepatocytes remain obscure, we performed physiological, molecular, and biochemical studies on hepatocytes to determine if they also contain water channels. Water permeability was studied by exposing isolated rat hepatocytes to buffers of different osmolarity and measuring cell volume by quantitative phase contrast, fluorescence and laser scanning confocal microscopy. Using this method, hepatocytes exposed to hypotonic buffers at 23 degrees C increased their cell volume in a time and osmolarity-dependent manner with an osmotic water permeability coefficient of 66.4 x 10(-4) cm/s. In studies done at 10 degrees C, the osmotic water permeability coefficient decreased by 55% (p < 0.001, at 23 degrees C; t test). The derived activation energy from these studies was 12.8 kcal/mol. After incubation of hepatocytes with amphotericin B at 10 degrees C, the osmotic water permeability coefficient increased by 198% (p < 0.001) and the activation energy value decreased to 3.6 kcal/mol, consistent with the insertion of artificial water channels into the hepatocyte plasma membrane. Reverse transcriptase polymerase chain reaction with hepatocyte RNA as template did not produce cDNAs for three of the known water channels. Both the cholesterol content and the cholesterol/phospholipid ratio of hepatocyte plasma membranes were significantly (p < 0.005) less than those of cholangiocytes; membrane fluidity of hepatocytes estimated by measuring steady-state anisotropy was higher than that of cholangiocytes. Our data suggests that the osmotic flow of

Hepatocyte Ploidy Is a Diversity Factor for Liver Homeostasis

PubMed Central

Kreutz, Clemens; MacNelly, Sabine; Follo, Marie; Wäldin, Astrid; Binninger-Lacour, Petra; Timmer, Jens; Bartolomé-Rodríguez, María M.

2017-01-01

Polyploidy, the existence of cells containing more than one pair of chromosomes, is a well-known feature of mammalian hepatocytes. Polyploid hepatocytes are found either as cells with a single polyploid nucleus or as multinucleated cells with diploid or even polyploid nuclei. In this study, we evaluate the degree of polyploidy in the murine liver by accounting both DNA content and number of nuclei per cell. We demonstrate that mouse hepatocytes with diploid nuclei have distinct metabolic characteristics compared to cells with polyploid nuclei. In addition to strong differential gene expression, comprising metabolic as well as signaling compounds, we found a strongly decreased insulin binding of nuclear polyploid cells. Our observations were associated with nuclear ploidy but not with total ploidy within a cell. We therefore suggest ploidy of the nuclei as an new diversity factor of hepatocytes and hypothesize that hepatocytes with polyploid nuclei may have distinct biological functions than mono-nuclear ones. This diversity is independent from the well-known heterogeneity related to the cells' position along the porto-central liver-axis. PMID:29163206

Effects of clofibric acid alone and in combination with 17β-estradiol on mRNA abundance in primary hepatocytes isolated from rainbow trout.

PubMed

Sovadinová, I; Liedtke, A; Schirmer, K

2014-09-01

Clofibric acid (CA) is the active substance of lipid lowering drugs. It is resistant to degradation, polar in nature, and has been found ubiquitously in the aquatic environment. Though CA is classified as a peroxisomal proliferator in rodents and is considered as a potential endocrine disruptor, little information exists on the effects of CA in aquatic organisms, such as fish. In the present study, we examined the mRNA levels of peroxisome proliferator- and estrogen-sensitive genes on the exposure of primary rainbow trout (Oncorhynchus mykiss) hepatocytes to CA alone and in combination with the natural female sex hormone, 17β-estradiol (E2). Our results demonstrate that rainbow trout hepatocytes are relatively refractory to the effects of CA on the PPAR signaling pathway and lipid metabolism. Moreover, CA did not show recognizable estrogenic activity, but after the induction of vitellogenesis by E2, CA significantly reduced vitellogenin (VTG) mRNA abundance. Apparently, the indirect repression of VTG transcription, independent of estrogen receptors, occurred. The mechanism is not yet clearly understood but may involve disruption of the stabilization of VTG mRNA known to be induced by E2. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hepatocyte attachment to laminin is mediated through multiple receptors

PubMed Central

1990-01-01

The interaction of hepatocytes with the basement membrane glycoprotein laminin was studied using synthetic peptides derived from laminin sequences. Rat hepatocytes bind to laminin and three different sites within the A and B1 chains of laminin were identified. Active laminin peptides include the PA22-2 peptide (close to the carboxyl end of the long arm in the A chain), the RGD-containing peptide, PA21 (in the short arm of the A chain) and the pentapeptide YIGSR (in the short arm of the B1 chain). PA22-2 was the most potent peptide, whereas the other two peptides had somewhat lower activity. Furthermore, hepatocyte attachment to laminin was inhibited by the three peptides, with PA22-2 being the most active. Various proteins from isolated membranes of cell- surface iodinated hepatocytes bound to a laminin affinity column including three immunologically related binding proteins : Mr = 67,000, 45,000, and 32,000. Several proteins--Mr = 80,000, 55,000, and 38,000- 36,000--with a lower affinity for laminin were also identified. Affinity chromatography on peptide columns revealed that the PA22-2 peptide specifically bound the Mr = 80,000, 67,000, 45,000, and 32,000 proteins, the PA21 peptide bound the Mr = 45,000 and 38,000-36,000 proteins and the YIGSR peptide column bound the 38,000-36,000 protein. Antisera to a bacterial fusion protein of the 32-kD laminin-binding protein (LBP-32) reacted strongly with the three laminin-binding proteins, Mr = 67,000, 45,000, and 32,000, showing that they are immunologically related. Immunoperoxidase microscopy studies confirmed that these proteins are present within the plasma membrane of the hepatocyte. The antisera inhibited the adhesion of hepatocytes to hepatocytes to laminin by 30%, supporting the finding that these receptors and others mediate the attachment of hepatocytes to several regions of laminin. PMID:2136861

Human hepatocyte growth factor promotes functional recovery in primates after spinal cord injury.

PubMed

Kitamura, Kazuya; Fujiyoshi, Kanehiro; Yamane, Jun-Ichi; Toyota, Fumika; Hikishima, Keigo; Nomura, Tatsuji; Funakoshi, Hiroshi; Nakamura, Toshikazu; Aoki, Masashi; Toyama, Yoshiaki; Okano, Hideyuki; Nakamura, Masaya

2011-01-01

Many therapeutic interventions for spinal cord injury (SCI) using neurotrophic factors have focused on reducing the area damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF), which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF) intrathecally. Motor function was assessed using an original open field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery. In vivo magnetic resonance imaging showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-human primates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in non-human primates after contusive cervical SCI.

Human Hepatocyte Growth Factor Promotes Functional Recovery in Primates after Spinal Cord Injury

PubMed Central

Kitamura, Kazuya; Fujiyoshi, Kanehiro; Yamane, Jun-ichi; Toyota, Fumika; Hikishima, Keigo; Nomura, Tatsuji; Funakoshi, Hiroshi; Nakamura, Toshikazu; Aoki, Masashi; Toyama, Yoshiaki; Okano, Hideyuki; Nakamura, Masaya

2011-01-01

Many therapeutic interventions for spinal cord injury (SCI) using neurotrophic factors have focused on reducing the area damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF), which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF) intrathecally. Motor function was assessed using an original open field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery. In vivo magnetic resonance imaging showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-human primates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in non-human primates after contusive cervical SCI. PMID:22140459

High Efficient Differentiation of Functional Hepatocytes from Porcine Induced Pluripotent Stem Cells

PubMed Central

Ao, Ying; Mich-Basso, Jocelyn Danielle; Lin, Bo; Yang, Lei

2014-01-01

Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs) by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM), and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP) 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications. PMID:24949734

A specific amino acid formula prevents alcoholic liver disease in rodents.

PubMed

Tedesco, Laura; Corsetti, Giovanni; Ruocco, Chiara; Ragni, Maurizio; Rossi, Fabio; Carruba, Michele O; Valerio, Alessandra; Nisoli, Enzo

2018-05-01

Chronic alcohol consumption promotes mitochondrial dysfunction, oxidative stress, defective protein metabolism, and fat accumulation in hepatocytes (liver steatosis). Inadequate amino acid metabolism is worsened by protein malnutrition, frequently present in alcohol-consuming patients, with reduced circulating branched-chain amino acids (BCAAs). Here we asked whether dietary supplementation with a specific amino acid mixture, enriched in BCAAs (BCAAem) and able to promote mitochondrial function in muscle of middle-aged rodents, would prevent mitochondrial dysfunction and liver steatosis in Wistar rats fed on a Lieber-DeCarli ethanol (EtOH)-containing liquid diet. Supplementation of BCAAem, unlike a mixture based on the amino acid profile of casein, abrogated the EtOH-induced fat accumulation, mitochondrial impairment, and oxidative stress in liver. These effects of BCAAem were accompanied by normalization of leucine, arginine, and tryptophan levels, which were reduced in liver of EtOH-consuming rats. Moreover, although the EtOH exposure of HepG2 cells reduced mitochondrial DNA, mitochondrial transcription factors, and respiratory chain proteins, the BCAAem but not casein-derived amino acid supplementation halted this mitochondrial toxicity. Nicotinamide adenine dinucleotide levels and sirtuin 1 (Sirt1) expression, as well as endothelial nitric oxide (eNOS) and mammalian/mechanistic target of rapamycin (mTOR) signaling pathways, were downregulated in the EtOH-exposed HepG2 cells. BCAAem reverted these molecular defects and the mitochondrial dysfunction, suggesting that the mitochondrial integrity obtained with the amino acid supplementation could be mediated through a Sirt1-eNOS-mTOR pathway. Thus a dietary activation of the mitochondrial biogenesis and function by a specific amino acid supplement protects against the EtOH toxicity and preserves the liver integrity in mammals. NEW & NOTEWORTHY Dietary supplementation of a specific amino acid formula prevents both

TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure.

PubMed

De Santis Puzzonia, Marco; Cozzolino, Angela Maria; Grassi, Germana; Bisceglia, Francesca; Strippoli, Raffaele; Guarguaglini, Giulia; Citarella, Franca; Sacchetti, Benedetto; Tripodi, Marco; Marchetti, Alessandra; Amicone, Laura

2016-01-01

In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA.

Measurement of Blood Coagulation Factor Synthesis in Cultures of Human Hepatocytes.

PubMed

Heinz, Stefan; Braspenning, Joris

2015-01-01

An important function of the liver is the synthesis and secretion of blood coagulation factors. Within the liver, hepatocytes are involved in the synthesis of most blood coagulation factors, such as fibrinogen, prothrombin, factor V, VII, IX, X, XI, XII, as well as protein C and S, and antithrombin, whereas liver sinusoidal endothelial cells produce factor VIII and von Willebrand factor. Here, we describe methods for the detection and quantification of most blood coagulation factors in hepatocytes in vitro. Hepatocyte cultures indeed provide a valuable tool to study blood coagulation factors. In addition, the generation and expansion of hepatocytes or hepatocyte-like cells may be used in future for cell-based therapies of liver diseases, including blood coagulation factor deficiencies.

Human but Not Mouse Hepatocytes Respond to Interferon-Lambda In Vivo

PubMed Central

Hermant, Pascale; Demarez, Céline; Mahlakõiv, Tanel; Staeheli, Peter; Meuleman, Philip; Michiels, Thomas

2014-01-01

The type III interferon (IFN) receptor is preferentially expressed by epithelial cells. It is made of two subunits: IFNLR1, which is specific to IFN-lambda (IFN-λ) and IL10RB, which is shared by other cytokine receptors. Human hepatocytes express IFNLR1 and respond to IFN-λ. In contrast, the IFN-λ response of the mouse liver is very weak and IFNLR1 expression is hardly detectable in this organ. Here we investigated the IFN-λ response at the cellular level in the mouse liver and we tested whether human and mouse hepatocytes truly differ in responsiveness to IFN-λ. When monitoring expression of the IFN-responsive Mx genes by immunohistofluorescence, we observed that the IFN-λ response in mouse livers was restricted to cholangiocytes, which form the bile ducts, and that mouse hepatocytes were indeed not responsive to IFN-λ. The lack of mouse hepatocyte response to IFN-λ was observed in different experimental settings, including the infection with a hepatotropic strain of influenza A virus which triggered a strong local production of IFN-λ. With the help of chimeric mice containing transplanted human hepatocytes, we show that hepatocytes of human origin readily responded to IFN-λ in a murine environment. Thus, our data suggest that human but not mouse hepatocytes are responsive to IFN-λ in vivo. The non-responsiveness is an intrinsic property of mouse hepatocytes and is not due to the mouse liver micro-environment. PMID:24498220

Effects of edaravone, a radical scavenger, on hepatocyte transplantation.

PubMed

Hayashi, Chihiro; Ito, Masahiro; Ito, Ryoutaro; Murakumo, Akiko; Yamamoto, Naoki; Hiramatsu, Noriko; Fox, Ira J; Horiguchi, Akihiko

2014-12-01

Hepatocyte transplantation (HTx) has yielded significant improvements in liver function and survival in experimentally induced acute liver failure and liver-based metabolic disease. However, transplantation is inefficient, and it is thought that transplanted hepatocytes have a shortened lifespan because of inflammation involving excess nitric oxide (NO). The present study aimed to clarify whether edaravone, a free radical scavenger used to treat ischemic stroke, could reduce ischemic changes in hepatocyte-transplanted livers. Edaravone (3 mg/kg) was administered intravenously 24 h before HTx to Nagase analbuminemic rats (NARs). Hepatocytes were isolated, and 30 × 10(6) cells were injected in a 1.0-ml volume directly into the spleens of NARs. All experimental groups studied received FK506 to control rejection. Animals in Group A received medium-only; Group B received HTx only; and Group C received HTx and edaravone. Forty-eight hours after transplantation, the hepatocytes from animals were isolated and analyzed for staining with propidium iodide- and annexin-V using flow cytometry. Liver sections were also studied by immunostaining for albumin, and TUNEL. Peripheral blood serum albumin levels were measured on post-transplant days 0, 3, 5, 7, 10 and 14 using ELISA. The edaravone-treated animals demonstrated an increased number of engrafted donor hepatocytes in the liver. The edaravone-treated liver sections also contained fewer TUNEL-positive cells and animals that received edaravone had higher serum albumin levels post-transplantation. Hepatocytes were also found to have increased in numbers 2 weeks following treatment with edaravone. Edaravone administration during HTx can suppress apoptosis near the transplanted cells, increasing engraftment. These studies indicate its potential usefulness for future clinical application. © 2014 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure

PubMed Central

Grassi, Germana; Bisceglia, Francesca; Strippoli, Raffaele; Guarguaglini, Giulia; Citarella, Franca; Sacchetti, Benedetto; Tripodi, Marco; Amicone, Laura

2016-01-01

In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA. PMID:27893804

Maximising the use of freshly isolated human hepatocytes.

PubMed

Evans, Peter J

2016-01-01

Freshly isolated human hepatocytes are the best model for predicting adverse drug reactions. However, their preparation and use present the investigator with many variables that are beyond their control. These include operation continuity and timing, size and number of cut surfaces on liver tissue and the prior history of the patient. To exploit the potential of freshly isolated human hepatocytes a method is required to preserve the cells in their initial in vivo like state. This experimental pausing allows experiments to be prioritised at convenient times of the day. A novel approach for selecting viable human hepatocytes by functional attachment to a gelatin gel is described rather than relying on their physical characteristics. The cells are preserved as a monolayer on the semi-solid support at 10°C as single spherical entities. The hepatocytes can be released into suspension, when required, by a temperature transition to 37°C for 20min. The cells can be used in suspension or as a monolayer. The length of preservation depends upon the source tissue. Hepatocytes from normal liver can be maintained for at least 4days and demonstrated to have the same level of CYP3A4 and the enzymes involved in glucuronidation and sulphation as freshly isolated cells. Cells from fatty liver, attached to gelatin, vary in their preservation time but it is at least 24h and so confluent monolayers, that survive at 37°C can be generated the following day. The technique enables freshly isolated human hepatocytes to be used more effectively. They can be preserved in times of plenty so more experimentation is possible. Alternatively, with poorer fatty cells the initial attachment on gelatin enables confluent monolayers of lipid rich cells to be studied. Copyright © 2015 Elsevier Inc. All rights reserved.

[Endoplasmic reticulum stress mediates lipopolysaccharide-induced apoptosis in rat hepatocyte].

PubMed

Ji, Ying-Lei; Yan, Jun; Wang, Yan-Sha; Liu, Yi-Chang; Gu, Zhen-Yong

2014-02-01

To investigate the role of endoplasmic reticulum stress (ERS) in lipopolysaccharide (LPS)-induced hepatocyte apoptosis. Cells of the rat hepatocyte line BRL were cultured. The hepatocytes were treated with LPS, ERS inducer thapsigargin (TG), and ERS inhibitor 4-phenylbutyric acid (4-PBA), respectively or in their different combination. The cell viability was measured by MTT assay. The cyto-nuclear morphological changes of apoptosis cells were detected by the fluorescent dye Hoechst 33258. The apoptosis rate was assessed by flow cytometry with Annexin V-FITC/PI double-staining. Expressions of GRP78 as ERS marker protein, CHOP, caspase-12 and cleaved-caspase-3 as ERS related protein were detected by Western blotting. LPS could cause a decrease in cell viability and an increase in apoptosis rate in a dose- and time-dependent manner. The expression of GRP78, CHOP, caspase-12 and cleaved-caspase-3 proteins were significantly increased with LPS treatment. TG led to a marked decrease in cell viability and an increase in apoptosis rate, which aggravated the hepatocyte injury induced by LPS; whereas 4-PBA alleviated LPS-induced apoptosis. ERS mediates LPS-induced hepatocyte injuries, indicating that ERS may play a vital role in the pathogenesis of LPS-induced hepatocyte injuries.

Rodent consumption in Khon Kaen Province, Thailand.

PubMed

Suwannarong, Kanokwan; Chapman, Robert S

2014-09-01

Rodents are important reservoirs of rodent-borne infections worldwide, including Southeast Asia and Northeast Thailand (Isaan), where rodent consumption may be a source of rodent-borne diseases. The behavior of consuming rodents is related to a population's traditions, knowledge, cultural, and household contexts, among other factors. This cross-sectional survey was conducted in Khon Kaen Province, Thailand during November-December 2011. It aimed to elicit information about rodent consumption among residents of this province, and to identify factors associated with rodent consumption there. Multiple logistic regression analysis indicated that male gender, large family size, and use of rainwater as the main source of drinking water were positively associated with reported rodent consumption in this province, while having proper knowledge/attitudes towards animal-borne disease was negatively associated. These results provide evidence-base information for further studies, such as participatory ac- tion research, to further explore how people interact with rodents in different contexts. Further research is also needed to characterize risk of zoonotic diseases in relation to rodent consumption.

Phosphorylation of hepatocyte growth factor receptor and epidermal growth factor receptor of human hepatocytes can be maintained in a (3D) collagen sandwich culture system.

PubMed

Engl, Tobias; Boost, Kim A; Leckel, Kerstin; Beecken, Wolf-Dietrich; Jonas, Dietger; Oppermann, Elsie; Auth, Marcus K H; Schaudt, André; Bechstein, Wolf-Otto; Blaheta, Roman A

2004-08-01

In vitro culture models that employ human liver cells could be potent tools for predictive studies on drug toxicity and metabolism in the pharmaceutical industry. However, an adequate receptor responsiveness is necessary to allow intracellular signalling and metabolic activity. We tested the ability of three-dimensionally arranged human hepatocytes to respond to the growth factors hepatocyte growth factor (HGF) or epidermal growth factor (EGF). Isolated adult human hepatocytes were cultivated within a three-dimensional collagen gel (sandwich) or on a two-dimensional collagen matrix. Cells were treated with HGF or EGF and expression and phosphorylative activity of HGF receptors (HGFr, c-met) or EGF receptors (EGFr) were measured by flow cytometry and Western blot. Increasing HGFr and EGFr levels were detected in hepatocytes growing two-dimensionally. However, both receptors were not activated in presence of growth factors. In contrast, when hepatocytes were plated within a three-dimensional matrix, HGFr and EGFr levels remained constantly low. However, both receptors became strongly phosphorylated by soluble HGF or EGF. We conclude that cultivation of human hepatocytes in a three-dimensionally arranged in vitro system allows the maintenance of specific functional activities. The necessity of cell dimensionality for HGFr and EGFr function should be considered when an adequate in vitro system has to be introduced for drug testing.

Hepatocytes express functional NOD1 and NOD2 receptors: A role for NOD1 in hepatocyte CC and CXC chemokine production

PubMed Central

Scott, Melanie J.; Chen, Christine; Sun, Qian; Billiar, Timothy R.

2010-01-01

Background & Aims NOD-like receptors are recently described cytosolic pattern recognition receptors. NOD1 and NOD2 are members of this family that recognize bacterial cell wall components, diaminopimelic acid and muramyl dipeptide, respectively. Both NOD1 and NOD2 have been associated with many inflammatory diseases, although their role in liver inflammation and infection has not been well studied. Materials and Methods We investigated the role of NOD receptors in mouse liver by assessing expression and activation of NOD1 and NOD2 in liver and primary isolated hepatocytes from C57BL/6 mice. Results Both NOD1 and NOD2 mRNA and protein were highly expressed in hepatocytes and liver. RIP2, the main signaling partner for NODs, was also expressed. Stimulation of hepatocytes with NOD1 ligand (C12-iEDAP) induced NFκB activation, activation of MAP kinases and expression of chemokines CCL5 (RANTES) and CXCL1 (KC). C12-iEDAP also synergized with interferon (IFN)γ to increase iNOS expression and production of nitric oxide. Despite activating NFκB, NOD1 ligand did not upregulate hepatocyte production of the acute phase proteins lipopolysaccharide binding protein, serum amyloid A, or soluble CD14 in cell culture supernatants, or upregulate mRNA expression of lipopolysaccharide binding protein, serum amyloid A, C-reactive protein, or serum amyloid P. NOD2 ligand (MDP) did not activate hepatocytes when given alone, but did synergize with Toll-like receptor ligands, lipopolysaccharide (LPS), and polyI:C to activate NFκB and MAPK. Conclusions All together these data suggest an important role for hepatocyte NOD1 in attracting leukocytes to the liver during infection and for hepatic NLRs to augment innate immune responses to pathogens. PMID:20615568

Increased hepatocyte fas expression and apoptosis in HIV and hepatitis C virus coinfection.

PubMed

Macias, Juan; Japón, Miguel A; Sáez, Carmen; Palacios, Rosa B; Mira, José A; García-García, José A; Merchante, Nicolás; Vergara, Salvador; Lozano, Fernando; Gómez-Mateos, Jesús; Pineda, Juan A

2005-11-01

Chronic hepatitis C disease (CHC) follows an accelerated course in human immunodeficiency virus (HIV) coinfection. The reasons for this are unclear. Fas-mediated hepatocyte apoptosis is involved in the pathogenesis of hepatitis C virus (HCV) infection. We sought to compare the expression of Fas on hepatocytes and irreversible apoptosis of hepatocytes among patients with CHC with and without HCV/HIV coinfection. Fas-immunostained hepatocytes were semiquantified, and apoptotic hepatocytes were detected by staining caspase-cleaved cytokeratin 18 filaments and counted across the entire section of liver-biopsy specimens from HCV-infected patients with and without HCV/HIV coinfection. One hundred thirty-four HCV/HIV-coinfected and 100 HCV-infected patients were included. HCV/HIV coinfection was associated with both diffuse distribution of Fas-stained hepatocytes (adjusted odds ratio [AOR], 7.4 [95% confidence interval {CI}, 3.8-14.4]) and with apoptotic hepatocyte counts greater than the median (AOR, 2.5 [95% CI, 1.5-4.5]). In HCV/HIV-coinfected patients, CD4+ cell nadir<200 cells/mL was associated with both Fas expression (AOR, 2.9 [95% CI, 1.3-6.8]) and hepatocyte apoptosis (AOR, 2.3 [95% CI, 1.1-4.9]). HCV/HIV-coinfected patients show higher levels of hepatocytes expressing Fas and undergoing irreversible apoptosis than do HCV-infected patients. However, low CD4+ cell nadirs in coinfected patients are associated with hepatocyte Fas expression and apoptosis.

Hepatocyte Produced Matrix Metalloproteinases Are Regulated by CD147 in Liver Fibrogenesis

PubMed Central

Morgan, Alison J.; Tu, Thomas; Wen, Victoria W.; Yee, Christine; Mridha, Auvro; Lee, Maggie; d'Avigdor, William; Locarnini, Stephen A.; McCaughan, Geoffrey W.; Warner, Fiona J.; McLennan, Susan V.; Shackel, Nicholas A.

2014-01-01

Background The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC. Methods Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention. Results In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls. Conclusion We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a

Hepatocyte produced matrix metalloproteinases are regulated by CD147 in liver fibrogenesis.

PubMed

Calabro, Sarah R; Maczurek, Annette E; Morgan, Alison J; Tu, Thomas; Wen, Victoria W; Yee, Christine; Mridha, Auvro; Lee, Maggie; d'Avigdor, William; Locarnini, Stephen A; McCaughan, Geoffrey W; Warner, Fiona J; McLennan, Susan V; Shackel, Nicholas A

2014-01-01

The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC. Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention. In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls. We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by

Rodents and Risk in the Mekong Delta of Vietnam: Seroprevalence of Selected Zoonotic Viruses in Rodents and Humans

PubMed Central

Van Cuong, Nguyen; Carrique-Mas, Juan; Vo Be, Hien; An, Nguyen Ngoc; Tue, Ngo Tri; Anh, Nguyet Lam; Anh, Pham Hong; Phuc, Nguyen The; Baker, Stephen; Voutilainen, Liina; Jääskeläinen, Anne; Huhtamo, Eili; Utriainen, Mira; Sironen, Tarja; Vaheri, Antti; Henttonen, Heikki; Vapalahti, Olli; Chaval, Yannick

2015-01-01

Abstract In the Mekong Delta in southern Vietnam, rats are commonly traded in wet markets and sold live for food consumption. We investigated seroprevalence to selected groups of rodent-borne viruses among human populations with high levels of animal exposure and among co-located rodent populations. The indirect fluorescence antibody test (IFAT) was used to determine seropositivity to representative reference strains of hantaviruses (Dobrava virus [DOBV], Seoul virus [SEOV]), cowpox virus, arenaviruses (lymphocytic choriomeningitis virus [LCMV]), flaviviruses (tick-borne encephalitis virus [TBEV]), and rodent parechoviruses (Ljungan virus), using sera from 245 humans living in Dong Thap Province and 275 rodents representing the five common rodent species sold in wet markets and present in peridomestic and farm settings. Combined seropositivity to DOBV and SEOV among the rodents and humans was 6.9% (19/275) and 3.7% (9/245), respectively; 1.1% (3/275) and 4.5% (11/245) to cowpox virus; 5.4% (15/275) and 47.3% (116/245) for TBEV; and exposure to Ljungan virus was 18.8% (46/245) in humans, but 0% in rodents. Very little seroreactivity was observed to LCMV in either rodents (1/275, 0.4%) or humans (2/245, 0.8%). Molecular screening of rodent liver tissues using consensus primers for flaviviruses did not yield any amplicons, whereas molecular screening of rodent lung tissues for hantavirus yielded one hantavirus sequence (SEOV). In summary, these results indicate low to moderate levels of endemic hantavirus circulation, possible circulation of a flavivirus in rodent reservoirs, and the first available data on human exposures to parechoviruses in Vietnam. Although the current evidence suggests only limited exposure of humans to known rodent-borne diseases, further research is warranted to assess public health implications of the rodent trade. PMID:25629782

Deletion of the rodent malaria ortholog for falcipain-1 highlights differences between hepatic and blood stage merozoites

PubMed Central

Lehmann, Christine; Hanson, Kirsten K.; Lin, Jing-wen; Rousseau, Kimberly; Carvalho, Filomena A.; van der Linden, Wouter A.; Santos, Nuno C.; Sinnis, Photini

2017-01-01

Proteases have been implicated in a variety of developmental processes during the malaria parasite lifecycle. In particular, invasion and egress of the parasite from the infected hepatocyte and erythrocyte, critically depend on protease activity. Although falcipain-1 was the first cysteine protease to be characterized in P. falciparum, its role in the lifecycle of the parasite has been the subject of some controversy. While an inhibitor of falcipain-1 blocked erythrocyte invasion by merozoites, two independent studies showed that falcipain-1 disruption did not affect growth of blood stage parasites. To shed light on the role of this protease over the entire Plasmodium lifecycle, we disrupted berghepain-1, its ortholog in the rodent parasite P. berghei. We found that this mutant parasite displays a pronounced delay in blood stage infection after inoculation of sporozoites. Experiments designed to pinpoint the defect of berghepain-1 knockout parasites found that it was not due to alterations in gliding motility, hepatocyte invasion or liver stage development and that injection of berghepain-1 knockout merosomes replicated the phenotype of delayed blood stage growth after sporozoite inoculation. We identified an additional role for berghepain-1 in preparing blood stage merozoites for infection of erythrocytes and observed that berghepain-1 knockout parasites exhibit a reticulocyte restriction, suggesting that berghepain-1 activity broadens the erythrocyte repertoire of the parasite. The lack of berghepain-1 expression resulted in a greater reduction in erythrocyte infectivity in hepatocyte-derived merozoites than it did in erythrocyte-derived merozoites. These observations indicate a role for berghepain-1 in processing ligands important for merozoite infectivity and provide evidence supporting the notion that hepatic and erythrocytic merozoites, though structurally similar, are not identical. PMID:28922424

LIVER REGENERATION STUDIES WITH RAT HEPATOCYTES IN PRIMARY CULTURE

EPA Science Inventory

Adult rat parenchymal hepatocytes in primary culture can be induced to enter into DNA synthesis and mitosis. The optimal conditions for hepatocyte replication are low plating density (less than 10,000 cells/sq cm) and 50% serum from two-thirds partially hepatectomized rats (48 hr...

The instant blood-mediated inflammatory reaction characterized in hepatocyte transplantation.

PubMed

Gustafson, Elisabet K; Elgue, Graciela; Hughes, Robin D; Mitry, Ragai R; Sanchez, Javier; Haglund, Ulf; Meurling, Staffan; Dhawan, Anil; Korsgren, Olle; Nilsson, Bo

2011-03-27

Hepatocyte transplantation (HcTx) has proven to be a safe procedure, although the functional results have been unsatisfactory, probably due to insufficient engraftment or a loss of transplanted mass or function. In this study, we investigate whether hepatocytes in contact with blood induce an inflammatory reaction leading to, similar to what happens in clinical islet transplantation, an instant blood-mediated inflammatory reaction (IBMIR) resulting in an early loss of transplanted cells. By using an experimental model that mimics the portal vein blood flow, we could study different parameters reflecting the effects on the innate immunity elicited by hepatocytes in contact with ABO-matched human blood. We report that all aspects of the IBMIR such as platelet and granulocyte consumption, coagulation, and complement activation were demonstrated. Addition of various specific inhibitors of coagulation allowed us to clearly delineate the various stages of the hepatocyte-triggered IBMIR and show that the reaction was triggered by tissue factor. Analysis of a case of clinical HcTx showed that hepatocyte-induced IBMIR also occurs in vivo. Both the inflammatory and the coagulation aspects were controlled by low-molecular-weight dextran sulfate. Isolated hepatocytes in contact with blood induce the IBMIR in vitro, and there are indications that these events are also relevant in vivo. According to these findings, HcTx would benefit from controlling a wider range of signals from the innate immune system.

The differentiation of hepatocyte-like cells from monkey embryonic stem cells.

PubMed

Ma, Xiaocui; Duan, Yuyou; Jung, Christine J; Wu, Jian; VandeVoort, Catherine A; Zern, Mark A

2008-12-01

Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as alpha-fetoprotein (AFP), albumin and alpha1-antitrypsin (alpha1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the alpha1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, alpha1-AT, AFP, hepatocyte nuclear factor 3beta, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and alpha1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 +/- 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage.

Effect of Immunosuppressive Agents on Hepatocyte Apoptosis Post-Liver Transplantation

PubMed Central

Lim, Eu Jin; Chin, Ruth; Nachbur, Ueli; Silke, John; Jia, Zhiyuan; Angus, Peter W.; Torresi, Joseph

2015-01-01

Introduction Immunosuppressants are used ubiquitously post-liver transplantation to prevent allograft rejection. However their effects on hepatocytes are unknown. Experimental data from non-liver cells indicate that immunosuppressants may promote cell death thereby driving an inflammatory response that promotes fibrosis and raises concerns that a similar effect may occur within the liver. We evaluated apoptosis within the liver tissue of post-liver transplant patients and correlated these findings with in vitro experiments investigating the effects of immunosuppressants on apoptosis in primary hepatocytes. Methods Hepatocyte apoptosis was assessed using immunohistochemistry for M30 CytoDEATH and cleaved PARP in human liver tissue. Primary mouse hepatocytes were treated with various combinations of cyclosporine, tacrolimus, sirolimus, or MMF. Cell viability and apoptosis were evaluated using crystal violet assays and Western immunoblots probed for cleaved PARP and cleaved caspase 3. Results Post-liver transplant patients had a 4.9-fold and 1.7-fold increase in M30 CytoDEATH and cleaved PARP compared to normal subjects. Cyclosporine and tacrolimus at therapeutic concentrations did not affect hepatocyte apoptosis, however when they were combined with MMF, cell death was significantly enhanced. Cell viability was reduced by 46% and 41%, cleaved PARP was increased 2.6-fold and 2.2-fold, and cleaved caspase 3 increased 2.2-fold and 1.8-fold following treatment with Cyclosporine/MMF and Tacrolimus/MMF respectively. By contrast, the sirolimus/MMF combination did not significantly reduce hepatocyte viability or promote apoptosis. Conclusion Commonly used immunosuppressive drug regimens employed after liver transplantation enhance hepatocyte cell death and may thus contribute to the increased liver fibrosis that occurs in a proportion of liver transplant recipients. PMID:26390404

DNA damage in lead-exposed hepatocytes: coexistence of apoptosis and necrosis?

PubMed

Narayana, Kilarkaje; Raghupathy, Raj

2012-04-01

The aim of the present study was to investigate the coexistence of oxidative DNA damage and apoptosis- and necrosis-related DNA damage, and to correlate with ultrastructural changes in hepatocyte nuclei in the lead-nitrate-exposed liver. Adult male Wistar rats were exposed to 0, 0.5, and 1% lead nitrate for 60 days, and the livers were sampled the next day. Ultrastructurally, hepatocyte nuclei showed no apoptosis-related morphological changes, but showed necrotic changes. Competitive enzyme-linked immunosorbent assay showed no change in 8-oxo-dG activity (P > 0.05), but immunohistochemistry showed its localization in hepatocytes, Kupffer cells, endothelium, and bile ductule epithelium. TUNEL-labeled DNA breaks presenting 3'-OH ends increased in hepatocytes in all functional zones of the portal acini and bile ductule epithelium (zones I>III>II). In situ oligo ligation revealed the existence of DNA breaks bearing duplex 3' overhangs and 5' P-blunt ends in hepatocytes of all functional zones and bile ductule epithelium. In conclusion, both apoptosis- and necrosis-related DNA damage coexist without significant oxidative DNA damage. Hepatocytes display changes related to necrosis, but not those related to apoptosis.

Fuzheng Huayu recipe alleviates hepatic fibrosis via inhibiting TNF-α induced hepatocyte apoptosis.

PubMed

Tao, Yan-yan; Yan, Xiu-chuan; Zhou, Tao; Shen, Li; Liu, Zu-long; Liu, Cheng-hai

2014-11-18

What was the relationship of Fuzheng Huayu recipe (FZHY) inhibiting hepatocyte apoptosis and HSC activation at different stage of liver fibrosis? In order to answer this question, the study was carried out to dynamically observe FZHY's effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis. Mice were randomly divided into four groups: normal, model, FZHY, and N-acetylcystein (NAC) groups. Acute hepatic injury and liver fibrosis in mice were induced by CCl4. Three days before the first CCl4 injection, treatment with FZHY powder or NAC respectively was started. In vitro, primary hepatocytes were pretreated with FZHY medicated serum or Z-VAD-FMK and then incubated with ActD and TNF-α. Primary HSCs were treated with DNA from apoptotic hepatocytes incubated by Act D/TNF-α or FZHY medicated. Liver sections were analyzed for HE staining and immunohistochemical evaluation of apoptosis. Serum ALT and AST, Alb content and TNF-α expression in liver tissue were detected. Hyp content was assayed and collagen deposition was visualized. Expressions of α-SMA and type I collagen were analyzed by immunofluorescence and immunoblotting. Flow cytometry, immunofluorescence, and DNA ladder for hepatocyte apoptosis and immunoblotting for TNF-R1, Bcl-2 and Bax were also analyzed. Mice showed characteristic features of massive hepatocytes apoptosis in early stage of liver injury and developed severe hepatic fibrosis in later phase. FZHY treatment significantly alleviated acute liver injury and hepatocyte apoptosis, and inhibited liver fibrosis by decreasing α-SMA expression and hepatic Hyp content. In vitro, primary hepatocytes were induced by TNF-α and Act D. The anti-apoptotic effect of FZHY was generated by reducing TNFR1 expression and balancing the expressions of Bcl-2 and Bax. Meanwhile, the nuclear DNA from apoptotic hepatocytes stimulated HSC activation in a dose dependent manner, and the DNA from

UDP-glucuronosyltransferase-dependent bioactivation of clofibric acid to a DNA-damaging intermediate in mouse hepatocytes.

PubMed

Ghaoui, Roula; Sallustio, Benedetta C; Burcham, Philip C; Fontaine, Frank R

2003-05-06

Glucuronidation of a number of carboxyl-containing drugs generates reactive acyl glucuronide metabolites. These electrophilic species alkylate cell proteins and may be implicated in the pathogenesis of a number of toxic syndromes seen in patients receiving the parent aglycones. Whether acyl glucuronides also attack nuclear DNA is unknown, although the acyl glucuronide formed from clofibric acid was recently found to decrease the transfection efficiency of phage DNA and generate strand breaks in plasmid DNA in vitro. To determine if such a DNA damage occurs within a cellular environment, the comet assay (i.e. single-cell gel electrophoresis) was used to detect DNA lesions in the nuclear genome of isolated mouse hepatocytes cultured with clofibric acid. Overnight exposure to 50 microM and higher concentrations of clofibric acid produced concentration-dependent increases in the comet areas of hepatocyte nuclei, with 1 mM clofibrate producing a 3.6-fold elevation over controls. These effects closely coincided with culture medium concentrations of the glucuronide metabolite formed from clofibric acid, 1-O-beta-clofibryl glucuronide. Consistent with a role for glucuronidation in the DNA damage observed, the glucuronidation inhibitor borneol diminished glucuronide formation from 100 microM clofibrate by 98% and returned comet areas to baseline levels. Collectively, these results suggest that the acyl glucuronide formed from clofibric acid is capable of migrating from its site of formation within the endoplasmic reticulum to generate strand nicks in nuclear DNA.

3D Cultivation Techniques for Primary Human Hepatocytes

PubMed Central

Bachmann, Anastasia; Moll, Matthias; Gottwald, Eric; Nies, Cordula; Zantl, Roman; Wagner, Helga; Burkhardt, Britta; Sánchez, Juan J. Martínez; Ladurner, Ruth; Thasler, Wolfgang; Damm, Georg; Nussler, Andreas K.

2015-01-01

One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device. PMID:27600213

Transplant of Hepatocytes, Undifferentiated Mesenchymal Stem Cells, and In Vitro Hepatocyte-Differentiated Mesenchymal Stem Cells in a Chronic Liver Failure Experimental Model: A Comparative Study.

PubMed

El Baz, Hanan; Demerdash, Zeinab; Kamel, Manal; Atta, Shimaa; Salah, Faten; Hassan, Salwa; Hammam, Olfat; Khalil, Heba; Meshaal, Safa; Raafat, Inas

2018-02-01

Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure. Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed. Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group. Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.

Caveolin-1 is enriched in the peroxisomal membrane of rat hepatocytes.

PubMed

Woudenberg, Jannes; Rembacz, Krzysztof P; van den Heuvel, Fiona A J; Woudenberg-Vrenken, Titia E; Buist-Homan, Manon; Geuken, Mariska; Hoekstra, Mark; Deelman, Leo E; Enrich, Carlos; Henning, Rob H; Moshage, Han; Faber, Klaas Nico

2010-05-01

Caveolae are a subtype of cholesterol-enriched lipid microdomains/rafts that are routinely detected as vesicles pinching off from the plasma membrane. Caveolin-1 is an essential component of caveolae. Hepatic caveolin-1 plays an important role in liver regeneration and lipid metabolism. Expression of caveolin-1 in hepatocytes is relatively low, and it has been suggested to also reside at other subcellular locations than the plasma membrane. Recently, we found that the peroxisomal membrane contains lipid microdomains. Like caveolin-1, hepatic peroxisomes are involved in lipid metabolism. Here, we analyzed the subcellular location of caveolin-1 in rat hepatocytes. The subcellular location of rat hepatocyte caveolin-1 was analyzed by cell fractionation procedures, immunofluorescence, and immuno-electron microscopy. Green fluorescent protein (GFP)-tagged caveolin-1 was expressed in rat hepatocytes. Lipid rafts were characterized after Triton X-100 or Lubrol WX extraction of purified peroxisomes. Fenofibric acid-dependent regulation of caveolin-1 was analyzed. Peroxisome biogenesis was studied in rat hepatocytes after RNA interference-mediated silencing of caveolin-1 and caveolin-1 knockout mice. Cell fractionation and microscopic analyses reveal that caveolin-1 colocalizes with peroxisomal marker proteins (catalase, the 70 kDa peroxisomal membrane protein PMP70, the adrenoleukodystrophy protein ALDP, Pex14p, and the bile acid-coenzyme A:amino acid N-acyltransferase BAAT) in rat hepatocytes. Artificially expressed GFP-caveolin-1 accumulated in catalase-positive organelles. Peroxisomal caveolin-1 is associated with detergent-resistant microdomains. Caveolin-1 expression is strongly repressed by the peroxisome proliferator-activated receptor-alpha agonist fenofibric acid. Targeting of peroxisomal matrix proteins and peroxisome number and shape were not altered in rat hepatocytes with 70%-80% reduced caveolin-1 levels and in livers of caveolin-1 knockout mice. Caveolin-1

Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats.

PubMed

Richert, Lysiane; Baze, Audrey; Parmentier, Céline; Gerets, Helga H J; Sison-Young, Rowena; Dorau, Martina; Lovatt, Cerys; Czich, Andreas; Goldring, Christopher; Park, B Kevin; Juhila, Satu; Foster, Alison J; Williams, Dominic P

2016-09-06

Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

21 CFR 1250.96 - Rodent control.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Rodent control. 1250.96 Section 1250.96 Food and... SANITATION Sanitation Facilities and Conditions on Vessels § 1250.96 Rodent control. Vessels shall be... of rodent control. ...

21 CFR 1250.96 - Rodent control.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Rodent control. 1250.96 Section 1250.96 Food and... SANITATION Sanitation Facilities and Conditions on Vessels § 1250.96 Rodent control. Vessels shall be... of rodent control. ...

21 CFR 1250.96 - Rodent control.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Rodent control. 1250.96 Section 1250.96 Food and... SANITATION Sanitation Facilities and Conditions on Vessels § 1250.96 Rodent control. Vessels shall be... of rodent control. ...

21 CFR 1250.96 - Rodent control.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Rodent control. 1250.96 Section 1250.96 Food and... SANITATION Sanitation Facilities and Conditions on Vessels § 1250.96 Rodent control. Vessels shall be... of rodent control. ...

21 CFR 1250.96 - Rodent control.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Rodent control. 1250.96 Section 1250.96 Food and... SANITATION Sanitation Facilities and Conditions on Vessels § 1250.96 Rodent control. Vessels shall be... of rodent control. ...

A refined characterisation of the NeoHepatocyte phenotype necessitates a reappraisal of the transdifferentiation hypothesis.

PubMed

Riquelme, Paloma; Wundt, Judith; Hutchinson, James A; Brulport, Marc; Jun, Yu; Sotnikova, Anna; Girreser, Ulrich; Braun, Felix; Gövert, Felix; Soria, Bernat; Nüssler, Andreas; Clement, Bernd; Hengstler, Jan G; Fändrich, Fred

2009-03-01

Under certain culture conditions human peripheral blood monocytes may be induced to express phenotypic markers of non-haematopoietic lineages, including hepatocyte-defining traits. One such example, the NeoHepatocyte, was previously shown to express a broad panel of hepatocyte-like marker antigens and metabolic activities, both in vitro and following engraftment in the liver of immunodeficient mice. In this report, a refined description of NeoHepatocytes, with regard to their expression of xenobiotic-metabolising enzymes, morphology, hepatocyte marker expression and cell surface phenotype, is presented in comparison with human macrophages in defined states of activation. Contrary to prior assertions, it would seem more likely that NeoHepatocytes express particular hepatocyte-defining genes during a normal programme of macrophage differentiation rather than undergoing a process of transdifferentiation to become hepatocyte-like cells.

In vitro culture of functionally active buffalo hepatocytes isolated by using a simplified manual perfusion method.

PubMed

Panda, Santanu; Bisht, Sonu; Malakar, Dhruba; Mohanty, Ashok K; Kaushik, Jai K

2015-01-01

In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes. Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3 ± 0.66)×107 cells per gram of liver tissue with a viability of 82.3 ± 3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies. We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes for studying gene

Hepatocytes traffic and export hepatitis B virus basolaterally by polarity-dependent mechanisms.

PubMed

Bhat, Purnima; Snooks, Michelle J; Anderson, David A

2011-12-01

Viruses commonly utilize the cellular trafficking machinery of polarized cells to effect viral export. Hepatocytes are polarized in vivo, but most in vitro hepatocyte models are either nonpolarized or have morphology unsuitable for the study of viral export. Here, we investigate the mechanisms of trafficking and export for the hepadnaviruses hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in polarized hepatocyte-derived cell lines and primary duck hepatocytes. DHBV export, but not replication, was dependent on the development of hepatocyte polarity, with export significantly abrogated over time as primary hepatocytes lost polarity. Using Transwell cultures of polarized N6 cells and adenovirus-based transduction, we observed that export of both HBV and DHBV was vectorially regulated and predominantly basolateral. Monitoring of polarized N6 cells and nonpolarized C11 cells during persistent, long-term DHBV infection demonstrated that newly synthesized sphingolipid and virus displayed significant colocalization and fluorescence resonance energy transfer, implying cotransportation from the Golgi complex to the plasma membrane. Notably, 15% of virus was released apically from polarized cells, corresponding to secretion into the bile duct in vivo, also in association with sphingolipids. We conclude that DHBV and, probably, HBV are reliant upon hepatocyte polarity to be efficiently exported and this export is in association with sphingolipid structures, possibly lipid rafts. This study provides novel insights regarding the mechanisms of hepadnavirus trafficking in hepatocytes, with potential relevance to pathogenesis and immune tolerance.

Lipid-induced Signaling Causes Release of Inflammatory Extracellular Vesicles from Hepatocytes

PubMed Central

Hirsova, Petra; Ibrahim, Samar H.; Krishnan, Anuradha; Verma, Vikas K.; Bronk, Steven F.; Werneburg, Nathan W.; Charlton, Michael R.; Shah, Vijay H.; Malhi, Harmeet; Gores, Gregory J.

2016-01-01

BACKGROUND & AIMS Hepatocyte cellular dysfunction and death induced by lipids, and macrophage-associated inflammation are characteristics of nonalcoholic steatohepatitis (NASH). The fatty acid palmitate can activate death receptor 5 (DR5) on hepatocytes, leading to their death, but little is known about how this process contributes to macrophage-associated inflammation. We investigated whether lipid-induced DR5 signaling results in release of extracellular vesicles (EV) from hepatocytes, and whether these can induce an inflammatory macrophage phenotype. METHODS Primary mouse and human hepatocytes and Huh7 cells were incubated with palmitate, its metabolite lysophosphatidylcholine, or diluent (control). The released EV were isolated, characterized, quantified, and applied to macrophages. C57BL/6 mice were placed on chow or a diet high in fat, fructose, and cholesterol to induce NASH. Some mice were also given the ROCK1 inhibitor fasudil; 2 weeks later, serum EVs were isolated and characterized by immunoblot and nanoparticle-tracking analyses. Livers were collected and analyzed by histology, immunohistochemistry, and quantitative PCR. RESULTS Incubation of primary hepatocytes and Huh7 cells with palmitate or lysophosphatidylcholine increased their release of EV, compared with control cells. This release was reduced by inactivating mediators of the DR5 signaling pathway or ROCK1 inhibition. Hepatocyte-derived EV contained TRAIL and induced expression of interleukin-1, beta (Il1b) and Il6 mRNAs in mouse bone marrow-derived macrophages. Activation of macrophages required DR5 and RIP1. Administration of the ROCK1 inhibitor fasudil to mice with NASH reduced serum levels of EV; this reduction was associated with decreased liver injury, inflammation, and fibrosis. CONCLUSIONS Lipids, which stimulate DR5, induce release of hepatocyte EV, which activate an inflammatory phenotype in macrophages. Strategies to inhibit ROCK1-dependent release of EV by hepatocytes might be

Aquaporins in desert rodent physiology.

PubMed

Pannabecker, Thomas L

2015-08-01

Desert rodents face a sizeable challenge in maintaining salt and water homeostasis due to their life in an arid environment. A number of their organ systems exhibit functional characteristics that limit water loss above that which occurs in non-desert species under similar conditions. These systems include renal, pulmonary, gastrointestinal, nasal, and skin epithelia. The desert rodent kidney preserves body water by producing a highly concentrated urine that reaches a maximum osmolality nearly three times that of the common laboratory rat. The precise mechanism by which urine is concentrated in any mammal is unknown. Insights into the process may be more apparent in species that produce highly concentrated urine. Aquaporin water channels play a fundamental role in water transport in several desert rodent organ systems. The role of aquaporins in facilitating highly effective water preservation in desert rodents is only beginning to be explored. The organ systems of desert rodents and their associated AQPs are described. © 2015 Marine Biological Laboratory.

Mature Hepatocytes Exhibit Unexpected Plasticity by Direct Dedifferentiation into Liver Progenitor Cells in Culture

PubMed Central

Chen, Yixin; Wong, Philip P.; Sjeklocha, Lucas; Steer, Clifford J.; Sahin, M. Behnan

2011-01-01

Although there have been numerous reports describing the isolation of liver progenitor cells from adult liver, their exact origin has not been clearly defined; and the role played by mature hepatocytes as direct contributors to the hepatic progenitor cell pool has remained largely unknown. Here we report strong evidence that mature hepatocytes in culture have the capacity to dedifferentiate into a population of adult liver progenitors without genetic or epigenetic manipulations. By using highly-purified mature hepatocytes, which were obtained from untreated, healthy rat liver and labeled with fluorescent dye PKH2, we found that hepatocytes in culture gave rise to a population of PKH2-positive liver progenitor cells. These cells, Liver Derived Progenitor Cells or LDPCS, which share phenotypic similarities with oval cells, were previously reported to be capable of forming mature hepatocytes both in culture and in animals. Studies done at various time points during the course of dedifferentiation cultures revealed that hepatocytes rapidly transformed into liver progenitors within one week through a transient oval cell-like stage. This finding was supported by lineage-tracing studies involving double-transgenic AlbuminCreXRosa26 mice expressing β-galactosidase exclusively in hepatocytes. Cultures set up with hepatocytes obtained from these mice resulted in generation of β-galactosidase-positive liver progenitor cells demonstrating that they were a direct dedifferentiation product of mature hepatocytes. Additionally, these progenitors differentiated into hepatocytes in vivo when transplanted into rats that had undergone retrorsine pretreatment and partial hepatectomy. Conclusion Our studies provide strong evidence for the unexpected plasticity of mature hepatocytes to dedifferentiate into progenitor cells in culture; and this may potentially have a significant impact on the treatment of liver diseases requiring liver or hepatocyte transplantation. PMID:21953633

[Effect of inducible nitric oxide on intracellular homeostasis of hepatocytes].

PubMed

Tang, Xi-Feng; Zhou, Dong-Yao; Kang, Ge-Fei

2002-02-01

To investigate the effects of inducible nitric oxide (NO) and exogenous NO on the intracellular homeostasis of the hepatocytes. Endogenous NO was induced by combined action of lipopolysaccharide (LPS) and cytokines in cultured rat hepatocytes, and exogenous NO was supplied by sodium nitroprusside (SNP) to stimulate the hepatocytes. The changes in intracellular malondialdehyde (MDA), reduced glutathione(GSH) and free calcium ([Ca2+]i) were observed. substantial increase by 7.97 times in intracellular MDA level and a decrease by 57.9% in GSH occurred in the hepatocytes after the cells had been incubated with LPS and cytokines for 24 h, which were reversed by 43.5% and 98.4% respectively by treatment with N(G)-monomethyl-L-arginine (NMMA), a competitive nitric oxide synthase (NOS) inhibitor. Verapamil significantly reduced both endogenous NO production and oxidative stress, while the effect of A23187 was not conspicuous. Incubation with chlorpromazine and Vitamine E (VitE), however, did not result in decreased release of NO by LPS- and cytokines-induced hepatocytes. After SNP exposure of the hepatocytes, the oxidative status was reversibly enhanced in a time-dependent manner. Short exposure to SNP led to a concentration-dependent inhibition of the rapid and transient increase in free calcium induced by K(+) depolarization and hepatopoietin-coupled calcium mobilization. Inducible NO may initiate and play a key role in the latter stages of metabolic and functional stress responses of hepatocytes against endotoxin and cytokines, when the reduction occurs in the capacity of NO to independently mediate lipid peroxidation and counteract oxidation. The inhibitory effect of NO on [Ca2+]i mobilization may be an important autoregulatory mechanism by means of negative feedback on protein kinase C-associated NOS induction.

Comparison of para-aminophenol cytotoxicity in rat renal epithelial cells and hepatocytes.

PubMed

Li, Ying; Bentzley, Catherine M; Tarloff, Joan B

2005-04-01

Several chemicals, including para-aminophenol (PAP), produce kidney damage in the absence of hepatic damage. Selective nephrotoxicity may be related to the ability of the kidney to reabsorb filtered water, thereby raising the intraluminal concentration of toxicants and exposing tubular epithelial cells to higher concentrations than would be present in other tissues. The present experiments tested the hypothesis that hepatocytes and renal epithelial cells exposed to equivalent concentrations of PAP would be equally susceptible to toxicity. Hepatocytes and renal epithelial cells were prepared by collagenase digestion of tissues obtained from female Sprague-Dawley rats. Toxicity was monitored using trypan blue exclusion, oxygen consumption and ATP content. We measured the rate of PAP clearance and formation of PAP-glutathione conjugate by HPLC. We found that renal epithelial cells accumulated trypan blue and showed declines in oxygen consumption and ATP content at significantly lower concentrations of PAP and at earlier time points than hepatocytes. The half-life of PAP in hepatocyte incubations was significantly shorter (0.71+/-0.07 h) than in renal epithelial cell incubations (1.33+/-0.23 h), suggesting that renal epithelial cells were exposed to PAP for longer time periods than hepatocytes. Renal epithelial cells formed significantly less glutathione conjugates of PAP (PAP-SG) than did hepatocytes, consistent with less efficient detoxification of reactive PAP intermediates by renal epithelial cells. Finally, hepatocytes contained significant more reduced glutathione (NPSH) than did renal epithelial cells, possibly explaining the enhanced formation of PAP-SG by this cell population. In conclusion, our data indicates that renal epithelial cells are intrinsically more susceptible to PAP cytotoxicity than are hepatocytes. This enhanced cytotoxicity may be due to longer exposure to PAP and/or reduced detoxification of reactive intermediates due to lower concentrations

Geomagnetic field detection in rodents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olcese, J.; Reuss, S.; Semm, P.

1988-01-01

In addition to behavioral evidence for the detection of earth-strength magnetic fields (MF) by rodents, recent investigations have revealed that electrophysiological and biochemical responses to MF occur in the pineal organ and retina of rodents. In addition, ferrimagnetic deposits have been identified in the ethmoidal regions of the rodent skull. These findings point to a new sensory phenomenon, which interfaces with many fields of biology, including neuroscience, psychophysics, behavioral ecology, chronobiology and sensory physiology.

Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dever, Joseph T.; Elfarra, Adnan A.

L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 {sup o}C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increasesmore » in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.« less

Hepatocyte transplantation for enzyme deficiency disease in congenic rats.

PubMed

Vroemen, J P; Buurman, W A; Heirwegh, K P; van der Linden, C J; Kootstra, G

1986-08-01

Long-term effects of hepatocyte transplantation (HTX) in the treatment of enzyme deficiency disease were studied. Congenic enzyme-deficient (R/APfd-j/j) and non-enzyme-deficient (R/APfd) rats were used as recipients and donors, respectively. The R/APfd-j/j rat strain is congenitally deficient of bilirubin uridyldiphosphate (UDP)-glucuronyl transferase. R/APfd-j/j rats underwent HTX by intrasplenic injection of 10(7) isolated R/APfd hepatocytes (group 1A). Another group of R/APfd-j/j rats was treated similarly, but underwent splenectomy after 11 weeks (group 1B). Controls consisted of R/APfd-j/j rats grafted with 10(7) R/APfd-j/j hepatocytes (group 2), and R/APfd-j/j rats that underwent a sham operation (group 3). Total plasma bilirubin (TB) levels were significantly reduced in groups 1A and 1B during the experiment (both P less than 0.01). In the control groups TB reduction was not observed. Bile analyses at 30 weeks after HTX showed that in group 1A 13.7 +/- 2.7% of total biliary bilirubin was conjugated. In group 1B a significantly lower fraction was conjugated: 6.6 +/- 1.1% (P less than 0.05). Conjugated bilirubin was not found in bile of groups 2 and 3. Histology showed survival of hepatocytes in all spleens of rats of groups 1A, 1B and 2. It is concluded that congenic hepatocytes from R/APfd donors are not rejected after transplantation into the R/APfd-j/j rat, and maintain long-term function. Splenectomy does not abolish, but does reduce, the therapeutic effect significantly, indicating that part of the transplanted hepatocytes maintains function in the enzyme-deficient host liver. The congenic R/APfd-j/j and R/APfd rat strains represent a new animal model for research in metabolic deficiency disease.

A Microfabricated Platform for Generating Physiologically-Relevant Hepatocyte Zonation

NASA Astrophysics Data System (ADS)

McCarty, William J.; Usta, O. Berk; Yarmush, Martin L.

2016-05-01

In vitro liver models have been important tools for more than 40 years for academic research and preclinical toxicity screening by the pharmaceutical industry. Hepatocytes, the highly metabolic parenchymal cells of the liver, are efficient at different metabolic chemistries depending on their relative spatial location along the sinusoid from the portal triad to the central vein. Although replicating hepatocyte metabolic zonation is vitally important for physiologically-relevant in vitro liver tissue and organ models, it is most often completely overlooked. Here, we demonstrate the creation of spatially-controlled zonation across multiple hepatocyte metabolism levels through the application of precise concentration gradients of exogenous hormone (insulin and glucagon) and chemical (3-methylcholanthrene) induction agents in a microfluidic device. Observed gradients in glycogen storage via periodic acid-Schiff staining, urea production via carbamoyl phosphatase synthetase I staining, and cell viability after exposure to allyl alcohol and acetaminophen demonstrated the in vitro creation of hepatocyte carbohydrate, nitrogen, alcohol degradation, and drug conjugation metabolic zonation. This type of advanced control system will be crucial for studies evaluating drug metabolism and toxicology using in vitro constructs.

Free Fatty Acids Shift Insulin-induced Hepatocyte Proliferation towards CD95-dependent Apoptosis*

PubMed Central

Sommerfeld, Annika; Reinehr, Roland; Häussinger, Dieter

2015-01-01

Insulin is known to induce hepatocyte swelling, which triggers via integrins and c-Src kinase an activation of the epidermal growth factor receptor (EGFR) and subsequent cell proliferation (1). Free fatty acids (FFAs) are known to induce lipoapoptosis in liver cells in a c-Jun-NH2-terminal kinase (JNK)-dependent, but death receptor-independent way (2). As non-alcoholic steatohepatitis (NASH) is associated with hyperinsulinemia and increased FFA-blood levels, the interplay between insulin and FFA was studied with regard to hepatocyte proliferation and apoptosis in isolated rat and mouse hepatocytes. Saturated long chain FFAs induced apoptosis and JNK activation in primary rat hepatocytes, but did not activate the CD95 (Fas, APO-1) system, whereas insulin triggered EGFR activation and hepatocyte proliferation. Coadministration of insulin and FFAs, however, abolished hepatocyte proliferation and triggered CD95-dependent apoptosis due to a JNK-dependent association of the activated EGFR with CD95, subsequent CD95 tyrosine phosphorylation and formation of the death-inducing signaling complex (DISC). JNK inhibition restored the proliferative insulin effect in presence of FFAs and prevented EGFR/CD95 association, CD95 tyrosine phosphorylation and DISC formation. Likewise, in presence of FFAs insulin increased apoptosis in hepatocytes from wild type but not from Alb-Cre-FASfl/fl mice, which lack functional CD95. It is concluded that FFAs can shift insulin-induced hepatocyte proliferation toward hepatocyte apoptosis by triggering a JNK signal, which allows activated EGFR to associate with CD95 and to trigger CD95-dependent apoptosis. Such phenomena may contribute to the pathogenesis of NASH. PMID:25548285

Experimental evaluation of rodent exclusion methods to reduce hantavirus transmission to residents in a Native American community in New Mexico.

PubMed

Hopkins, Andrew S; Whitetail-Eagle, Joe; Corneli, Amy L; Person, Bobbie; Ettestad, Paul J; DiMenna, Mark; Norstog, Jon; Creswell, Jacob; Khan, Ali S; Olson, James G; Cavallaro, Kathleen F; Bryan, Ralph T; Cheek, James E; Begay, Bruce; Hoddenbach, Gerard A; Ksiazek, Thomas G; Mills, James N

2002-01-01

We conducted a pilot study to evaluate the efficacy of rodent proofing continuously occupied homes as a method for lowering the risk for hantavirus pulmonary syndrome (HPS) among residents of a Native American community in northwestern New Mexico. Rodent proofing of dwellings was paired with culturally appropriate health education. Seventy homes were randomly assigned to treatment or control categories. Treatment homes were rodent-proofed by sealing openings around foundations, doors, roofs, and pipes and repairing screens and windows. Repairs to each dwelling were limited to $500 US. After repairs were completed, 15-20 snap traps were placed in each treatment and control home and checked approximately every 2 days for an average of 3-4 weeks. During 23,373 trap nights, one house mouse (Mus musculus) was captured in one treatment home, and 20 mice (16 deer mice, Peromyscus maniculatus, two Pinyon mice, Peromyscus truei, and two unidentified mice) were captured in five control homes (one house had 14 captures, two had two captures, and two had one capture). Trap success was 0.01% in treatment homes and 0.15% in controls. Intensity of infestation (mean number of mice captured per infested home) was 1 in treatment homes and 4 in controls. Observations of evidence of infestation (feces, nesting material, gnaw marks, or reports of infestation by occupant) per 100 days of observation were 1.2 in treatment homes and 3.1 in controls. Statistical power of the experiment was limited because it coincided with a period of low rodent abundance (August-November 2000). Nevertheless, these results suggest that inexpensive rodent proofing of occupied rural homes can decrease the frequency and intensity of rodent intrusion, thereby reducing the risk of HPS among rural residents in the southwestern United States.

Primary hepatocytes and their cultures in liver apoptosis research

PubMed Central

Vinken, Mathieu; Maes, Michaël; Oliveira, André G.; Cogliati, Bruno; Marques, Pedro E.; Menezes, Gustavo B.; Dagli, Maria Lúcia Zaidan; Vanhaecke, Tamara; Rogiers, Vera

2014-01-01

Apoptosis not only plays a key role in physiological demise of defunct hepatocytes, but is also associated with a plethora of acute and chronic liver diseases as well as with hepatotoxicity. The present paper focuses on the modelling of this mode of programmed cell death in primary hepatocyte cultures. Particular attention is paid to the activation of spontaneous apoptosis during the isolation of hepatocytes from the liver, its progressive manifestation upon the subsequent establishment of cell cultures and simultaneously to strategies to counteract this deleterious process. In addition, currently applied approaches to experimentally induce controlled apoptosis in this in vitro setting for mechanistic research purposes and thereby its detection using relevant biomarkers are reviewed. PMID:24013573

Soluble asialoglycoprotein receptors reflect the apoptosis of hepatocytes.

PubMed

Kakegawa, Tetsuji; Ise, Hirohiko; Sugihara, Nobuhiro; Nikaido, Toshio; Negishi, Naoki; Akaike, Toshihiro; Tanaka, Eiji

2002-01-01

Cell death is thought to take place through at least two distinct processes: apoptosis and necrosis. There is increasing evidence that dysregulation of the apoptotic program is involved in liver diseases. However, there is no method to simply evaluate apoptosis in the liver tissue at present. It has been reported that the expression of asialoglycoprotein receptors (AGPRs) increases with apoptosis, but there is no report until now that investigates the influence of soluble AGPRs on apoptosis of hepatocytes. Soluble AGPRs have been reported to be present in human serum under physiological conditions. In the present study, in order to investigate the correlation between apoptosis of hepatocytes and soluble AGPR, mouse soluble AGPRs were detected using SDS-PAGE and Western blot analysis was conducted using anti-extracellular mouse hepatic lectin-1 (Ex-MHL-1) antiserum (polyclonal rabbit serum). The mouse soluble AGPRs were present in culture medium and mouse serum when hepatocytes were damaged. The soluble AGPRs increased proportionately, as the number of dead hepatocytes increased. In addition, soluble AGPRs existed more when apoptotic cell death was observed in in vitro and in vivo than when necrotic cell death was observed. The extracellular moiety of MHL-1 exists in the culture medium and mouse serum as a soluble AGPR, but the detailed mechanism of releasing soluble AGPR from hepatocytes has not been revealed yet. We described the first evidence for the relation between quantity of soluble AGPRs with two kinds of cell death: necrosis and apoptosis. Based on the results of our study, soluble AGPRs might become a new marker of apoptosis in the liver tissue and be useful for clinical diagnosis and treatment for liver diseases.

Interspecies differences in metabolism of arsenic by cultured primary hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Drobna, Zuzana; Walton, Felecia S.; Harmon, Anne W.

2010-05-15

Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAsmore » methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [{sup 73}As]arsenite (iAs{sup III}; 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs{sup III} to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs{sup III} than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs{sup III} was associated with inhibition of DMAs production by moderate concentrations of iAs{sup III} and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also

Gene expression analysis of a porcine hepatocyte/bile duct in vitro differentiaion model

USDA-ARS?s Scientific Manuscript database

A serum-free, feeder-cell-dependent, inductive differentiation culture system of porcine hepatocytes and bile ductules was analyzed for differential gene expression on a porcine genome microarray. Primary cultures of baby pig hepatocytes (BPH) were matured in culture as a monolayer of hepatocytes w...

Superior In vivo Transduction of Human Hepatocytes Using Engineered AAV3 Capsid.

PubMed

Vercauteren, Koen; Hoffman, Brad E; Zolotukhin, Irene; Keeler, Geoffrey D; Xiao, Jing W; Basner-Tschakarjan, Etiena; High, Katherine A; Ertl, Hildegund Cj; Rice, Charles M; Srivastava, Arun; de Jong, Ype P; Herzog, Roland W

2016-06-01

Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in "humanized" mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.

Virtual reality systems for rodents

PubMed Central

Ayaz, Aslı

2017-01-01

Abstract Over the last decade virtual reality (VR) setups for rodents have been developed and utilized to investigate the neural foundations of behavior. Such VR systems became very popular since they allow the use of state-of-the-art techniques to measure neural activity in behaving rodents that cannot be easily used with classical behavior setups. Here, we provide an overview of rodent VR technologies and review recent results from related research. We discuss commonalities and differences as well as merits and issues of different approaches. A special focus is given to experimental (behavioral) paradigms in use. Finally we comment on possible use cases that may further exploit the potential of VR in rodent research and hence inspire future studies. PMID:29491968

Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities.

PubMed

Sakai, Yusuke; Koike, Makiko; Kawahara, Daisuke; Hasegawa, Hideko; Murai, Tomomi; Yamanouchi, Kosho; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Fujita, Fumihiko; Kuroki, Tamotsu; Eguchi, Susumu

2018-03-05

Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1×10 3  cells/cm 2 . Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Differential Lectin Agglutination of Fetal, Dividing-Postnatal, and Malignant Hepatocytes

PubMed Central

Becker, F. F.

1974-01-01

Numerous studies have reported the capacity of the lectin, concanavalin A, to agglutinate selected cell-types. The finding that cells transformed in culture, embryonic cells, and malignant cells are all agglutinated by this substance, may contribute to our understanding of the oncogenic process. The present study compared the response to concanavalin A of rat hepatocytes derived from livers of differing developmental and mitotic-status as well as those derived from malignant liver tumors (hepatomas). Fetal hepatocytes and hepatoma cells were highly susceptible to agglutination while hepatocytes from post-natal livers, whether dividing or quiescent, were not. Treatment with protease(s) did not make the interphase hepatocyte agglutinable. These data emphasize the importance of examining a wide variety of cells in attempting to understand the interaction of lectins on cell surfaces, and further, demonstrate the value of obtaining cells directly from tissue(s) during differing physiologic and pathologic states. Images PMID:4373708

TECHNIQUES IN ASEPTIC RODENT SURGERY

PubMed Central

Hoogstraten-Miller, Shelley L.; Brown, Patricia A.

2008-01-01

Performing aseptic survival surgery in rodents can be challenging. This unit describes some basic principles to assist clinicians, researchers, and technicians in becoming proficient in performing aseptic rodent surgery. PMID:18729061

Property of hepatitis B virus replication in Tupaia belangeri hepatocytes.

PubMed

Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

2016-01-08

The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3-6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10(5) copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10(4)-10(6) copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10(3) copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Variable responses of small and large human hepatocytes to hypoxia and hypoxia/reoxygenation (H–R)

PubMed Central

Bhogal, Ricky H.; Weston, Christopher J.; Curbishley, Stuart M.; Bhatt, Anand N.; Adams, David H.; Afford, Simon C.

2011-01-01

Hypoxia and hypoxia–reoxygenation (H–R) regulate human hepatocyte cell death by mediating the accumulation of reactive oxygen species (ROS). Hepatocytes within the liver are organised into peri-portal (PP) and peri-venous (PV) subpopulations. PP and PV hepatocytes differ in size and function. We investigated whether PP and PV human hepatocytes exhibit differential susceptibility to hypoxic stress. Isolated hepatocytes were used in an in vitro model of hypoxia and H–R. ROS production and cell death were assessed using flow cytometry. PV, and not PP hepatocytes, accumulate intracellular ROS in a mitochondrial dependent manner during hypoxia and H–R. This increased ROS regulates hepatocyte apoptosis and necrosis via a mitochondrial pathway. These findings have implications on the understanding of liver injury and application of potential therapeutic strategies. PMID:21356211

Rodent Research-1 Validation of Rodent Hardware

NASA Technical Reports Server (NTRS)

Globus, Ruth; Beegle, Janet

2013-01-01

To achieve novel science objectives, validation of a rodent habitat on ISS will enable - In-flight analyses during long duration spaceflight- Use of genetically altered animals- Application of modern analytical techniques (e.g. genomics, proteomics, and metabolomics)

Epidermal growth factor-stimulated protein phosphorylation in rat hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Connelly, P.A.; Sisk, R.B.; Johnson, R.M.

1987-05-01

Epidermal growth factor (EGF) causes a 6-fold increase in the phosphorylation state of a cytosolic protein (pp36, M/sub r/ = 36,000, pI = 5.5) in hepatocytes isolated from fasted, male, Wistar rats. Stimulation of /sup 32/P incorporation is observed as early as 1 min following treatment of hepatocytes with EGF and is still present at 30 min after exposure to the growth factor. The phosphate incorporated into pp36 in response to EGF is located predominantly in serine but not tyrosine residues. Phosphorylation of pp36 does not occur in response to insulin or to agents which specifically activate the cAMP-dependent proteinmore » kinase (S/sub p/ -cAMPS), protein kinase C (PMA) or Ca/sup 2 +//calmodulin-dependent protein kinases (A23187) in these cells. Prior treatment of hepatocytes with the cAMP analog, S/sub p/-cAMPS, or ADP-ribosylation of N/sub i/, the inhibitory GTP-binding protein of the adenylate cyclase complex, does not prevent EGF-stimulated phosphorylation of pp36. However, as seen in other cell types, pretreatment of hepatocytes with PMA abolishes all EGF-mediated responses including phosphorylation of pp36. These results suggest that EGP specifically activates an uncharacterized, serine protein kinase in hepatocytes that is distal to the intrinsic EGF receptor tyrosine protein kinase. The rapid activation of this kinase suggests that it may play an important role in the early response of the cell to EGF.« less

Solubilized liver extracellular matrix maintains primary rat hepatocyte phenotype in-vitro.

PubMed

Loneker, Abigail E; Faulk, Denver M; Hussey, George S; D'Amore, Antonio; Badylak, Stephen F

2016-04-01

Whole organ engineering and cell-based regenerative medicine approaches are being investigated as potential therapeutic options for end-stage liver failure. However, a major challenge of these strategies is the loss of hepatic specific function after hepatocytes are removed from their native microenvironment. The objective of the present study was to determine if solubilized liver extracellular matrix (ECM), when used as a media supplement, can better maintain hepatocyte phenotype compared to type I collagen alone or solubilized ECM harvested from a non-liver tissue source. Liver extracellular matrix (LECM) from four different species was isolated via liver tissue decellularization, solubilized, and then used as a media supplement for primary rat hepatocytes (PRH). The four species of LECM investigated were human, porcine, canine and rat. Cell morphology, albumin secretion, and ammonia metabolism were used to assess maintenance of hepatocyte phenotype. Biochemical and mechanical characterization of each LECM were also conducted. Results showed that PRH's supplemented with canine and porcine LECM maintained their phenotype to a greater extent compared to all other groups. PRH's supplemented with canine and porcine LECM showed increased bile production, increased albumin production, and the formation of multinucleate cells. The findings of the present study suggest that solubilized liver ECM can support in-vitro hepatocyte culture and should be considered for therapeutic and diagnostic techniques that utilize hepatocytes. © 2016 Wiley Periodicals, Inc.

In vitro differentiation of rat bone marrow mesenchymal stem cells into hepatocytes.

PubMed

Feng, Zhihui; Li, Changying; Jiao, Shuxian; Hu, Bin; Zhao, Lin

2011-01-01

To investigate the mechanism and regulation of differentiation from bone marrow mesenchymal stem cells (BMSCs) into hepatocytes and to find a new source for therapies of hepatic diseases. We isolated BMSCs for subsequent differentiation in the presence of hepatocyte growth factor (HGF) or beta-nerve growth factor (beta-NGF). Cell morphology was observed and cell surface phenotypings were detected by flow cytometry. a1-antitrypsin (AAT) expression of the hepatocytes was confirmed by immunocytochemistry and albumin expression was validated by real time PCR and western blotting. The expression of high-affinity nerve growth factor receptor (TrkA) and the activation of Erk pathway were detected by western blotting. Hepatocyte functional activity was confirmed by uptake of indocyanine green (ICG) assay. Small round cells appeared in the presence of HGF on day 10 or beta-NGF on day 12. Differentiated cells expressed albumin and had functional characteristics of hepatocytes, such as uptake of ICG. BMSCs were positive for TrkA. HGF and beta-NGF significantly upregulated the protein levels of phospho-Erk. BMSCs could differentiate into hepatocytes in the differentiation media including HGF or beta-NGF. Combination of HGF and beta-NGF significantly increased the efficiency of hepatic differentiation.

YAP Inhibition Restores Hepatocyte Differentiation in Advanced HCC, Leading to Tumor Regression.

PubMed

Fitamant, Julien; Kottakis, Filippos; Benhamouche, Samira; Tian, Helen S; Chuvin, Nicolas; Parachoniak, Christine A; Nagle, Julia M; Perera, Rushika M; Lapouge, Marjorie; Deshpande, Vikram; Zhu, Andrew X; Lai, Albert; Min, Bosun; Hoshida, Yujin; Avruch, Joseph; Sia, Daniela; Campreciós, Genís; McClatchey, Andrea I; Llovet, Josep M; Morrissey, David; Raj, Lakshmi; Bardeesy, Nabeel

2015-03-10

Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC). Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat in maintaining metabolic specialization, differentiation, and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs that control hepatocyte quiescence and differentiation. Importantly, treatment with small interfering RNA-lipid nanoparticles (siRNA-LNPs) targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of the positional identity of hepatocytes, supports targeting of YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype that is potentially responsive to this approach. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Rifampicin exacerbates isoniazid-induced toxicity in human but not in rat hepatocytes in tissue-like cultures

PubMed Central

Shen, C; Meng, Q; Zhang, G; Hu, W

2007-01-01

Background and purpose: Rifampicin has been extensively reported to exacerbate the hepatotoxicity of isoniazid in patients with tuberculosis. However, this was controversially claimed by previous reports using rat models. This study evaluated the effect of rifampicin on isoniazid-induced hepatocyte toxicity by using human and rat hepatocytes in tissue-like culture. Experimental approach: Hepatocytes in tissue-like gel entrapment were used to examine isoniazid toxicity, as shown by cell viability, intracellular glutathione content and albumin secretion. For demonstration of the differential effects of rifampicin on human and rat hepatocytes, induction by rifampicin of cytochrome P450 (CYP) 2E1, a major enzyme associated with isoniazid hepatotoxicity, was detected by 4-nitrocatechol formation and RT-PCR analysis. Key results: Rifampicin (12 μM) enhanced isoniazid-induced toxicity in human hepatocytes but not in rat hepatocytes. Enhanced CYP 2E1 enzymic activity and mRNA expression were similarly detected in human hepatocytes but not in rat hepatocytes. Both rat and human hepatocytes in gel entrapment were more sensitive to isoniazid treatment compared with the corresponding hepatocytes in a monolayer culture. Conclusions and implications: The difference in induction of CYP 2E1 by rifampicin between rat and human hepatocytes accounted for the difference in exacerbation of isoniazid hepatocyte toxicity by rifampicin, with more significant toxicity in gel entrapment than in monolayer cultures. Thus, human hepatocytes in tissue-like cultures (gel entrapment) could be an effective model for hepatotoxicity research in vitro, closer to the in vivo situation. PMID:18071298

HBV life cycle is restricted in mouse hepatocytes expressing human NTCP.

PubMed

Li, Hanjie; Zhuang, Qiuyu; Wang, Yuze; Zhang, Tianying; Zhao, Jinghua; Zhang, Yali; Zhang, Junfang; Lin, Yi; Yuan, Quan; Xia, Ningshao; Han, Jiahuai

2014-03-01

Recent studies have revealed that human sodium taurocholate cotransporting polypeptide (SLC10A1 or NTCP) is a functional cellular receptor for hepatitis B virus (HBV). However, whether human NTCP can support HBV infection in mouse hepatocyte cell lines has not been clarified. Because an HBV-permissible mouse model would be helpful for the study of HBV pathogenesis, it is necessary to investigate whether human NTCP supports the susceptibility of mouse hepatocyte cell lines to HBV. The results show that exogenous human NTCP expression can render non-susceptible HepG2 (human), Huh7 (human), Hepa1-6 (mouse), AML-12 (mouse) cell lines and primary mouse hepatocyte (PMH) cells susceptible to hepatitis D virus (HDV) which employs HBV envelope proteins. However, human NTCP could only introduce HBV susceptibility in human-derived HepG2 and Huh7 cells, but not in mouse-derived Hepa1-6, AML-12 or PMH cells. These data suggest that although human NTCP is a functional receptor that mediates HBV infection in human cells, it cannot support HBV infection in mouse hepatocytes. Our study indicated that the restriction of HBV in mouse hepatocytes likely occurs after viral entry but prior to viral transcription. We have excluded the role of mouse hepatocyte nuclear factors in the restriction of the HBV life cycle and showed that knockdown or inhibition of Sting, TBK1, IRF3 or IRF7, the components of the anti-viral signaling pathways, had no effect on HBV infection in mouse hepatocytes. Therefore, murine restriction factors that limit HBV infection need to be identified before a HBV-permissible mouse line can be created.

The establishment and characterization of immortal hepatocyte cell lines from a mouse liver injury model.

PubMed

Risal, Prabodh; Cho, Baik Hwan; Sylvester, Karl G; Kim, Jae-Chun; Kim, Hyoung Tae; Jeong, Yeon Jun

2011-09-01

Hepatocytes are an important research tool used for numerous applications. However, a short life span and a limited capacity to replicate in vitro limit the usefulness of primary hepatocyte cultures. We have hypothesized that in vivo priming of hepatocyte could make them more susceptible to growth factors in the medium for continuous proliferation in vitro. Here, a novel approach used to establish hepatocyte cell lines that included hepatocyte priming in vivo prior to culture with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet was attempted. The cell line grew in a monolayer while maintaining a granular cytoplasm and a round nucleus. Electron microscopy displayed hepatocyte-like features including mitochondria, glycogen granules, and the presence of bile canaliculi. This cell line expressed many mature hepatocyte-specific genes including albumin, alpha1-antitrypsin, glucose 6-phosphatase, and tyrosine aminotransferase. Functional characteristic of hepatocytes like the ability to store glycogen, lipid, and synthesis of urea is well demonstrated by this cell line. These cells demonstrated anchorage dependent growth properties in soft agar and did not form tumors after transplantation into nude mice. This cell line can be sustained in culture for more than 100 passages (>1.5 years) without undergoing noticeable morphological changes or transformation. This novel method resulted in the establishment of an immortal, non-transformed hepatocyte cell line with functional characteristics that may aid research of cell metabolism, toxicology, and hepatocyte transplantation.

The largest fossil rodent

PubMed Central

Rinderknecht, Andrés; Blanco, R. Ernesto

2008-01-01

The discovery of an exceptionally well-preserved skull permits the description of the new South American fossil species of the rodent, Josephoartigasia monesi sp. nov. (family: Dinomyidae; Rodentia: Hystricognathi: Caviomorpha). This species with estimated body mass of nearly 1000 kg is the largest yet recorded. The skull sheds new light on the anatomy of the extinct giant rodents of the Dinomyidae, which are known mostly from isolated teeth and incomplete mandible remains. The fossil derives from San José Formation, Uruguay, usually assigned to the Pliocene–Pleistocene (4–2 Myr ago), and the proposed palaeoenvironment where this rodent lived was characterized as an estuarine or deltaic system with forest communities. PMID:18198140

Sensitivity to coincidences and paranormal belief.

PubMed

Hadlaczky, Gergö; Westerlund, Joakim

2011-12-01

Often it is difficult to find a natural explanation as to why a surprising coincidence occurs. In attempting to find one, people may be inclined to accept paranormal explanations. The objective of this study was to investigate whether people with a lower threshold for being surprised by coincidences have a greater propensity to become believers compared to those with a higher threshold. Participants were exposed to artificial coincidences, which were formally defined as less or more probable, and were asked to provide remarkability ratings. Paranormal belief was measured by the Australian Sheep-Goat Scale. An analysis of the remarkability ratings revealed a significant interaction effect between Sheep-Goat score and type of coincidence, suggesting that people with lower thresholds of surprise, when experiencing coincidences, harbor higher paranormal belief than those with a higher threshold. The theoretical aspects of these findings were discussed.

Tim2 is expressed in mouse fetal hepatocytes and regulates their differentiation.

PubMed

Watanabe, Natsumi; Tanaka, Minoru; Suzuki, Kaori; Kumanogoh, Atsushi; Kikutani, Hitoshi; Miyajima, Atsushi

2007-05-01

Liver development is regulated by various extracellular molecules such as cytokines and cell surface proteins. Although several such regulators have been identified, additional molecules are likely to be involved in liver development. To identify such molecules, we employed the signal sequence trap (SST) method to screen cDNAs encoding a secreted or membrane protein from fetal liver and obtained a number of clones. Among them, we found that T cell immunoglobulin and mucin domain 2 (Tim2) was expressed specifically on immature hepatocytes in the fetal liver. Tim2 has been shown to regulate immune responses, but its role in liver development had not been studied. We have examined the possible role of Tim2 in hepatocyte differentiation. At first, we prepared a soluble Tim2 fusion protein consisting of its extracellular domain and the Fc domain of human IgG (Tim2-hFc) and found that it bound to fetal and adult hepatocytes, suggesting that there are Tim2-binding molecules on hepatocytes. Second, Tim2-hFc inhibited the differentiation of hepatocytes in fetal liver primary culture, i.e., the expression of mature hepatic enzymes and accumulation of glycogen were severely reduced. Third, Tim2-hFc also inhibited proliferation of fetal hepatocytes. Fourth, down-regulation of Tim2 expression by small interfering RNA (siRNA) enhanced the expression of liver differentiation marker genes. It is strongly suggested that Tim2 is involved in the differentiation of fetal hepatocytes.

Dexamethasone treatment induces the reprogramming of pancreatic acinar cells to hepatocytes and ductal cells.

PubMed

Al-Adsani, Amani; Burke, Zoë D; Eberhard, Daniel; Lawrence, Katherine L; Shen, Chia-Ning; Rustgi, Anil K; Sakaue, Hiroshi; Farrant, J Mark; Tosh, David

2010-10-27

The pancreatic exocrine cell line AR42J-B13 can be reprogrammed to hepatocytes following treatment with dexamethasone. The question arises whether dexamethasone also has the capacity to induce ductal cells as well as hepatocytes. AR42J-B13 cells were treated with and without dexamethasone and analyzed for the expression of pancreatic exocrine, hepatocyte and ductal markers. Addition of dexamethasone inhibited pancreatic amylase expression, induced expression of the hepatocyte marker transferrin as well as markers typical of ductal cells: cytokeratin 7 and 19 and the lectin peanut agglutinin. However, the number of ductal cells was low compared to hepatocytes. The proportion of ductal cells was enhanced by culture with dexamethasone and epidermal growth factor (EGF). We established several features of the mechanism underlying the transdifferentiation of pancreatic exocrine cells to ductal cells. Using a CK19 promoter reporter, we show that a proportion of the ductal cells arise from differentiated pancreatic exocrine-like cells. We also examined whether C/EBPβ (a transcription factor important in the conversion of pancreatic cells to hepatocytes) could alter the conversion from acinar cells to a ductal phenotype. Overexpression of an activated form of C/EBPβ in dexamethasone/EGF-treated cells provoked the expression of hepatocyte markers and inhibited the expression of ductal markers. Conversely, ectopic expression of a dominant-negative form of C/EBPβ, liver inhibitory protein, inhibited hepatocyte formation in dexamethasone-treated cultures and enhanced the ductal phenotype. These results indicate that hepatocytes and ductal cells may be induced from pancreatic exocrine AR42J-B13 cells following treatment with dexamethasone. The conversion from pancreatic to hepatocyte or ductal cells is dependent upon the expression of C/EBPβ.

Epigallocatechin-3-gallate ameliorates insulin resistance in hepatocytes.

PubMed

Ma, Shan-Bo; Zhang, Rui; Miao, Shan; Gao, Bin; Lu, Yang; Hui, Sen; Li, Long; Shi, Xiao-Peng; Wen, Ai-Dong

2017-06-01

Hyperglycemia is a typical pathogenic factor in a series of complications among patients with type II diabetes. Epigallocatechin-3-gallate (EGCG) is the major polyphenol extracted from green tea and is reported to be an antioxidant. The aim of the present study was to examine the effect of EGCG on insulin resistance in human HepG2 cells pretreated with high concentrations of glucose. The protein kinase B (AKT)/glycogen synthase kinase (GSK) pathways were analyzed using western blot analysis in HepG2 cells and primary mouse hepatocytes treated with high glucose and/or EGCG. Cellular glycogen content was determined using a glycogen assay kit. Reactive oxygen species (ROS) production was determined using dihydroethidium staining and flow cytometry. c‑JUN N‑terminal kinase (JNK)/insulin receptor substrate 1 (IRS1)/AKT/GSK signaling was explored using western blot analysis in HepG2 cells treated with high glucose and/or EGCG or N-acetyl-cysteine. High glucose significantly decreased the levels of phosphorylated AKT and GSK in HepG2 cells and mouse primary hepatocytes. Pretreatment with EGCG significantly restored the activation of AKT and GSK in HepG2 cells and primary hepatocytes exposed to high glucose. In HepG2 cells and primary hepatocytes, glycogen synthesis was improved by EGCG treatment in a dose‑dependent manner. High glucose significantly stimulated the production of ROS while EGCG protected high glucose‑induced ROS production. ROS is known to serve a major role in high glucose induced‑insulin resistance by increasing JNK and IRS1 serine phosphorylation. In the present study, EGCG was observed to enhance the insulin‑signaling pathway. EGCG ameliorated high glucose‑induced insulin resistance in the hepatocytes by potentially decreasing ROS‑induced JNK/IRS1/AKT/GSK signaling.

Metabolism and disposition of 2-ethylhexyl-p-methoxycinnamate following oral gavage and dermal exposure in Harlan Sprague Dawley rats and B6C3F1/N mice and in hepatocytes in vitro.

PubMed

Fennell, Timothy R; Mathews, James M; Snyder, Rodney W; Hong, Yan; Watson, Scott L; Black, Sherry R; McIntyre, Barry S; Waidyanatha, Suramya

2017-11-23

1. 2-Ethylhexyl-p-methoxycinnamate (EHMC) is commonly used as an ingredient in sunscreens, resulting in potential oral and dermal exposure in humans. 2. Clearance and metabolism of EHMC in hepatocytes and disposition and metabolism of EHMC in rodents following oral (8-800 mg/kg) intravenous (IV) (8 mg/kg) or dermal (0.8-80 mg/kg representing 0.1-10% formulation concentration) exposure to [ 14 C]EHMC were investigated in rats and mice. 3. EHMC was rapidly cleared from rat and mouse hepatocytes (half-life ≤3.16 min) and less rapidly (half-life ≤48 min) from human hepatocytes. 4. [ 14 C]EHMC was extensively absorbed and excreted primarily in urine by 72 h after oral administration to rats (65-80%) and mice (63-72%). Oral doses to rats were excreted to a lesser extent (3-8%) in feces and as CO 2 (1-4%). Radioactive residues in tissues were <1% of the dose. There were no sex or species differences in disposition in rats. 5. Following dermal application, 34-42% of an 8-mg/kg dose was absorbed in rats, and 54-62% in mice in 72-h. 6. Among numerous urinary metabolites associated with hydrolysis of the ester, two potential reproductive and developmental toxicants, 2-ethylhexanol and 2-ethylhexanoic acid were produced by metabolism of EHMC.

Rapid generation of functional hepatocyte-like cells from human adipose-derived stem cells.

PubMed

Fu, Yanli; Deng, Jie; Jiang, Qingyuan; Wang, Yuan; Zhang, Yujing; Yao, Yunqi; Cheng, Fuyi; Chen, Xiaolei; Xu, Fen; Huang, Meijuan; Yang, Yang; Zhang, Shuang; Yu, Dechao; Zhao, Robert Chunhua; Wei, Yuquan; Deng, Hongxin

2016-08-05

Liver disease is a major cause of death worldwide. Orthotropic liver transplantation (OLT) represents the only effective treatment for patients with liver failure, but the increasing demand for organs is unfortunately so great that its application is limited. Hepatocyte transplantation is a promising alternative to OLT for the treatment of some liver-based metabolic disorders or acute liver failure. Unfortunately, the lack of donor livers also makes it difficult to obtain enough viable hepatocytes for hepatocyte-based therapies. Currently, a fundamental solution to this key problem is still lacking. Here we show a novel non-transgenic protocol that facilitates the rapid generation of functional induced hepatocytes (iHeps) from human adipose-derived stem cells (hADSCs), providing a source of available cells for autologous hepatocytes to treat liver disease. We used collagenase digestion to isolate hADSCs. The surface marker was detected by flow cytometry. The multipotential differentiation potency was detected by induction into adipocytes, osteocytes, and chondrocytes. Passage 3-7 hADSCs were induced into iHeps using an induction culture system composed of small molecule compounds and cell factors. Primary cultured hADSCs presented a fusiform or polygon appearance that became fibroblast-like after passage 3. More than 95 % of the cells expressed the mesenchymal cell markers CD29, CD44, CD166, CD105, and CD90. hADSCs possessed multipotential differentiation towards adipocytes, osteocytes, and chondrocytes. We rapidly induced hADSCs into iHeps within 10 days in vitro; the cellular morphology changed from fusiform to close-connected cubiform, which was similar to hepatocytes. After induction, most of the iHeps co-expressed albumin and alpha-1 antitrypsin; they also expressed mature hepatocyte special genes and achieved the basic functions of hepatocyte. Moreover, iHep transplantation could improve the liver function of acute liver-injured NPG mice and prolong life. We

Variable responses of small and large human hepatocytes to hypoxia and hypoxia/reoxygenation (H-R).

PubMed

Bhogal, Ricky H; Weston, Christopher J; Curbishley, Stuart M; Bhatt, Anand N; Adams, David H; Afford, Simon C

2011-03-23

Hypoxia and hypoxia-reoxygenation (H-R) regulate human hepatocyte cell death by mediating the accumulation of reactive oxygen species (ROS). Hepatocytes within the liver are organised into peri-portal (PP) and peri-venous (PV) subpopulations. PP and PV hepatocytes differ in size and function. We investigated whether PP and PV human hepatocytes exhibit differential susceptibility to hypoxic stress. Isolated hepatocytes were used in an in vitro model of hypoxia and H-R. ROS production and cell death were assessed using flow cytometry. PV, and not PP hepatocytes, accumulate intracellular ROS in a mitochondrial dependent manner during hypoxia and H-R. This increased ROS regulates hepatocyte apoptosis and necrosis via a mitochondrial pathway. These findings have implications on the understanding of liver injury and application of potential therapeutic strategies. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Vectorial Entry and Release of Hepatitis A Virus in Polarized Human Hepatocytes ▿

PubMed Central

Snooks, Michelle J.; Bhat, Purnima; Mackenzie, Jason; Counihan, Natalie A.; Vaughan, Nicola; Anderson, David A.

2008-01-01

Hepatitis A virus (HAV) is an enterically transmitted virus that replicates predominantly in hepatocytes within the liver before excretion via bile through feces. Hepatocytes are polarized epithelial cells, and it has been assumed that the virus load in bile results from direct export of HAV via the apical domain of polarized hepatocytes. We have developed a subclone of hepatocyte-derived HepG2 cells (clone N6) that maintains functional characteristics of polarized hepatocytes but displays morphology typical of columnar epithelial cells, rather than the complex morphology that is typical of hepatocytes. N6 cells form microcolonies of polarized cells when grown on glass and confluent monolayers of polarized cells on semipermeable membranes. When N6 microcolonies were exposed to HAV, infection was restricted to peripheral cells of polarized colonies, whereas all cells could be infected in colonies of nonpolarized HepG2 cells (clone C11) or following disruption of tight junctions in N6 colonies with EGTA. This suggests that viral entry occurs predominantly via the basolateral plasma membrane, consistent with uptake of virus from the bloodstream after enteric exposure, as expected. Viral export was also found to be markedly vectorial in N6 but not C11 cells. However, rather than being exported from the apical domain as expected, more than 95% of HAV was exported via the basolateral domain of N6 cells, suggesting that virus is first excreted from infected hepatocytes into the bloodstream rather than to the biliary tree. Enteric excretion of HAV may therefore rely on reuptake and transcytosis of progeny HAV across hepatocytes into the bile. These studies provide the first example of the interactions between viruses and polarized hepatocytes. PMID:18579610

Nanofabricated Collagen-Inspired Synthetic Elastomers for Primary Rat Hepatocyte Culture

PubMed Central

Bettinger, Christopher J.; Kulig, Katherine M.; Vacanti, Joseph P.

2009-01-01

Synthetic substrates that mimic the properties of extracellular matrix proteins hold significant promise for use in systems designed for tissue engineering applications. In this report, we designed a synthetic polymeric substrate that is intended to mimic chemical, mechanical, and topological characteristics of collagen. We found that elastomeric poly(ester amide) substrates modified with replica-molded nanotopographic features enhanced initial attachment, spreading, and adhesion of primary rat hepatocytes. Further, hepatocytes cultured on nanotopographic substrates also demonstrated reduced albumin secretion and urea synthesis, which is indicative of strongly adherent hepatocytes. These results suggest that these engineered substrates can function as synthetic collagen analogs for in vitro cell culture. PMID:18847357

Highly Dynamic Host Actin Reorganization around Developing Plasmodium Inside Hepatocytes

PubMed Central

Gomes-Santos, Carina S. S.; Itoe, Maurice A.; Afonso, Cristina; Henriques, Ricardo; Gardner, Rui; Sepúlveda, Nuno; Simões, Pedro D.; Raquel, Helena; Almeida, António Paulo; Moita, Luis F.; Frischknecht, Friedrich; Mota, Maria M.

2012-01-01

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver. PMID:22238609

Human Hepatocyte Isolation: Does Portal Vein Embolization Affect the Outcome?

PubMed

Kluge, Martin; Reutzel-Selke, Anja; Napierala, Hendrik; Hillebrandt, Karl Herbert; Major, Rebeka Dalma; Struecker, Benjamin; Leder, Annekatrin; Siefert, Jeffrey; Tang, Peter; Lippert, Steffen; Sallmon, Hannes; Seehofer, Daniel; Pratschke, Johann; Sauer, Igor M; Raschzok, Nathanael

2016-01-01

Primary human hepatocytes are widely used for basic research, pharmaceutical testing, and therapeutic concepts in regenerative medicine. Human hepatocytes can be isolated from resected liver tissue. Preoperative portal vein embolization (PVE) is increasingly used to decrease the risk of delayed postoperative liver regeneration by induction of selective hypertrophy of the future remnant liver tissue. The aim of this study was to investigate the effect of PVE on the outcome of hepatocyte isolation. Primary human hepatocytes were isolated from liver tissue obtained from partial hepatectomies (n = 190) using the two-step collagenase perfusion technique followed by Percoll purification. Of these hepatectomies, 27 isolations (14.2%) were performed using liver tissue obtained from patients undergoing PVE before surgery. All isolations were characterized using parameters that had been described in the literature as relevant for the outcome of hepatocyte isolation. The isolation outcomes of the PVE and the non-PVE groups were then compared before and after Percoll purification. Metabolic parameters (transaminases, urea, albumin, and vascular endothelial growth factor secretion) were measured in the supernatant of cultured hepatocytes for more than 6 days (PVE: n = 4 and non-PVE: n = 3). The PVE and non-PVE groups were similar in regard to donor parameters (sex, age, and indication for surgery), isolation parameters (liver weight and cold ischemia time), and the quality of the liver tissue. The mean initial viable cell yield did not differ between the PVE and non-PVE groups (10.16 ± 2.03 × 10(6) cells/g vs. 9.70 ± 0.73 × 10(6) cells/g, p = 0.499). The initial viability was slightly better in the PVE group (77.8% ± 2.03% vs. 74.4% ± 1.06%). The mean viable cell yield (p = 0.819) and the mean viability (p = 0.141) after Percoll purification did not differ between the groups. PVE had no effect on enzyme leakage and metabolic

Lipopolysaccharide Stimulates p62-Dependent Autophagy-Like Aggregate Clearance in Hepatocytes

PubMed Central

Deng, Meihong; Sun, Qian; Loughran, Patricia; Billiar, Timothy R.; Scott, Melanie J.

2014-01-01

Impairment of autophagy has been associated with liver injury. TLR4-stimulation by LPS upregulates autophagy in hepatocytes, although the signaling pathways involved remain elusive. The objective of this study was to determine the signaling pathway leading to LPS-stimulated autophagy in hepatocytes. Cell lysates from livers of wild type (WT; C57BL/6) mice given LPS (5 mg/kg-IP) and hepatocytes from WT, TLR4ko, and MyD88ko mice treated with LPS (100 ng/mL) up to 24 h were collected. LC3II, p62/SQSTM1, Nrf2, and beclin1 levels were determined by immunoblot, immunofluorescence, and qPCR. Autophagy-like activation was measured by GFP-LC3-puncta formation and LC3II-expression. Beclin1, Nrf2, p62, MyD88, and TIRAP were knocked-down using siRNA. LC3II-expression increased in both liver and hepatocytes after LPS and was dependent on TLR4. Beclin1 expression did not increase after LPS in hepatocytes and beclin1-knockdown did not affect LC3II levels. In hepatocytes given LPS, expression of p62 increased and p62 colocalized with LC3. p62-knockdown prevented LC3II puncta formation. LPS-induced LC3II/p62-puncta also required MyD88/TIRAP signaling and localization of both Nrf2 and NFκB transcription factors to the nucleus to upregulate p62-expression. Therefore, TLR4-activation by LPS in hepatocytes induces a p62-mediated, not beclin1-mediated, autophagy-like clearance pathway that is hepatoprotective by clearing aggregate-prone or misfolded proteins from the cytosol and preserving energy homeostasis under stress. PMID:24683544

Lipopolysaccharide stimulates p62-dependent autophagy-like aggregate clearance in hepatocytes.

PubMed

Chen, Christine; Deng, Meihong; Sun, Qian; Loughran, Patricia; Billiar, Timothy R; Scott, Melanie J

2014-01-01

Impairment of autophagy has been associated with liver injury. TLR4-stimulation by LPS upregulates autophagy in hepatocytes, although the signaling pathways involved remain elusive. The objective of this study was to determine the signaling pathway leading to LPS-stimulated autophagy in hepatocytes. Cell lysates from livers of wild type (WT; C57BL/6) mice given LPS (5 mg/kg-IP) and hepatocytes from WT, TLR4ko, and MyD88ko mice treated with LPS (100 ng/mL) up to 24 h were collected. LC3II, p62/SQSTM1, Nrf2, and beclin1 levels were determined by immunoblot, immunofluorescence, and qPCR. Autophagy-like activation was measured by GFP-LC3-puncta formation and LC3II-expression. Beclin1, Nrf2, p62, MyD88, and TIRAP were knocked-down using siRNA. LC3II-expression increased in both liver and hepatocytes after LPS and was dependent on TLR4. Beclin1 expression did not increase after LPS in hepatocytes and beclin1-knockdown did not affect LC3II levels. In hepatocytes given LPS, expression of p62 increased and p62 colocalized with LC3. p62-knockdown prevented LC3II puncta formation. LPS-induced LC3II/p62-puncta also required MyD88/TIRAP signaling and localization of both Nrf2 and NF κ B transcription factors to the nucleus to upregulate p62-expression. Therefore, TLR4-activation by LPS in hepatocytes induces a p62-mediated, not beclin1-mediated, autophagy-like clearance pathway that is hepatoprotective by clearing aggregate-prone or misfolded proteins from the cytosol and preserving energy homeostasis under stress.

Depot-dependent effects of adipose tissue explants on co-cultured hepatocytes.

PubMed

Du, Zhen-Yu; Ma, Tao; Lock, Erik-Jan; Hao, Qin; Kristiansen, Karsten; Frøyland, Livar; Madsen, Lise

2011-01-01

We have developed an in vitro hepatocyte-adipose tissue explant (ATE) co-culture model enabling examination of the effect of visceral and subcutaneous adipose tissues on primary rat hepatocytes. Initial analyses of inflammatory marker genes were performed in fractionated epididymal or inguinal adipose tissues. Expressions of inflammation related genes (IL-6, TNF-α, COX-2) were higher in the inguinal than the epididymal ATE. Similarly, expressions of marker genes of macrophage and monocyte (MPEG-1, CD68, F4/80, CD64) were higher in the stromal vascular fraction (SVF) isolated from inguinal ATE than that from epididymal ATE. However, expressions of lipolysis related genes (ATGL, HSL, perilipin-1) were higher in the epididymal adipocytes than inguinal adipocytes. Moreover, secretion of IL-6 and PGE(2) was higher from inguinal ATEs than from epididymal ATEs. There was a trend that the total levels of IL-6, TNF-α and PGE(2) in the media from inguinal ATEs co-cultured with primary rat hepatocytes were higher than that in the media from epididymal ATEs co-cultured with hepatocytes, although the significant difference was only seen in PGE(2). Lipolysis, measured as glycerol release, was similar in the ATEs isolated from inguinal and epididymal adipose tissues when cultured alone, but the glycerol release was higher in the ATEs isolated from epididymal than from inguinal adipose tissue when co-cultured with hepatocytes. Compared to epididymal ATEs, the ATEs from inguinal adipose tissue elicited a stronger cytotoxic response and higher level of insulin resistance in the co-cultured hepatocytes. In conclusion, our results reveal depot-dependent effects of ATEs on co-cultured primary hepatocytes, which in part may be related to a more pronounced infiltration of stromal vascular cells (SVCs), particularly macrophages, in inguinal adipose tissue resulting in stronger responses in terms of hepatotoxicity and insulin-resistance.

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

PubMed

Wigg, Alan J; Phillips, John W; Wheatland, Loretta; Berry, Michael N

2003-06-01

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

Functional expression and regulation of drug transporters in monolayer- and sandwich-cultured mouse hepatocytes.

PubMed

Noel, Gregory; Le Vee, Marc; Moreau, Amélie; Stieger, Bruno; Parmentier, Yannick; Fardel, Olivier

2013-04-11

Primary hepatocyte cultures are now considered as convenient models for in vitro analyzing liver drug transport. However, if primary human and rat hepatocytes have been well-characterized with respect to drug transporter expression and regulation, much less is known for primary mouse hepatocytes. The present study was therefore designed to gain insights about this point. The profile of sinusoidal and canalicular drug transporter mRNA expression in short time (4h)-cultured mouse hepatocytes was found to be highly correlated with that of freshly isolated hepatocytes; by contrast, those of counterparts cultured for a longer time (until 4 days) either in monolayer configurations on plastic or collagen or in sandwich configuration with matrigel were profoundly altered: uptake drug transporters such as Oct1, Oatps and Oat2 were thus down-regulated, whereas most of efflux transporters such as Mdr1a/b, Mrp3, Mrp4 and Bcrp were induced. Moreover, short time-cultured hepatocytes exhibited the highest levels of sinusoidal influx transporter activities. Transporter-mediated drug secretion into canalicular networks was however only observed in sandwich-cultured hepatocytes. Mouse hepatocytes cultured either in monolayer or sandwich configurations were finally shown to exhibit up-regulation of referent transporters in response to exposure to prototypical activators of the drug sensing receptors pregnane X receptor, aryl hydrocarbon receptor or constitutive androstane receptor. Taken together, these data demonstrate the feasibility of using primary mouse hepatocytes for investigating potential interactions of xenobiotics with hepatic transporter activity or regulation, provided that adequate culture conditions are retained. Copyright © 2013 Elsevier B.V. All rights reserved.

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes

PubMed Central

Ding, Jianqiang; Yannam, Govardhana R.; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I.; Wong, Ronald J.; Avsar, Yesim; Guha, Chandan; Perlmutter, David H.; Fox, Ira J.; Roy-Chowdhury, Jayanta

2011-01-01

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z–expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%–98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z–expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals. PMID:21505264

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes.

PubMed

Ding, Jianqiang; Yannam, Govardhana R; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I; Wong, Ronald J; Avsar, Yesim; Guha, Chandan; Perlmutter, David H; Fox, Ira J; Roy-Chowdhury, Jayanta

2011-05-01

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

DGAT2 Inhibition Alters Aspects of Triglyceride Metabolism in Rodents but Not in Non-human Primates.

PubMed

McLaren, David G; Han, Seongah; Murphy, Beth Ann; Wilsie, Larissa; Stout, Steven J; Zhou, Haihong; Roddy, Thomas P; Gorski, Judith N; Metzger, Daniel E; Shin, Myung K; Reilly, Dermot F; Zhou, Heather H; Tadin-Strapps, Marija; Bartz, Steven R; Cumiskey, Anne-Marie; Graham, Thomas H; Shen, Dong-Ming; Akinsanya, Karen O; Previs, Stephen F; Imbriglio, Jason E; Pinto, Shirly

2018-06-05

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triglyceride (TG) synthesis and has been shown to play a role in regulating hepatic very-low-density lipoprotein (VLDL) production in rodents. To explore the potential of DGAT2 as a therapeutic target for the treatment of dyslipidemia, we tested the effects of small-molecule inhibitors and gene silencing both in vitro and in vivo. Consistent with prior reports, chronic inhibition of DGAT2 in a murine model of obesity led to correction of multiple lipid parameters. In contrast, experiments in primary human, rhesus, and cynomolgus hepatocytes demonstrated that selective inhibition of DGAT2 has only a modest effect. Acute and chronic inhibition of DGAT2 in rhesus primates recapitulated the in vitro data yielding no significant effects on production of plasma TG or VLDL apolipoprotein B. These results call into question whether selective inhibition of DGAT2 is sufficient for remediation of dyslipidemia. Copyright © 2018 Elsevier Inc. All rights reserved.

Parvovirus B19-Induced Apoptosis of Hepatocytes

PubMed Central

Poole, Brian D.; Karetnyi, Yuory V.; Naides, Stanley J.

2004-01-01

Parvovirus B19 (B19 virus) can persist in multiple tissues and has been implicated in a variety of diseases, including acute fulminant liver failure. The mechanism by which B19 virus induces liver failure remains unknown. Hepatocytes are nonpermissive for B19 virus replication. We previously reported that acute fulminant liver failure associated with B19 virus infection was characterized by hepatocellular dropout. We inoculated both primary hepatocytes and the hepatocellular carcinoma cell line Hep G2 with B19 virus and assayed for apoptosis by using annexin V staining. Reverse transcriptase PCR analysis and immunofluorescence demonstrated that B19 virus was able to infect the cells and produce its nonstructural protein but little or no structural capsid protein. Infection with B19 virus induced means of 28% of Hep G2 cells and 10% of primary hepatocytes to undergo apoptosis, which were four- and threefold increases, respectively, over background levels. Analysis of caspase involvement showed that B19 virus-inoculated cultures had a significant increase in the number of cells with active caspase 3. Inhibition studies demonstrated that caspases 3 and 9, but not caspase 8, are required for B19 virus-induced apoptosis. PMID:15220451

The rodent ultrasound production mechanism.

PubMed

Roberts, L H

1975-03-01

Rodents produce two types of sounds, audible and ultrasonic, that differ markedly in physical structure. Studies of sound production in light gases show that whereas the audible cries appear to be produced, as in the case of most other mammals, by vibrating structures in the larynx, the ultrasonic cries are produced by a different mechanism, probably a whistle. 'Bird-call' whistles are shown to have all the properties of rodent ultrasonic cries and to mimic them in almost every detail. Thus it is concluded that rodents have two distinct sound production mechanisms, one for audible cries and one for ultrasonic cries.

Cadmium exposure exacerbates hyperlipidemia in cholesterol-overloaded hepatocytes via autophagy dysregulation.

PubMed

Rosales-Cruz, Patricia; Domínguez-Pérez, Mayra; Reyes-Zárate, Elizabeth; Bello-Monroy, Oscar; Enríquez-Cortina, Cristina; Miranda-Labra, Roxana; Bucio, Leticia; Gómez-Quiroz, Luis Enrique; Rojas-Del Castillo, Emilio; Gutiérrez-Ruíz, María Concepción; Souza-Arroyo, Verónica

2018-04-01

Metabolic factors are the major risk of non-alcoholic fatty liver disease, although other factors may contribute steatosis. Cadmium exposure produces histopathological and molecular changes in liver, which are consistent with steatosis. In the present study, we describe the effect of low cadmium acute treatment on hepatocytes obtained from mice fed with a high cholesterol diet. Our data suggest that hepatocytes with cholesterol overload promote an adaptive response against cadmium-induced acute toxicity by up-regulating anti-apoptotic proteins, managing ROS overproduction, increasing GSH synthesis and MT-II content to avoid protein oxidation. Cadmium treatment increases lipid content in cholesterol-fed mice hepatocytes because of an impaired autophagy process. Our data suggest an essential function of macroautophagy in the regulation of lipid storage induced by Cd on hepatocytes, that implies that alterations in this pathway may be a mechanism that aggravates hepatic steatosis. Copyright © 2018. Published by Elsevier B.V.

HEPATOCYTE GROWTH FACTOR ACTS AS A MITOGEN AND CHEMOATTRACTANT FOR POSTNATAL SUBVENTRICULAR ZONE-OLFACTORY BULB NEUROGENESIS

PubMed Central

Wang, Tsu-Wei; Zhang, Huailin; Gyetko, Margaret R.; Parent, Jack M.

2011-01-01

Neural progenitor cells persist throughout life in the forebrain subventricular zone (SVZ). They generate neuroblasts that migrate to the olfactory bulb and differentiate into interneurons, but mechanisms underlying these processes are poorly understood. Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic factor that influences cell motility, proliferation and morphogenesis in neural and non-neural tissues. HGF and its receptor, c-Met, are present in the rodent SVZ-olfactory bulb pathway. Using in vitro neurogenesis assays and in vivo studies of partially HGF-deficient mice, we find that HGF promotes SVZ cell proliferation and progenitor cell maintenance, while slowing differentiation and possibly altering cell fate choices. HGF also acts as a chemoattractant for SVZ neuroblasts in co-culture assays. Decreased HGF signaling induces ectopic SVZ neuroblast migration and alters the timing of migration to the olfactory bulb. These results suggest that HGF influences multiple steps in postnatal forebrain neurogenesis. HGF is a mitogen for SVZ neural progenitors, and regulates their differentiation and olfactory bulb migration. PMID:21683144

Hepatocyte transplantation and advancements in alternative cell sources for liver-based regenerative medicine.

PubMed

Lee, Charlotte A; Sinha, Siddharth; Fitzpatrick, Emer; Dhawan, Anil

2018-06-01

Human hepatocyte transplantation has been actively perused as an alternative to liver replacement for acute liver failure and liver-based metabolic defects. Current challenges in this field include a limited cell source, reduced cell viability following cryopreservation and poor engraftment of cells into the recipient liver with consequent limited life span. As a result, alternative stem cell sources such as pluripotent stem cells, fibroblasts, hepatic progenitor cells, amniotic epithelial cells and mesenchymal stem/stromal cells (MSCs) can be used to generate induced hepatocyte like cells (HLC) with each technique exhibiting advantages and disadvantages. HLCs may have comparable function to primary human hepatocytes and could offer patient-specific treatment. However, long-term functionality of transplanted HLCs and the potential oncogenic risks of using stem cells have yet to be established. The immunomodulatory effects of MSCs are promising, and multiple clinical trials are investigating their effect in cirrhosis and acute liver failure. Here, we review the current status of hepatocyte transplantation, alternative cell sources to primary human hepatocytes and their potential in liver regeneration. We also describe recent clinical trials using hepatocytes derived from stem cells and their role in improving the phenotype of several liver diseases.

Cadmium supplement triggers endoplasmic reticulum stress response and cytotoxicity in primary chicken hepatocytes.

PubMed

Shao, Cheng-Cheng; Li, Nan; Zhang, Zi-Wei; Su, Jian; Li, Shu; Li, Jin-Long; Xu, Shi-Wen

2014-08-01

Cadmium (Cd), a potent hepatotoxin, has been reported to induce endoplasmic reticulum (ER) stress in various cell types. However, whether such effect exists in bird is still unclear. To delineate the effects of Cd exposure on ER stress response, we examined the expression of 78-kDa glucose-regulated protein (GRP78) and alteration in calcium homeostasis in primary chicken hepatocytes treated with 2-22 µM Cd for 24 h. A significant decrease of cell viability was observed in chicken hepatocytes following Cd administration. In cells treated with Cd, GRP78 protein levels increased in a dose-dependent manner. In addition, GRP78 and GRP94mRNA levels were elevated in response to Cd exposure. The increase of the intracellular Ca(2+) concentration in chicken hepatocytes was found during Cd exposure. Cd significantly decreased the CaM mRNA levels in hepatocytes. These results show that Cd regulates the expression of GRP78 and calcium homeostasis in chicken hepatocytes, suggesting that ER stress induced by Cd plays an important role in the mechanisms of Cd cytotoxicity to the bird hepatocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

Coincident disruptive coloration

PubMed Central

Cuthill, Innes C.; Székely, Aron

2008-01-01

Even if an animal matches its surroundings perfectly in colour and texture, any mismatch between the spatial phase of its pattern and that of the background, or shadow created by its three-dimensional relief, is potentially revealing. Nevertheless, for camouflage to be fully broken, the shape must be recognizable. Disruptive coloration acts against object recognition by the use of high-contrast internal colour boundaries to break up shape and form. As well as the general outline, characteristic features such as eyes and limbs must also be concealed; this can be achieved by having the colour patterns on different, but adjacent, body parts aligned to match each other (i.e. in phase). Such ‘coincident disruptive coloration’ ensures that there is no phase disjunction where body parts meet, and causes different sections of the body to blend perceptually. We tested this theory using field experiments with predation by wild birds on artificial moth-like targets, whose wings and (edible pastry) bodies had colour patterns that were variously coincident or not. We also carried out an experiment with humans searching for analogous targets on a computer screen. Both experiments show that coincident disruptive coloration is an effective mechanism for concealing an otherwise revealing body form. PMID:18990668

[Cadmium citotoxicity in mice hepatocytes and implications on tropical environments].

PubMed

Marcano, Letty; Faría, Clarisa de R; Carruyo, Ingrid; Montiel, Xiomara

2006-06-01

We analyzed phenotypic, structural and ultrastructural alterations induced by Cd+2 in hepatocytes extracted from Swiss Albino mice. Cadmium was given orally in watery solution of CdCl2 during 100 days at concentrations of 50 ppm, 100 ppm and 150 ppm. In controls, distilled water alone was used. The samples were processed with the paraffin inclusion and hematoxilin-eosin coloration techniques for light microscopy. For transmission electron microscopy we used the conventional technique. We found phenotypic (size and weight differences) and physiologic changes (muscular weakness, unrest); at the structural level we noticed loss of trabecular disposition and of lobulillar architecture, lymphocyte agglomeration, vacuolization, dilatation of sinusoid and central vein, among others. The ultrastructural study evidenced alterations coincident with those seen with light microscopy, which were accentuated with the increase of metal concentration: nucleolus with a high number of fibrillar centers (50 ppm); voluminous lipidic drops in the cytoplasm, loose endoplasmic rough reticulum, citoplasmatic vacuolization, altered lisosomes and peroxisomes (100 ppm); contracted nuclei with condensed cromatine, dilatation of intracellular space and mitochondria, and loss of fibrillar areas (150 ppm). Cadmium produces a toxic effect in the hepatic cells; the effect is more severe at higher concentration, leading to cellular necrosis.

Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance.

PubMed

Zeigerer, Anja; Wuttke, Anne; Marsico, Giovanni; Seifert, Sarah; Kalaidzidis, Yannis; Zerial, Marino

2017-01-01

Exploring the cell biology of hepatocytes in vitro could be a powerful strategy to dissect the molecular mechanisms underlying the structure and function of the liver in vivo. However, this approach relies on appropriate in vitro cell culture systems that can recapitulate the cell biological and metabolic features of the hepatocytes in the liver whilst being accessible to experimental manipulations. Here, we adapted protocols for high-resolution fluorescence microscopy and quantitative image analysis to compare two primary hepatocyte culture systems, monolayer and collagen sandwich, with respect to the distribution of two distinct populations of early endosomes (APPL1 and EEA1-positive), endocytic capacity, metabolic and signaling activities. In addition to the re-acquisition of hepatocellular polarity, primary hepatocytes grown in collagen sandwich but not in monolayer culture recapitulated the apico-basal distribution of EEA1 endosomes observed in liver tissue. We found that such distribution correlated with the organization of the actin cytoskeleton in vitro and, surprisingly, was dependent on the nutritional state in vivo. Hepatocytes in collagen sandwich also exhibited faster kinetics of low-density lipoprotein (LDL) and epidermal growth factor (EGF) internalization, showed improved insulin sensitivity and preserved their ability for glucose production, compared to hepatocytes in monolayer cultures. Although no in vitro culture system can reproduce the exquisite structural features of liver tissue, our data nevertheless highlight the ability of the collagen sandwich system to recapitulate key structural and functional properties of the hepatocytes in the liver and, therefore, support the usage of this system to study aspects of hepatocellular biology in vitro. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Kukoamine B promotes TLR4-independent lipopolysaccharide uptake in murine hepatocytes.

PubMed

Yang, Dong; Zheng, Xinchuan; Wang, Ning; Fan, Shijun; Yang, Yongjun; Lu, Yongling; Chen, Qian; Liu, Xin; Zheng, Jiang

2016-09-06

Free bacterial lipopolysaccharide (LPS) is generally removed from the bloodstream through hepatic uptake via TLR4, the LPS pattern recognition receptor, but mechanisms for internalization and clearance of conjugated LPS are less clear. Kukoamine B (KB) is a novel cationic alkaloid that interferes with LPS binding to TLR4. In this study, KB accelerated blood clearance of LPS. KB also enhanced LPS distribution in the hepatic tissues of C57 BL/6 mice, along with LPS uptake in primary hepatocytes and HepG2 cells. By contrast, KB inhibited LPS internalization in Kupffer and RAW 264.7 cells. Loss of TLR4 did not affect LPS uptake into KB-treated hepatocytes. We also detected selective upregulation of the asialoglycoprotein receptor (ASGPR) upon KB treatment, and ASGPR colocalized with KB in cultured hepatocytes. Molecular docking showed that KB bound to ASGPR in a manner similar to GalNAc, a known ASGPR agonist. GalNAc dose-dependently reduced KB internalization, suggesting it competes with KB for ASGPR binding, and ASGPR knockdown also impaired LPS uptake into hepatocytes. Finally, while KB enhanced LPS uptake, it was protective against LPS-induced inflammation and hepatocyte injury. Our study provides a new mechanism for conjugated LPS hepatic uptake induced by the LPS neutralizer KB and mediated by membrane ASGPR binding.

Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure.

PubMed

Kim, Yohan; Kang, Kyojin; Jeong, Jaemin; Paik, Seung Sam; Kim, Ji Sook; Park, Su A; Kim, Wan Doo; Park, Jisun; Choi, Dongho

2017-02-01

The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6-8 weeks old mice by a 2-step collagenase method. Samples of 4 × 10 7 hepatocytes with 80%-90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin , HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.

Interleukin-1β induces tumor necrosis factor-α secretion from rat hepatocytes.

PubMed

Yoshigai, Emi; Hara, Takafumi; Inaba, Hiroyuki; Hashimoto, Iwao; Tanaka, Yoshito; Kaibori, Masaki; Kimura, Tominori; Okumura, Tadayoshi; Kwon, A-Hon; Nishizawa, Mikio

2014-05-01

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine involved in various inflammatory diseases. The only production of TNF-α in the liver is thought to be from hepatic macrophages known as Kupffer cells, predominantly in response to bacterial lipopolysaccharide (LPS). Primary cultured rat hepatocytes were used to analyze TNF-α expression in response to the pro-inflammatory cytokine, interleukin-1β (IL-1β). Livers of rats subjected to LPS-induced endotoxemia were analyzed. Immunocytochemistry and enzyme-linked immunosorbent assays demonstrated that IL-1β-treated rat hepatocytes secreted TNF-α, and RNA analyses indicated that TNF-α mRNA was induced specifically by IL-1β. Northern blot analysis showed that not only mRNA, but also a natural antisense transcript (asRNA), was transcribed from the rat Tnf gene in IL-1β-treated hepatocytes. TNF-α was detected in the hepatocytes of LPS-treated rats. Both TNF-α mRNA and asRNA were expressed in the hepatocytes of LPS-treated rats, human hepatocellular carcinoma and human monocyte/macrophage cells. To disrupt the interaction between TNF-α asRNA and TNF-α mRNA, sense oligonucleotides corresponding to TNF-α mRNA were introduced into rat hepatocytes resulting in significantly increased levels of TNF-α mRNA. One of these sense oligonucleotides increased a half-life of TNF-α mRNA, suggesting that the TNF-α asRNA may reduce the stability of TNF-α mRNA. IL-1β-stimulated rat hepatocytes are a newly identified source of TNF-α in the liver. TNF-α mRNA and asRNA are expressed in rats and humans, and the TNF-α asRNA reduces the stability of the TNF-α mRNA. Hepatocytes and TNF-α asRNA may be therapeutic targets to regulate levels of TNF-α mRNA. © 2013 The Japan Society of Hepatology.

Gait Analysis Methods for Rodent Models of Osteoarthritis

PubMed Central

Jacobs, Brittany Y.; Kloefkorn, Heidi E.; Allen, Kyle D.

2014-01-01

Patients with osteoarthritis (OA) primarily seek treatment due to pain and disability, yet the primary endpoints for rodent OA models tend to be histological measures of joint destruction. The discrepancy between clinical and preclinical evaluations is problematic, given that radiographic evidence of OA in humans does not always correlate to the severity of patient-reported symptoms. Recent advances in behavioral analyses have provided new methods to evaluate disease sequelae in rodents. Of particular relevance to rodent OA models are methods to assess rodent gait. While obvious differences exist between quadrupedal and bipedal gait sequences, the gait abnormalities seen in humans and in rodent OA models reflect similar compensatory behaviors that protect an injured limb from loading. The purpose of this review is to describe these compensations and current methods used to assess rodent gait characteristics, while detailing important considerations for the selection of gait analysis methods in rodent OA models. PMID:25160712

3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for drug toxicity testing.

PubMed

Takayama, Kazuo; Kawabata, Kenji; Nagamoto, Yasuhito; Kishimoto, Keisuke; Tashiro, Katsuhisa; Sakurai, Fuminori; Tachibana, Masashi; Kanda, Katsuhiro; Hayakawa, Takao; Furue, Miho Kusuda; Mizuguchi, Hiroyuki

2013-02-01

Although it is expected that hepatocyte-like cells differentiated from human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells will be utilized in drug toxicity testing, the actual applicability of hepatocyte-like cells in this context has not been well examined so far. To generate mature hepatocyte-like cells that would be applicable for drug toxicity testing, we established a hepatocyte differentiation method that employs not only stage-specific transient overexpression of hepatocyte-related transcription factors but also a three-dimensional spheroid culture system using a Nanopillar Plate. We succeeded in establishing protocol that could generate more matured hepatocyte-like cells than our previous protocol. In addition, our hepatocyte-like cells could sensitively predict drug-induced hepatotoxicity, including reactive metabolite-mediated toxicity. In conclusion, our hepatocyte-like cells differentiated from human ES cells or iPS cells have potential to be applied in drug toxicity testing. Copyright © 2012 Elsevier Ltd. All rights reserved.

Hepatocyte transplants improve liver function and encephalopathy in portacaval shunted rats.

PubMed

Fogel, Wieslawa Agnieszka; Stasiak, Anna; Maksymowicz, Michał; Kobos, Jozef; Unzeta, Mercedes; Mussur, Miroslaw

2014-07-01

Rats with portacaval shunt (PCS) are useful experimental models of human hepatic encephalopathy in chronic liver dysfunction. We have previously shown that PCS modifies amine neurotransmitter systems in the CNS and increases voluntary alcohol intake by rats. Hepatocyte transplantation, used in acute liver failure, has recently also been applied to chronic liver diseases, which prompted us to investigate whether the altered brain amine system and the drinking behavior in long-term shunted rats could be normalized by hepatocyte transplants. Hepatocytes, isolated from syngeneic donors by collagenase digestion, were injected (3 × 10(6) cells/rat) into the pancreatic tail region, 6 months after PCS. Hepatic function was evaluated by measuring urine urea and plasma L-histidine concentrations. A free choice test with two bottles (tap water and 10% ethyl alcohol) was performed for 3 days to assess the rats' preference for alcohol. The rats were euthanized 2 months posttransplantation. Brain histamine and 5-hydroxyindoleacetic acid (5-HIAA) levels were measured by radioenzymatic assay and by HPLC-EC, respectively, N-tele-methylhistamine by GC/MS while MAOA and MAOB activities by isotopic procedures. Portacaval shunt rats with hepatocyte transplants gave more urea than before transplantation, with lower plasma L-His levels and higher body weight versus the PCS counterparts. Also, those rats consumed less alcohol. The CNS amines and 5-HIAA concentrations, as well as MAO-B activity, being abnormally high in untreated PCS rats, significantly reduced after PCS hepatocyte treatment. The results support the therapeutic values of hepatocyte transplants in chronic liver diseases and the temporary character of PCS-exerted CNS dysfunctions. © 2014 John Wiley & Sons Ltd.

Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition.

PubMed

Ishihara, Yasuhiro; Shiba, Dai; Shimamoto, Norio

2006-07-15

Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as alpha-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress.

Comparative cytotoxicity of alachlor, acetochlor, and metolachlor herbicides in isolated rat and cryopreserved human hepatocytes.

PubMed

Kale, Vijay M; Miranda, Sonia R; Wilbanks, Mitchell S; Meyer, Sharon A

2008-02-01

Noncancerous adverse effects observed at the lowest dose for chloroacetanilide herbicides alachlor [2-chloro-2',6'-diethyl-N-(methoxymethyl)-acetanilide] and acetochlor [2-chloro-2'-methyl-6'-ethyl-N-(ethoxymethyl)acetanilide], but not metolachlor [2-chloro-2'-ethyl-6'-methyl-N-(1-methyl-2-methoxymethyl)acetanilide], are hepatotoxicity in rats and dogs. Liver microsomal N-dealkylation, a step in the putative activating pathway, of acetochlor exceeds that of alachlor and is negligible for metolachlor. In the present investigation, cytotoxicity of the three chloroacetanilides was ranked using isolated rat and cryopreserved human hepatocytes to correlate this endpoint with CYP3A-dependent metabolism. Chloroacetanilide cytotoxicity in rat hepatocyte suspensions was time dependent (e.g., LC(50 - alachlor/2 h) vs. LC(50 - alachlor/4 h) = 765 vs. 325 muM). Alachlor and acetochlor were more potent than metolachlor after 2 and 4 h, times when N-dealkylated alachlor product 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) formation was readily detectable. Alachlor and acetochlor potencies with cryopreserved human hepatocytes at 2 h were comparable to freshly isolated rat hepatocytes, and alachlor metabolism to CDEPA was likewise detectable. Unlike rat hepatocytes, metolachlor potency was equivalent to acetochlor and alachlor in human hepatocytes. Furthermore, chloroacetanilide cytotoxicity from two sources of human hepatocytes varied inversely with CYP3A4 activity. Collectively, while cytotoxicity in rat hepatocytes was consistent with chloroacetanilide activation by CYP3A, an activating role for CYP3A4 was not supported with human hepatocytes. (c) 2008 Wiley Periodicals, Inc.

Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation

PubMed Central

Weerasinghe, Sujith V.W.; Singla, Amika; Leonard, Jessica M.; Hanada, Shinichiro; Andrews, Philip C.; Lok, Anna S.; Omary, M. Bishr

2011-01-01

Genetic factors impact liver injury susceptibility and disease progression. Prominent histological features of some chronic human liver diseases are hepatocyte ballooning and Mallory-Denk bodies. In mice, these features are induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in a strain-dependent manner, with the C57BL and C3H strains showing high and low susceptibility, respectively. To identify modifiers of DDC-induced liver injury, we compared C57BL and C3H mice using proteomic, biochemical, and cell biological tools. DDC elevated reactive oxygen species (ROS) and oxidative stress enzymes preferentially in C57BL livers and isolated hepatocytes. C57BL livers and hepatocytes also manifested significant down-regulation, aggregation, and nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH knockdown depleted bioenergetic and antioxidant enzymes and elevated hepatocyte ROS, whereas GAPDH overexpression decreased hepatocyte ROS. On the other hand, C3H livers had higher expression and activity of the energy-generating nucleoside-diphosphate kinase (NDPK), and knockdown of hepatocyte NDPK augmented DDC-induced ROS formation. Consistent with these findings, cirrhotic, but not normal, human livers contained GAPDH aggregates and NDPK complexes. We propose that GAPDH and NDPK are genetic modifiers of murine DDC-induced liver injury and potentially human liver disease. PMID:22006949

β-Adrenergic induction of lipolysis in hepatocytes is inhibited by ethanol exposure.

PubMed

Schott, Micah B; Rasineni, Karuna; Weller, Shaun G; Schulze, Ryan J; Sletten, Arthur C; Casey, Carol A; McNiven, Mark A

2017-07-14

In liver steatosis ( i.e. fatty liver), hepatocytes accumulate many large neutral lipid storage organelles known as lipid droplets (LDs). LDs are important in the maintenance of energy homeostasis, but the signaling mechanisms that stimulate LD metabolism in hepatocytes are poorly defined. In adipocytes, catecholamines target the β-adrenergic (β-AR)/cAMP pathway to activate cytosolic lipases and induce their recruitment to the LD surface. Therefore, the goal of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP-mediated lipolysis in response to β-AR stimulation. Using primary rat hepatocytes and human hepatoma cells, we found that treatment with the β-AR agent isoproterenol caused substantial LD loss via activation of cytosolic lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). β-Adrenergic stimulation rapidly activated PKA, which led to the phosphorylation of ATGL and HSL and their recruitment to the LD surface. To test whether this β-AR-dependent lipolysis pathway was altered in a model of alcoholic fatty liver, primary hepatocytes from rats fed a 6-week EtOH-containing Lieber-DeCarli diet were treated with cAMP agonists. Compared with controls, EtOH-exposed hepatocytes showed a drastic inhibition in β-AR/cAMP-induced LD breakdown and the phosphorylation of PKA substrates, including HSL. This observation was supported in VA-13 cells, an EtOH-metabolizing human hepatoma cell line, which displayed marked defects in both PKA activation and isoproterenol-induced ATGL translocation to the LD periphery. In summary, these findings suggest that β-AR stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic EtOH insult leading to fatty liver.

DISTINCT FUNCTIONS OF JNK AND C-JUN IN OXIDANT-INDUCED HEPATOCYTE DEATH

PubMed Central

Amir, Muhammad; Liu, Kun; Zhao, Enpeng; Czaja, Mark J.

2013-01-01

Overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling is a central mechanism of hepatocyte injury and death including that from oxidative stress. However, the functions of JNK and c-Jun are still unclear, and this pathway also inhibits hepatocyte death. Previous studies of menadione-induced oxidant stress demonstrated that toxicity resulted from sustained JNK/c-Jun activation as death was blocked by the c-Jun dominant negative TAM67. To further delineate the function of JNK/c-Jun signaling in hepatocyte injury from oxidant stress, the effects of direct JNK inhibition on menadione-induced death were examined. In contrast to the inhibitory effect of TAM67, pharmacological JNK inhibition by SP600125 sensitized the rat hepatocyte cell line RALA255-10G to death from menadione. SP600125 similarly sensitized mouse primary hepatocytes to menadione toxicity. Death from SP600125/menadione was c-Jun dependent as it was blocked by TAM67, but independent of c-Jun phosphorylation. Death occurred by apoptosis and necrosis and activation of the mitochondrial death pathway. Short hairpin RNA knockdowns of total JNK or JNK2 sensitized to death from menadione, whereas a jnk1 knockdown was protective. Jnk2 null mouse primary hepatocytes were also sensitized to menadione death. JNK inhibition magnified decreases in cellular ATP content and β-oxidation induced by menadione. This effect mediated cell death as chemical inhibition of β-oxidation also sensitized cells to death from menadione, and supplementation with the β-oxidation substrate oleate blocked death. Components of the JNK/c-Jun signaling pathway have opposing functions in hepatocyte oxidant stress with JNK2 mediating resistance to cell death and c-Jun promoting death. PMID:22644775

Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xue, Tao; Luo, Peihua; Zhu, Hong

2012-06-15

Dasatinib, a multitargeted inhibitor of BCR–ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague–Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 andmore » cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity. -- Highlights: ►Dasatinib shows potential hepatotoxicity both in vitro and in vivo. ►Apoptosis plays a vital role in

Curcumin attenuates insulin resistance in hepatocytes by inducing Nrf2 nuclear translocation.

PubMed

Zhao, Shu-Guang; Li, Qiang; Liu, Zhen-Xiong; Wang, Jing-Jie; Wang, Xv-Xia; Qin, Ming; Wen, Qin-Sheng

2011-01-01

NF-E2-Related Factor-2 (Nrf2) is a transcription factor that plays a crucial role in the cellular protection against oxidative stress. Curcumin has been reported to induce Nrf2 nuclear translocation and upregulate the expression of numerous reactive oxygen species (ROS) detoxifying and antioxidant genes in hepatocytes. This study was designed to investigate whether curcumin-induced Nrf2 nuclear translocation could reduce ROS-mediated insulin resistance in cultured LO2 hepatocytes. Human LO2 hepatocytes were incubated with curcumine and glucose oxidase (GO) in the presence/absence of wortmannin (a phosphatidyinositol 3-kinase (PI3K) inhibitor), oxidative stress, cellular damage, Nrf2 nuclear translocation and insulin resistance were measured. GO exposure significantly increased intracellular ROS, glutathione (GSH) depletion, malondialdehyde (MDA) formation, and increased activities of cellular lactate dehydrogenase (LDH) and aspartate amino transferase (AST), as well as causing insulin resistance. Curcumin pretreatment significantly attenuated these disturbances in intracellular ROS, liver enzyme activity and significantly antagonized the lipid peroxidation, GSH depletion and insulin resistance induced by GO in LO2 hepatocytes. These effects paralleled Nrf2 nuclear translocation induced by curcumin. Wortmannin partially blocked curcumin-induced Nrf2 nuclear translocation. In addition, wortmannin prevented curcumin-induced improvements in intracellular ROS, MDA formation, GSH depletion, liver enzyme activity and insulin resistance in cultured LO2 hepatocytes. These findings suggest that curcumin could reduce ROS-mediated insulin resistance in hepatocytes, at least in part through nuclear translocation of Nrf2.

Determination of metabolic stability using cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss)

EPA Science Inventory

Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with procedures for using fresh or cryopreserved hepatocytes to assess chemical metabolic stability in fish by means of a substrate depletion approach. Variations on thes...

Serum-Free Medium and Mesenchymal Stromal Cells Enhance Functionality and Stabilize Integrity of Rat Hepatocyte Spheroids

PubMed Central

Bao, Ji; Fisher, James E.; Lillegard, Joseph B.; Wang, William; Amiot, Bruce; Yu, Yue; Dietz, Allan B.; Nahmias, Yaakov; Nyberg, Scott L.

2013-01-01

Long-term culture of hepatocyte spheroids with high ammonia clearance is valuable for therapeutic applications, especially the bioartificial liver. However, the optimal conditions are not well studied. We hypothesized that liver urea cycle enzymes can be induced by high protein diet and maintain on a higher expression level in rat hepatocyte spheroids by serum-free medium (SFM) culture and coculture with mesenchymal stromal cells (MSCs). Rats were feed normal protein diet (NPD) or high protein diet (HPD) for 7 days before liver digestion and isolation of hepatocytes. Hepatocyte spheroids were formed and maintained in a rocked suspension culture with or without MSCs in SFM or 10% serum-containing medium (SCM). Spheroid viability, kinetics of spheroid formation, hepatic functions, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. We observed that urea cycle enzymes of hepatocyte spheroids can be induced by high protein diet. SFM and MSCs enhanced ammonia clearance and ureagenesis and stabilized integrity of hepatocyte spheroids compared to control conditions over 14 days. Hepatocytes from high protein diet-fed rats formed spheroids and maintained a high level of ammonia detoxification for over 14 days in a novel SFM. Hepatic functionality and spheroid integrity were further stabilized by coculture of hepatocytes with MSCs in the spheroid microenvironment. These findings have direct application to development of the spheroid reservoir bioartificial liver. PMID:23006214

Dynamic regulation of EZH2 from HPSc to hepatocyte-like cell fate

PubMed Central

Helsen, Nicky; Vanhove, Jolien; Boon, Ruben; Xu, Zhuofei; Ordovas, Laura; Verfaillie, Catherine M.

2017-01-01

Currently, drug metabolization and toxicity studies rely on the use of primary human hepatocytes and hepatoma cell lines, which both have conceivable limitations. Human pluripotent stem cell (hPSC)—derived hepatocyte-like cells (HLCs) are an alternative and valuable source of hepatocytes that can overcome these limitations. EZH2 (enhancer of zeste homolog 2), a transcriptional repressor of the polycomb repressive complex 2 (PRC2), may play an important role in hepatocyte development, but its role during in vitro hPSC-HLC differentiation has not yet been assessed. We here demonstrate dynamic regulation of EZH2 during hepatic differentiation of hPSC. To enhance EZH2 expression, we inducibly overexpressed EZH2 between d0 and d8, demonstrating a significant improvement in definitive endoderm formation, and improved generation of HLCs. Despite induction of EZH2 overexpression until d8, EZH2 transcript and protein levels decreased from d4 onwards, which might be caused by expression of microRNAs predicted to inhibit EZH2 expression. In conclusion, our studies demonstrate that EZH2 plays a role in endoderm formation and hepatocyte differentiation, but its expression is tightly post-transcriptionally regulated during this process. PMID:29091973

DNA adducts of the tobacco carcinogens 2-amino-9H-pyrido[2,3-b]indole and 4-aminobiphenyl are formed at environmental exposure levels and persist in human hepatocytes.

PubMed

Nauwelaërs, Gwendoline; Bellamri, Medjda; Fessard, Valérie; Turesky, Robert J; Langouët, Sophie

2013-09-16

Aromatic amines and structurally related heterocyclic aromatic amines (HAAs) are produced during the combustion of tobacco or during the high-temperature cooking of meat. Exposure to some of these chemicals may contribute to the etiology of several common types of human cancers. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant HAA formed in mainstream tobacco smoke: it arises in amounts that are 25-100 times greater than the levels of the arylamine, 4-aminobiphenyl (4-ABP), a human carcinogen. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a prevalent HAA formed in cooked meats. AαC and MeIQx are rodent carcinogens; however, their carcinogenic potency in humans is unknown. A preliminary assessment of the carcinogenic potential of these HAAs in humans was conducted by examining the capacity of primary human hepatocytes to form DNA adducts of AαC and MeIQx, in comparison to 4-ABP, followed by the kinetics of DNA adduct removal by cellular enzyme repair systems. The principal DNA adducts formed were N-(deoxyguanosin-8-yl) (dG-C8) adducts. Comparable levels of DNA adducts were formed with AαC and 4-ABP, whereas adduct formation was ∼5-fold lower for MeIQx. dG-C8-AαC and dG-C8-4-ABP were formed at comparable levels in a concentration-dependent manner in human hepatocytes treated with procarcinogens over a 10,000-fold concentration range (1 nM-10 μM). Pretreatment of hepatocytes with furafylline, a selective inhibitor of cytochrome P450 1A2, resulted in a strong diminution of DNA adducts signifying that P450 1A2 is a major P450 isoform involved in bioactivation of these procarcinogens. The kinetics of adduct removal varied for each hepatocyte donor. Approximately half of the DNA adducts were removed within 24 h of treatment; however, the remaining lesions persisted over 5 days. The high levels of AαC present in tobacco smoke and its propensity to form persistent DNA adducts in human hepatocytes suggest that AαC can contribute to DNA damage

DNA Adducts of the Tobacco Carcinogens 2-Amino-9H-pyrido[2,3-b]indole and 4-Aminobiphenyl are Formed at Environmental Exposure levels and Persist in Human Hepatocytes

PubMed Central

Nauwelaërs, Gwendoline; Bellamri, Medjda; Fessard, Valérie; Turesky, Robert J.; Langouët, Sophie

2013-01-01

Aromatic amines and structurally related heterocyclic aromatic amines (HAAs) are produced during the combustion of tobacco or during the high-temperature cooking of meat. Exposure to some of these chemicals may contribute to the etiology of several common types of human cancers. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant HAA formed in mainstream tobacco smoke: it arises in amounts that are 25–100 times greater than the levels of the arylamine, 4-aminobiphenyl (4-ABP), a human carcinogen. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a prevalent HAA formed in cooked meats. AαC and MeIQx are rodent carcinogens; however, their carcinogenic potency in humans is unknown. A preliminary assessment of the carcinogenic potential of these HAAs in humans was conducted by examining the capacity of primary human hepatocytes to form DNA adducts of AαC and MeIQx, in comparison to 4-ABP, followed by the kinetics of DNA adduct removal by cellular enzyme repair systems. The principal DNA adducts formed were N-(deoxyguanosin-8-yl) (dG-C8) adducts. Comparable levels of DNA adducts were formed with AαC and 4-ABP, whereas adduct formation was ~5-fold lower for MeIQx. dG-C8-AαC and dG-C8-4-ABP were formed at comparable levels in a concentration-dependent manner in human hepatocytes treated with procarcinogens over a ten thousand-fold concentration range (1 nM – 10 µM). Pretreatment of hepatocytes with furafylline, a selective inhibitor of cytochrome P450 1A2, resulted in a strong diminution of DNA adducts signifying that P450 1A2 is a major P450 isoform involved in bioactivation of these procarcinogens. The kinetics of adduct removal varied for each hepatocyte donor. Approximately half of the DNA adducts were removed within 24 h of treatment; however, the remaining lesions persisted over 5 days. The high levels of AαC present in tobacco smoke and its propensity to form persistent DNA adducts in human hepatocytes, suggests that AαC can contribute to

Ultrastructure of the hepatocytes in a vertebrate liver without bile ducts.

PubMed Central

Youson, J H; Sidon, E W; Peek, W D; Shivers, R R

1985-01-01

Thin sections and freeze fracture replicas were used to study the structure of the hepatocytes of the parasitic adult lamprey (Petromyzon marinus L.). Despite the absence of bile ducts and bile canaliculi, the hepatocytes have some features which resemble those of cells in the livers of other vertebrates. Hepatocytes are characterised by large gap junctions, many cytoplasmic inclusions, and large deposits of iron. The latter is present throughout the cytoplasmic matrix and within large inclusion bodies which may arise through sequestration of parts of the cytoplasm by membrane isolation. There is no evidence for the involvement of hepatocytes in glucose metabolism but their fine structure reflects the production of bile products and the processing of lipoproteins. The accumulation of bile products within cytoplasmic inclusions resembles the situation resulting from biliary atresia or other cholestatic conditions in higher organisms. There is little folding of the plasma membrane facing the perivascular space (of Dissé), perhaps indicating limited involvement of this surface in the transport of bile products. Nerve endings in close apposition to hepatocytes suggest possible nervous control or metabolic function or the presence of sensory receptors in lamprey liver. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 15 Fig. 16 PMID:2999046

Miniature enzyme-based electrodes for detection of hydrogen peroxide release from alcohol-injured hepatocytes.

PubMed

Matharu, Zimple; Enomoto, James; Revzin, Alexander

2013-01-15

Alcohol insult to the liver sets off a complex sequence of inflammatory and fibrogenic responses. There is increasing evidence that hepatocytes play a key role in triggering these responses by producing inflammatory signals such as cytokines and reactive oxygen species (ROS). In the present study, we employed a cell culture/biosensor platform consisting of electrode arrays integrated with microfluidics to monitor extracellular H(2)O(2), one of the major ROS types, produced by primary rat hepatocytes during alcohol injury. The biosensor consisted of hydrogel microstructures with entrapped horseradish peroxidase (HRP) immobilized on an array of miniature gold electrodes. These arrays of sensing electrodes were integrated into microfluidic devices and modified with collagen (I) to promote hepatocyte adhesion. Once seeded into the microfluidic devices, hepatocytes were exposed to 100 mM ethanol and the signal at the working electrode was monitored by cyclic voltammetry (CV) over the course of 4 h. The CV experiments revealed that hepatocytes secreted up to 1.16 μM H(2)O(2) after 3 h of stimulation. Importantly, when hepatocytes were incubated with antioxidants or alcohol dehydrogenase inhibitor prior to alcohol exposure, the H(2)O(2) signal was decreased by ~5-fold. These experiments further confirmed that the biosensor was indeed monitoring oxidative stress generated by the hepatocytes and also pointed to one future use of this technology for screening hepatoprotective effects of antioxidants.

A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brückner, Sandra, E-mail: sandra.brueckner@medizin.uni-leipzig.de; Tautenhahn, Hans-Michael, E-mail: hans-michael.tautenhahn@medizin.uni-leipzig.de; TRM, Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Str. 55, Leipzig D-04103

Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention inmore » the pig model. Methods: Mesenchymal surface antigens and multi-lineage differentiation potential of porcine MSC isolated by collagenase digestion either from bone marrow or adipose tissue (subcutaneous/visceral) were assessed by flow cytometry. Morphology and functional properties (urea-, glycogen synthesis and cytochrome P450 activity) were determined during culture under differentiation conditions and compared with primary porcine hepatocytes. Results: MSC from porcine adipose tissue and from bone marrow express the typical mesenchymal markers CD44, CD29, CD90 and CD105 but not haematopoietic markers. MSC from both sources displayed differentiation into the osteogenic as well as adipogenic lineage. After hepatocyte differentiation, expression of CD105 decreased significantly and cells adopted the typical polygonal morphology of hepatocytes. Glycogen storage was comparable in adipose tissue- and bone marrow-derived cells. Urea synthesis was about 35% lower in visceral than in subcutaneous adipose tissue-derived MSC. Cytochrome P450 activity increased significantly during differentiation and was twice as high in hepatocyte-like cells generated from bone marrow as from adipose tissue. Conclusion: The

Hepatocyte heterogeneity in the metabolism of carbohydrates.

PubMed

Jungermann, K; Thurman, R G

1992-01-01

Periportal and perivenous hepatocytes possess different amounts and activities of the rate-generating enzymes of carbohydrate and oxidative energy metabolism and thus different metabolic capacities. This is the basis of the model of metabolic zonation, according to which periportal cells catalyze predominantly the oxidative catabolism of fatty and amino acids as well as glucose release and glycogen formation via gluconeogenesis, and perivenous cells carry out preferentially glucose uptake for glycogen synthesis and glycolysis coupled to liponeogenesis. The input of humoral and nervous signals into the periportal and perivenous zones is different; gradients of oxygen, substrates and products, hormones and mediators and nerve densities exist which are important not only for the short-term regulation of carbohydrate metabolism but also for the long-term regulation of zonal gene expression. The specialization of periportal and perivenous hepatocytes in carbohydrate metabolism has been well characterized. In vivo evidence is provided by the complex metabolic situation termed the 'glucose paradox' and by zonal flux differences calculated on the basis of the distribution of enzymes and metabolites. In vitro evidence is given by the different flux rates determined with classical invasive techniques, e.g. in periportal-like and perivenous-like hepatocytes in cell culture, in periportal- and perivenous-enriched hepatocyte populations and in perfused livers during orthograde and retrograde flow, as well as with noninvasive techniques using miniature oxygen electrodes, e.g. in livers perfused in either direction. Differences of opinion in the interpretation of studies with invasive and noninvasive techniques by the authors are discussed. The declining gradient in oxygen concentrations, the decreasing glucagon/insulin ratio and the different innervation could be important factors in the zonal expression of the genes of carbohydrate-metabolizing enzymes. While it is clear that

Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

PubMed

Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

2018-01-01

Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

Macrophage activation by factors released from acetaminophen-injured hepatocytes: Potential role of HMGB1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dragomir, Ana-Cristina; Laskin, Jeffrey D.; Laskin, Debra L., E-mail: laskin@eohsi.rutgers.edu

2011-06-15

Toxic doses of acetaminophen (AA) cause hepatocellular necrosis. Evidence suggests that activated macrophages contribute to the pathogenic process; however, the factors that activate these cells are unknown. In these studies, we assessed the role of mediators released from AA-injured hepatocytes in macrophage activation. Treatment of macrophages with conditioned medium (CM) collected 24 hr after treatment of mouse hepatocytes with 5 mM AA (CM-AA) resulted in increased production of reactive oxygen species (ROS). Macrophage expression of heme oxygenase-1 (HO-1) and catalase mRNA was also upregulated by CM-AA, as well as cyclooxygenase (COX)-2 and 12/15-lipoxygenase (LOX). CM-AA also upregulated expression of themore » proinflammatory chemokines, MIP-1{alpha} and MIP-2. The effects of CM-AA on expression of COX-2, MIP-1{alpha} and MIP-2 were inhibited by blockade of p44/42 MAP kinase, suggesting a biochemical mechanism mediating macrophage activation. Hepatocytes injured by AA were found to release HMGB1, a potent macrophage activator. This was inhibited by pretreatment of hepatocytes with ethyl pyruvate (EP), which blocks HMGB1 release. EP also blocked CM-AA induced ROS production and antioxidant expression, and reduced expression of COX-2, but not MIP-1{alpha} or MIP-2. These findings suggest that HMGB1 released by AA-injured hepatocytes contributes to macrophage activation. This is supported by our observation that expression of the HMGB1 receptor RAGE is upregulated in macrophages in response to CM-AA. These data indicate that AA-injured hepatocytes contribute to the inflammatory environment in the liver through the release of mediators such as HMGB1. Blocking HMGB1/RAGE may be a useful approach to limiting classical macrophage activation and AA-induced hepatotoxicity. - Research Highlights: > These studies analyze macrophage activation by mediators released from acetaminophen-damaged hepatocytes. > Factors released from acetaminophen-injured hepatocytes induce

[Zoo-anthroponoses involving rodents: epidemiologic considerations].

PubMed

Rodhain, F

1996-01-01

A large number of zoo-anthroponoses is known, in the natural cycle of which are involved rodents or lagomorphs. This is mainly related to the numerical importance of rodents, their ecological plasticity, their relationships with man. Thus, it is important, for the epidemiologist, to differentiate synanthropic rodents living in close contact with man and often spread by him, which are at the origine of frequently urban diseases, from wild rodents usually having only few contacts with man in rural areas only. The events allowing the passage of an infectious agent from rodent to man appear varied, as are epidemiological schemes in which these animals can play different roles: they can act, alone or together with other animals, as natural reservoirs, or, on the contrary, they are only occasionally infected, and the transmission can occur by direct contact, by bite, through the environment, or with the help of an arthropod vector. It is particularly interesting to follow the evolution of some of these epidemiological systems, in order to evaluate the impact of human activities, such as deforestations, new technics for agriculture and cattle breeding, urbanization, increasing transportation, development of leisure activities. In order to reduce this infectious risk, we should look for the means of a correct balance between rodent and human populations, by joining the control of epidemiologically dangerous species, modification of some of our behaviours, a relevant health information, together with the care of the protection of natural ecosystems.

Fetal liver-derived mesenchymal stromal cells augment engraftment of transplanted hepatocytes

PubMed Central

Joshi, Meghnad; Patil, Pradeep B.; He, Zhong; Holgersson, Jan; Olausson, Michael; Sumitran-Holgersson, Suchitra

2012-01-01

Background aims One important problem commonly encountered after hepatocyte transplantation is the low numbers of transplanted cells found in the graft. If hepatocyte transplantation is to be a viable therapeutic approach, significant liver parenchyma repopulation is required. Mesenchymal stromal cells (MSC) produce high levels of various growth factors, cytokines and metalloproteinases, and have immunomodulatory effects. We therefore hypothesized that co-transplantation of MSC with human fetal hepatocytes (hFH) could augment in vivo expansion after transplantation. We investigated the ability of human fetal liver MSC (hFLMSC) to augment expansion of phenotypically and functionally well-characterized hFH. Methods Two million hFH (passage 6) were either transplanted alone or together (1:1 ratio) with green fluorescence protein-expressing hFLMSC into the spleen of C57BL/6 nude mice with retrorsine-induced liver injury. Results After 4 weeks, engraftment of cells was detected by fluorescence in situ hybridization using a human-specific DNA probe. Significantly higher numbers of cells expressing human cytokeratin (CK)8, CK18, CK19, Cysteine-rich MNNG HOS Transforming gene (c-Met), alpha-fetoprotein (AFP), human nuclear antigen, mitochondrial antigen, hepatocyte-specific antigen and albumin (ALB) were present in the livers of recipient animals co-transplanted with hFLMSC compared with those without. Furthermore, expression of human hepatocyte nuclear factor (HNF)-4α and HNF-1β, and cytochrome P450 (CYP) 3A7 mRNA was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) in these animals. In addition, significantly increased amounts of human ALB were detected. Importantly, hFLMSC did not transdifferentiate into hepatocytes. Conclusions Our study reports the use of a novel strategy for enhanced liver repopulation and thereby advances this experimental procedure closer to clinical liver cell therapy. PMID:22424216

Control of Domestic Rats & Mice, Training Guide--Rodent Control Series.

ERIC Educational Resources Information Center

Bjornson, Bayard F.; And Others

As one booklet in a series on rodent control, this training guide has been developed to assist administrators, rodent-control operators, and others responsible for rodent-control operations in the training of employees in this field. Topics covered include rodents and human welfare, description and habits of domestic rats and mice, rodent-borne…

LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.

PubMed

Homolya, László; Fu, Dong; Sengupta, Prabuddha; Jarnik, Michal; Gillet, Jean-Pierre; Vitale-Cross, Lynn; Gutkind, J Silvio; Lippincott-Schwartz, Jennifer; Arias, Irwin M

2014-01-01

Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation.

Rodent-vegetation relationships in southeastern Montana

Treesearch

James G. MacCracken; Daniel W. Uresk; Hansen; Richard M.

1985-01-01

Plant communities of southeastern Montana were surveyed for rodents over a two year period. Deer mice (Peromyscus maniculatus) were the most abundant rodent species found on the study area. Prairie voles (Microtus ochrogaster), meadow voles (M. pennsylvanicus), sagebrush voles (Lagurus curtatus...

Lurking in the Shadows: Emerging Rodent Infectious Diseases

PubMed Central

Besselsen, David G.; Franklin, Craig L.; Livingston, Robert S.; Riley, Lela K.

2013-01-01

Rodent parvoviruses, Helicobacter spp., murine norovirus, and several other previously unknown infectious agents have “emerged” in laboratory rodents relatively recently. These agents have been discovered serendipitously or through active investigation of atypical serology results, cell culture contamination, unexpected histopathology, or previously unrecognized clinical disease syndromes. The potential research impact of these agents is not fully known. Infected rodents have demonstrated immunomodulation, tumor suppression, clinical disease (particularly in immunodeficient rodents), and histopathology. Perturbations of organismal and cellular physiology also likely occur. These agents posed unique challenges to laboratory animal resource programs once discovered; it was necessary to develop specific diagnostic assays and an understanding of their epidemiology and transmission routes before attempting eradication, and then evaluate eradication methods for efficacy. Even then management approaches varied significantly, from apathy to total exclusion, and such inconsistency has hindered the sharing and transfer of rodents among institutions, particularly for genetically modified rodent models that may not be readily available. As additional infectious agents are discovered in laboratory rodents in coming years, much of what researchers have learned from experiences with the recently identified pathogens will be applicable. This article provides an overview of the discovery, detection, and research impact of infectious agents recently identified in laboratory rodents. We also discuss emerging syndromes for which there is a suspected infectious etiology, and the unique challenges of managing newly emerging infectious agents. PMID:18506061

Ductular reaction in hereditary hemochromatosis: the link between hepatocyte senescence and fibrosis progression.

PubMed

Wood, Marnie J; Gadd, Victoria L; Powell, Lawrie W; Ramm, Grant A; Clouston, Andrew D

2014-03-01

The development of portal fibrosis following the iron loading of hepatocytes is the first stage of fibrogenesis in hereditary hemochromatosis. In other chronic liver diseases it has been shown that a ductular reaction (DR) appears early, correlates with fibrosis progression, and is a consequence of activation of an alternative pathway of hepatocyte replication. This study was designed to investigate the presence of the DR in hemochromatosis and describe its associations. Liver biopsies from 63 C282Y homozygous patients were assessed for hepatic iron concentration (HIC) and graded for iron loading, fibrosis stage, steatosis, and inflammation. Immunostaining allowed quantification of the DR, hepatocyte senescence and proliferation, and analysis incorporated clinical data. Hepatocyte senescence was positively correlated with HIC, serum ferritin, and oxidative stress. A DR was demonstrated and occurred prior to histological fibrosis. HIC, age, hepatocyte senescence and proliferation, portal inflammation, and excessive alcohol consumption all had significant associations with the extent of the DR. In multivariate analysis, iron loading, hepatocyte replicative arrest, and portal inflammation remained independently and significantly associated with the DR. Of factors associated with fibrosis progression, the DR (odds ratio [OR] 10.86 P<0.0001) and the presence of portal inflammation (OR 4.31, P=0.028) remained significant after adjustment for cofactors. The extent of the DR regressed following therapeutic venesection. Iron loading of hepatocytes leads to impaired replication, stimulating the development of the DR in hemochromatosis and this correlates strongly with hepatic fibrosis. Portal inflammation occurs in hemochromatosis and is independently associated with the DR and fibrosis, and thus its role in this disease should be evaluated further. © 2014 by the American Association for the Study of Liver Diseases.

Digital coincidence counting

NASA Astrophysics Data System (ADS)

Buckman, S. M.; Ius, D.

1996-02-01

This paper reports on the development of a digital coincidence-counting system which comprises a custom-built data acquisition card and associated PC software. The system has been designed to digitise the pulse-trains from two radiation detectors at a rate of 20 MSamples/s with 12-bit resolution. Through hardware compression of the data, the system can continuously record both individual pulse-shapes and the time intervals between pulses. Software-based circuits are used to process the stored pulse trains. These circuits are constructed simply by linking together icons representing various components such as coincidence mixers, time delays, single-channel analysers, deadtimes and scalers. This system enables a pair of pulse trains to be processed repeatedly using any number of different methods. Some preliminary results are presented in order to demonstrate the versatility and efficiency of this new method.

20 CFR 654.415 - Insect and rodent control.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Insect and rodent control. 654.415 Section 654.415 Employees' Benefits EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF LABOR SPECIAL... Insect and rodent control. Housing and facilities shall be free of insects, rodents, and other vermin. ...

20 CFR 654.415 - Insect and rodent control.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 20 Employees' Benefits 3 2011-04-01 2011-04-01 false Insect and rodent control. 654.415 Section 654.415 Employees' Benefits EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF LABOR SPECIAL... Insect and rodent control. Housing and facilities shall be free of insects, rodents, and other vermin. ...

Non-alcoholic steatohepatitis pathogenesis: sublethal hepatocyte injury as a driver of liver inflammation

PubMed Central

Ibrahim, Samar H; Hirsova, Petra; Gores, Gregory J

2018-01-01

A subset of patients with non-alcoholic fatty liver disease develop an inflammatory condition, termed nonalcoholic steatohepatitis (NASH). NASH is characterised by hepatocellular injury, innate immune cell-mediated inflammation and progressive liver fibrosis. The mechanisms whereby hepatic inflammation occurs in NASH remain incompletely understood, but appear to be linked to the proinflammatory microenvironment created by toxic lipid-induced hepatocyte injury, termed lipotoxicity. In this review, we discuss the signalling pathways induced by sublethal hepatocyte lipid overload that contribute to the pathogenesis of NASH. Furthermore, we will review the role of proinflammatory, proangiogenic and profibrotic hepatocyte-derived extracellular vesicles as disease biomarkers and pathogenic mediators during lipotoxicity. We also review the potential therapeutic strategies to block the feed-forward loop between sublethal hepatocyte injury and liver inflammation. PMID:29367207

Comparative analysis of the oral mucosae from rodents and non-rodents: Application to the nonclinical evaluation of sublingual immunotherapy products

PubMed Central

Thirion-Delalande, Catherine; Gervais, Frédéric; Fisch, Cécile; Cuiné, Jean; Baron-Bodo, Véronique; Moingeon, Philippe

2017-01-01

Background A comparative characterization of the oral mucosa in various animals is needed to identify the best animal model(s) for nonclinical evaluation of sublingual immunotherapy products. With this aim, we studied the histological characteristics and immune cell infiltrates of oral mucosae from common animal species. Methods Three oral regions (i.e. ventral surface of the tongue, mouth floor and cheek) obtained from eight animal species, including rodents (i.e. mice, rats, hamsters, guinea pigs) and non-rodents (i.e. rabbits, dogs, minipigs and monkeys) were characterized by histology and immunohistology in comparison with a human tongue. Results Rodents exhibit a thin keratinized epithelium with low epithelial extensions, whereas non-rodents, most particularly minipigs and monkeys, display a non-keratinized epithelium with larger rete ridges, similarly to humans. Glycogen-rich cells in the superficial epithelial layers are observed in samples from both minipigs, monkeys and humans. Comparable immune subpopulations detected in the 3 oral regions from rodent and non-rodent species include MHC-II+ antigen presenting cells, mostly CD163+ macrophages, located in the lamina propria (LP) and muscle tissue in the vicinity of resident CD3+CD4+ T cells. Limited numbers of mast cells are also detected in the LP and muscle tissue from all species. Conclusion The oral mucosae of minipigs and monkeys are closest to that of humans, and the immune networks are quite similar between all rodents and non-rodents. Taking into account the ethical and logistical difficulties of performing research in the latter species, rodents and especially mice, should preferentially be used for pharmacodynamics/efficacy studies. Our data also support the use of minipigs to perform biodistribution and safety studies of sublingual immunotherapy products. PMID:28886055

Recovery and normalization of triple coincidences in PET

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lage, Eduardo, E-mail: elage@mit.edu; Parot, Vicente; Dave, Shivang R.

2015-03-15

Purpose: Triple coincidences in positron emission tomography (PET) are events in which three γ-rays are detected simultaneously. These events, though potentially useful for enhancing the sensitivity of PET scanners, are discarded or processed without special consideration in current systems, because there is not a clear criterion for assigning them to a unique line-of-response (LOR). Methods proposed for recovering such events usually rely on the use of highly specialized detection systems, hampering general adoption, and/or are based on Compton-scatter kinematics and, consequently, are limited in accuracy by the energy resolution of standard PET detectors. In this work, the authors propose amore » simple and general solution for recovering triple coincidences, which does not require specialized detectors or additional energy resolution requirements. Methods: To recover triple coincidences, the authors’ method distributes such events among their possible LORs using the relative proportions of double coincidences in these LORs. The authors show analytically that this assignment scheme represents the maximum-likelihood solution for the triple-coincidence distribution problem. The PET component of a preclinical PET/CT scanner was adapted to enable the acquisition and processing of triple coincidences. Since the efficiencies for detecting double and triple events were found to be different throughout the scanner field-of-view, a normalization procedure specific for triple coincidences was also developed. The effect of including triple coincidences using their method was compared against the cases of equally weighting the triples among their possible LORs and discarding all the triple events. The authors used as figures of merit for this comparison sensitivity, noise-equivalent count (NEC) rates and image quality calculated as described in the NEMA NU-4 protocol for the assessment of preclinical PET scanners. Results: The addition of triple-coincidence events with

Recovery and normalization of triple coincidences in PET.

PubMed

Lage, Eduardo; Parot, Vicente; Moore, Stephen C; Sitek, Arkadiusz; Udías, Jose M; Dave, Shivang R; Park, Mi-Ae; Vaquero, Juan J; Herraiz, Joaquin L

2015-03-01

Triple coincidences in positron emission tomography (PET) are events in which three γ-rays are detected simultaneously. These events, though potentially useful for enhancing the sensitivity of PET scanners, are discarded or processed without special consideration in current systems, because there is not a clear criterion for assigning them to a unique line-of-response (LOR). Methods proposed for recovering such events usually rely on the use of highly specialized detection systems, hampering general adoption, and/or are based on Compton-scatter kinematics and, consequently, are limited in accuracy by the energy resolution of standard PET detectors. In this work, the authors propose a simple and general solution for recovering triple coincidences, which does not require specialized detectors or additional energy resolution requirements. To recover triple coincidences, the authors' method distributes such events among their possible LORs using the relative proportions of double coincidences in these LORs. The authors show analytically that this assignment scheme represents the maximum-likelihood solution for the triple-coincidence distribution problem. The PET component of a preclinical PET/CT scanner was adapted to enable the acquisition and processing of triple coincidences. Since the efficiencies for detecting double and triple events were found to be different throughout the scanner field-of-view, a normalization procedure specific for triple coincidences was also developed. The effect of including triple coincidences using their method was compared against the cases of equally weighting the triples among their possible LORs and discarding all the triple events. The authors used as figures of merit for this comparison sensitivity, noise-equivalent count (NEC) rates and image quality calculated as described in the NEMA NU-4 protocol for the assessment of preclinical PET scanners. The addition of triple-coincidence events with the authors' method increased peak

Transformation of primary human hepatocytes in hepatocellular carcinoma.

PubMed

Montalbano, Mauro; Rastellini, Cristiana; Wang, Xiaofu; Corsello, Tiziana; Eltorky, Mahmoud A; Vento, Renza; Cicalese, Luca

2016-03-01

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Currently, there is limited knowledge of neoplastic transformation of hepatocytes in HCC. In clinical practice, the high rate of HCC local recurrence suggests the presence of different hepatocyte populations within the liver and particularly in the tumor proximity. The present study investigated primary human hepatocyte cultures obtained from liver specimens of patients affected by cirrhosis and HCC, their proliferation and transformation. Liver samples were obtained from seven HCC cirrhotic patients and from three patients with normal liver (NL). Immediately after surgery, cell outgrowth and primary cultures were obtained from the HCC lesion, the cirrhotic tissue proximal (CP, 1-3 cm) and distal (CD, >5 cm) to the margin of the neoplastic lesion, or from NL. Cells were kept in culture for 16 weeks. Morphologic analyses were performed and proliferation rate of the different cell populations compared over time. Glypican-3, Heppar1, Arginase1 and CD-44 positivity were tested. The degree of invasiveness of cells acquiring neoplastic characteristics was studied with a transwell migration assay. We observed that HCC cells maintained their morphology and unmodified neoplastic characteristics when cultured. Cells isolated from CP, showed a progressive morphologic transformation in HCC-like cells accompanied by modification of markers expression with signs of invasiveness. Absence of HCC contamination in the CP isolates was confirmed. In CD samples some of these characteristics were present and at significantly lower levels. With the present study, we are the first to have identified and describe the existence of human hepatocytes near the cancerous lesion that can transform in HCC in vitro.

Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeom, Chul-gon; Kim, Dong-il; Park, Min-jung

Previously, we reported that CARM1 undergoes ubiquitination-dependent degradation in renal podocytes. It was also reported that CARM1 is necessary for fasting-induced hepatic gluconeogenesis. Based on these reports, we hypothesized that treatment with insulin, a hormone typically present under the ‘fed’ condition, would inhibit gluconeogenesis via CARM1 degradation. HepG2 cells, AML-12 cells, and rat primary hepatocytes were treated with insulin to confirm CARM1 downregulation. Surprisingly, insulin treatment increased CARM1 expression in all cell types examined. Furthermore, treatment with insulin increased histone 3 methylation at arginine 17 and 26 in HepG2 cells. To elucidate the role of insulin-induced CARM1 upregulation, the HA-CARM1more » plasmid was transfected into HepG2 cells. CARM1 overexpression did not increase the expression of lipogenic proteins generally increased by insulin signaling. Moreover, CARM1 knockdown did not influence insulin sensitivity. Insulin is known to facilitate hepatic proliferation. Like insulin, CARM1 overexpression increased CDK2 and CDK4 expression. In addition, CARM1 knockdown reduced the number of insulin-induced G2/M phase cells. Moreover, GFP-CARM1 overexpression increased the number of G2/M phase cells. Based on these results, we concluded that insulin-induced CARM1 upregulation facilitates hepatocyte proliferation. These observations indicate that CARM1 plays an important role in liver pathophysiology. - Highlights: • Insulin treatment increases CARM1 expression in hepatocytes. • CARM1 overexpression does not increase the expression of lipogenic proteins. • CARM1 knockdown does not influence insulin sensitivity. • Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation.« less

In Vitro Metabolism of 3,4-Methylenedioxymethamphetamine in Human Hepatocytes

PubMed Central

Ramaley, Corinne; Leonard, Susan C.; Miller, Jeffrey D.; Wilson, Denita Takesha-Mashia; Chang, Sai Y.; Chen, Qingyu; Li, Feng; Du, Chengan

2014-01-01

Users of the illicit drug, 3,4-methylenedioxymethamphetamine (MDMA), show signs of neurotoxicity. However, the precise mechanism of neurotoxicity caused by use of MDMA has not yet been elucidated. Synthetic glutathione (GSH) conjugates of MDMA are transported into the brain by the GSH transporter and subsequently produce neurotoxicity. The objective of this research is to show direct evidence of the formation of GSH adducts of MDMA in human hepatocytes. High-performance liquid chromatography coupled with tandem mass spectrometry was utilized to examine in vitro incubations of MDMA with cryopreserved human hepatocytes. The use of hydrophilic liquid chromatography in combination with linear ion trap mass spectrometry permitted the identification of two possible GSH metabolites. Enhanced product ion scans of m/z = 499 and 487 amu of extracts from hepatocytes treated with 1.0 mM MDMA show a distinct fragmentation pattern (m/z 194.2, 163, 135, 105), suggesting the formation of MDMA–GSH conjugate, MDMA-SG and 3,4-dihydroxymethamphetamine-SG. The formation of an MDMA–GSH conjugate was further supported by the apparent lack of the same fragmentation pattern from hepatocyte samples without MDMA treatment. The results generated from this study yield valuable qualitative and quantitative information about the neurotoxic thioether metabolites formed from MDMA in humans. PMID:24682111

Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes.

PubMed

Rojas, Joselyn; Teran-Angel, Guillermo; Barbosa, Luisa; Peterson, Darrell L; Berrueta, Lisbeth; Salmen, Siham

2016-05-01

Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttling of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection. Copyright © 2016 Elsevier Inc. All rights reserved.

Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes.

PubMed

Yanhong, Fan; Chenghua, He; Guofang, Liu; Haibin, Zhang

2008-10-01

The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion, and pancreatin digestion. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll gradient centrifugation). The harvested hepatocytes were suspended in DMEM, M199 (cultured in 5% CO(2)), or L-15 (cultured without 5% CO(2)) medium then cultured at 17, 27, or 37 degrees C. Cell yield was counted by use of a hemocytometer, and the viability of the cells was assessed by use of the Trypan blue exclusion test. Results from these studies showed that the best method of isolation was pancreatin digestion (the cell yield was 2.7 x 10(8) per g (liver weight) and the viability was 98.4%) and the best medium was M199 (cultured in 5% CO(2)) or L-15 (cultured without 5% CO(2)). The optimum culture temperature was 27 degrees C. The primary hepatocytes culture of Cyprimus carpio grew well and satisfied requirements for most toxicological experiments in this condition.

Comparative studies on fatty acid synthesis, glycogen metabolism, and gluconeogenesis by hepatocytes isolated from lean and obese Zucker rats.

PubMed

McCune, S A; Durant, P J; Jenkins, P A; Harris, R A

1981-12-01

Hepatocytes isolated from genetically obese female Zucker rats and lean female Zucker rats were compared. Hepatocytes from fed obese rats exhibited greater rates of fatty acid synthesis, more extensive accumulation of lactate and pyruvate from their glycogen stores, increased rates of net glucose utilization but produced less ketone bodies from exogenous fatty acids and had lower citrate levels than hepatocytes from lean rats. Lipogenesis was not as sensitive to dibutyryl cyclic AMP (DBcAMP) inhibition in hepatocytes from obese rats but glycogenolysis was stimulated to the same extent by this nucleotide in both preparations. Ketogenesis was less sensitive to stimulation by DBcAMP in hepatocytes from obese rats. A difference in sensitivity of lipogenesis to DBcAMP was not found when lactate plus pyruvate was added to the incubation medium, suggesting that a greater rate of glycolysis by hepatocytes from obese rats accounts for their relative insensitivity to DBcAMP. Citrate levels were elevated by DBcAMP to a greater extent in hepatocytes from obese rats. Hepatocytes prepared from lean rats starved for 48 hr were glycogen depleted and lacked significant capacity for lipogenesis and glycogen synthesis. In contrast, hepatocytes isolated from starved obese rats retained considerable amounts of liver glycogen and exhibited detectable rates of lipogenesis and glycogen synthesis. Hepatocytes prepared from starved lean rats gave faster apparent rates of lactate gluconeogenesis than hepatocytes prepared from starved obese rats. Thus, hepatocytes prepared from obese Zucker rats are more glycogenic, glycolytic, and lipogenic but less ketogenic and glucogenic than hepatocytes prepared from lean rats.

Alternative Cell Sources to Adult Hepatocytes for Hepatic Cell Therapy.

PubMed

Pareja, Eugenia; Gómez-Lechón, María José; Tolosa, Laia

2017-01-01

Adult hepatocyte transplantation is limited by scarce availability of suitable donor liver tissue for hepatocyte isolation. New cell-based therapies are being developed to supplement whole-organ liver transplantation, to reduce the waiting-list mortality rate, and to obtain more sustained and significant metabolic correction. Fetal livers and unsuitable neonatal livers for organ transplantation have been proposed as potential useful sources of hepatic cells for cell therapy. However, the major challenge is to use alternative cell sources for transplantation that can be derived from reproducible methods. Different types of stem cells with hepatic differentiation potential are eligible for generating large numbers of functional hepatocytes for liver cell therapy to treat degenerative disorders, inborn hepatic metabolic diseases, and organ failure. Clinical trials are designed to fully establish the safety profile of such therapies and to define target patient groups and standardized protocols.

Spectroscopic signature of mouse embryonic stem cell-derived hepatocytes using synchrotron Fourier transform infrared microspectroscopy

NASA Astrophysics Data System (ADS)

Thumanu, Kanjana; Tanthanuch, Waraporn; Ye, Danna; Sangmalee, Anawat; Lorthongpanich, Chanchao; Parnpai, Rangsun; Heraud, Philip

2011-05-01

Stem cell-based therapy for liver regeneration has been proposed to overcome the persistent shortage in the supply of suitable donor organs. A requirement for this to succeed is to find a rapid method to detect functional hepatocytes, differentiated from embryonic stem cells. We propose Fourier transform infrared (FTIR) microspectroscopy as a versatile method to identify the early and last stages of the differentiation process leading to the formation of hepatocytes. Using synchrotron-FTIR microspectroscopy, the means of identifying hepatocytes at the single-cell level is possible and explored. Principal component analysis and subsequent partial least-squares (PLS) discriminant analysis is applied to distinguish endoderm induction from hepatic progenitor cells and matured hepatocyte-like cells. The data are well modeled by PLS with endoderm induction, hepatic progenitor cells, and mature hepatocyte-like cells able to be discriminated with very high sensitivity and specificity. This method provides a practical tool to monitor endoderm induction and has the potential to be applied for quality control of cell differentiation leading to hepatocyte formation.

Resveratrol Inhibited Hydroquinone-Induced Cytotoxicity in Mouse Primary Hepatocytes

PubMed Central

Wang, Da-Hong; Ootsuki, Yoshie; Fujita, Hirofumi; Miyazaki, Masahiro; Yie, Qinxia; Tsutsui, Ken; Sano, Kuniaki; Masuoka, Noriyoshi; Ogino, Keiki

2012-01-01

Hydroquinone (1,4-benzenediol) has been widely used in clinical situations and the cosmetic industry because of its depigmenting effects. Most skin-lightening hydroquinone creams contain 4%–5% hydroquinone. We have investigated the role of resveratrol in prevention of hydroquinone induced cytotoxicity in mouse primary hepatocytes. We found that 400 µM hydroquinone exposure alone induced apoptosis of the cells and also resulted in a significant drop of cell viability compared with the control, and pretreatment of resveratrol to a final concentration of 0.5 mM 1 h before hydroquinone exposure did not show a significant improvement in the survival rate of the hepatocytes, however, relatively higher concentrations of resveratrol (≥1 mM) inhibited apoptosis of the mouse primary hepatocytes and increased cell viability in a dose-dependent manner, and in particular the survival rate of the hepatocytes was recovered from 28% to near 100% by 5 mM resveratrol. Interestingly, pretreatment with resveratrol for longer time (24 h), even in very low concentrations (50 µM, 100 µM), blocked the damage of hydroquinone to the cells. We also observed that resveratrol pretreatment suppressed hydroquinone-induced expression of cytochrome P450 2E1 mRNA dose-dependently. The present study suggests that resveratrol protected the cells against hydroquinone-induced toxicity through its antioxidant function and possibly suppressive effect on the expression of cytochrome P450 2E1. PMID:23202692

Resveratrol inhibited hydroquinone-induced cytotoxicity in mouse primary hepatocytes.

PubMed

Wang, Da-Hong; Ootsuki, Yoshie; Fujita, Hirofumi; Miyazaki, Masahiro; Yie, Qinxia; Tsutsui, Ken; Sano, Kuniaki; Masuoka, Noriyoshi; Ogino, Keiki

2012-09-19

Hydroquinone (1,4-benzenediol) has been widely used in clinical situations and the cosmetic industry because of its depigmenting effects. Most skin-lightening hydroquinone creams contain 4%-5% hydroquinone. We have investigated the role of resveratrol in prevention of hydroquinone induced cytotoxicity in mouse primary hepatocytes. We found that 400 µM hydroquinone exposure alone induced apoptosis of the cells and also resulted in a significant drop of cell viability compared with the control, and pretreatment of resveratrol to a final concentration of 0.5 mM 1 h before hydroquinone exposure did not show a significant improvement in the survival rate of the hepatocytes, however, relatively higher concentrations of resveratrol (≥1 mM) inhibited apoptosis of the mouse primary hepatocytes and increased cell viability in a dose-dependent manner, and in particular the survival rate of the hepatocytes was recovered from 28% to near 100% by 5 mM resveratrol. Interestingly, pretreatment with resveratrol for longer time (24 h), even in very low concentrations (50 µM, 100 µM), blocked the damage of hydroquinone to the cells. We also observed that resveratrol pretreatment suppressed hydroquinone-induced expression of cytochrome P450 2E1 mRNA dose-dependently. The present study suggests that resveratrol protected the cells against hydroquinone-induced toxicity through its antioxidant function and possibly suppressive effect on the expression of cytochrome P450 2E1.

Hypoxia promotes liver-stage malaria infection in primary human hepatocytes in vitro.

PubMed

Ng, Shengyong; March, Sandra; Galstian, Ani; Hanson, Kirsten; Carvalho, Tânia; Mota, Maria M; Bhatia, Sangeeta N

2014-02-01

Homeostasis of mammalian cell function strictly depends on balancing oxygen exposure to maintain energy metabolism without producing excessive reactive oxygen species. In vivo, cells in different tissues are exposed to a wide range of oxygen concentrations, and yet in vitro models almost exclusively expose cultured cells to higher, atmospheric oxygen levels. Existing models of liver-stage malaria that utilize primary human hepatocytes typically exhibit low in vitro infection efficiencies, possibly due to missing microenvironmental support signals. One cue that could influence the infection capacity of cultured human hepatocytes is the dissolved oxygen concentration. We developed a microscale human liver platform comprised of precisely patterned primary human hepatocytes and nonparenchymal cells to model liver-stage malaria, but the oxygen concentrations are typically higher in the in vitro liver platform than anywhere along the hepatic sinusoid. Indeed, we observed that liver-stage Plasmodium parasite development in vivo correlates with hepatic sinusoidal oxygen gradients. Therefore, we hypothesized that in vitro liver-stage malaria infection efficiencies might improve under hypoxia. Using the infection of micropatterned co-cultures with Plasmodium berghei, Plasmodium yoelii or Plasmodium falciparum as a model, we observed that ambient hypoxia resulted in increased survival of exo-erythrocytic forms (EEFs) in hepatocytes and improved parasite development in a subset of surviving EEFs, based on EEF size. Further, the effective cell surface oxygen tensions (pO2) experienced by the hepatocytes, as predicted by a mathematical model, were systematically perturbed by varying culture parameters such as hepatocyte density and height of the medium, uncovering an optimal cell surface pO2 to maximize the number of mature EEFs. Initial mechanistic experiments revealed that treatment of primary human hepatocytes with the hypoxia mimetic, cobalt(II) chloride, as well as a HIF-1�

Merging bioreactor technology with 3D hepatocyte-fibroblast culturing approaches: Improved in vitro models for toxicological applications.

PubMed

Leite, Sofia B; Teixeira, Ana P; Miranda, Joana P; Tostões, Rui M; Clemente, João J; Sousa, Marcos F; Carrondo, Manuel J T; Alves, Paula M

2011-06-01

During the last years an increasing number of in vitro models have been developed for drug screening and toxicity testing. Primary cultures of hepatocytes are, by far, the model of choice for those high-throughput studies but their spontaneous dedifferentiation after some time in culture hinders long-term studies. Thus, novel cell culture systems allowing extended hepatocyte maintenance and more predictive long term in vitro studies are required. It has been shown that hepatocytes functionality can be improved and extended in time when cultured as 3D-cell aggregates in environmental controlled stirred bioreactors. In this work, aiming at further improving hepatocytes functionality in such 3D cellular structures, co-cultures with fibroblasts were performed. An inoculum concentration of 1.2×10(5) cell/mL and a 1:2 hepatocyte:mouse embryonic fibroblast ratio allowed to improve significantly the albumin secretion rate and both ECOD (phase I) and UGT (phase II) enzymatic activities in 3D co-cultures, as compared to the routinely used 2D hepatocyte monocultures. Significant improvements were also observed in relation to 3D monocultures of hepatocytes. Furthermore, hepatocytes were able to respond to the addition of beta-Naphtoflavone by increasing ECOD activity showing CYP1A inducibility. The dependence of CYP activity on oxygen concentration was also observed. In summary, the improved hepatocyte specific functions during long term incubation of 3D co-cultures of hepatocytes with fibroblasts indicate that this system is a promising in vitro model for long term toxicological studies. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Ron Receptor Regulates Kupffer Cell-Dependent Cytokine Production and Hepatocyte Survival Following Endotoxin Exposure in Mice

PubMed Central

Stuart, William D.; Kulkarni, Rishikesh M.; Gray, Jerilyn K.; Vasiliauskas, Juozas; Leonis, Mike A.; Waltz, Susan E.

2011-01-01

Previous studies demonstrated that targeted deletion of the Ron receptor tyrosine kinase (TK) domain in mice leads to marked hepatocyte protection in a well-characterized model of lipopolysaccharide (LPS)-induced acute liver failure in D-galactosamine (GalN)-sensitized mice. Hepatocyte protection in TK−/− mice was observed despite paradoxically elevated serum levels of tumor necrosis factor alpha (TNFα). To understand the role of Ron in the liver, purified populations of Kupffer cells and hepatocytes from wild-type (TK+/+) and TK−/− mice were studied. Utilizing quantitative RT-PCR, we demonstrated that Ron is expressed in these cell-types. Moreover, we also recapitulated the protected hepatocyte phenotype and exaggerated cytokine production observed in the TK−/− mice in vivo through the use of purified cultured cells ex vivo. We show that isolated TK−/− Kupffer cells produce increased levels of TNFα and select cytokines compared to TK+/+ cells following LPS stimulation. We also show that conditioned media from LPS-treated TK−/− Kupffer cells was more toxic to hepatocytes than control media, suggesting the exaggerated levels of cytokines produced from the TK−/− Kupffer cells are detrimental to wild type hepatocytes. In addition, we observed that TK−/− hepatocytes were more resistant to cell death compared to TK+/+ hepatocytes, suggesting that Ron functions in both the epithelial and inflammatory cell compartments to regulate acute liver injury. These findings were confirmed in vivo in mice with hepatocyte and macrophage cell-type-specific conditional Ron deletions. Mice with Ron loss selectively in hepatocytes exhibited less liver damage and increased survival compared to mice with Ron loss in macrophages. In conclusion, we have dissected cell-type-specific roles for Ron such that this receptor modulates cytokine production from Kupffer cells and inhibits hepatocyte survival in response to injury. PMID:21520175

V1-receptor mediated GSH efflux by vasopressin from rat hepatocytes.

PubMed

Sato, C; Liu, J H; Uchihara, M; Izumi, N; Yauchi, T; Sakaj, Y; Asahina, Y; Fukuma, T; Takano, T; Marumo, F

1992-01-01

Vasopression increases sinusoidal efflux of GSH in the perfused rat liver. The mechanism of this effect was studied in the perfused rat liver and in isolated rat hepatocytes. Vasopressin stimulated GSH efflux in both systems and a V1-receptor antagonist (OPC-21268) significantly inhibited the effect of vasopressin suggesting that vasopressin stimulates GSH efflux from rat hepatocytes via V1-receptor.

Carbon Tetrachloride Increases Intracellular Calcium in Rat Liver and Hepatocyte Cultures

DTIC Science & Technology

1986-05-12

to Phenyl- ephrine Figure 21 . Quin2-loaded Hepatocyte& Exposed to Carbon Tetrachloride or Phenylephrine Figure 22. Quin2-loaded Hepatocyte...HEPATOTOXIN Carbon tetrachloride (CC14 ) is an historically important hepato- toxin that has been investigated since before the turn of the century ...through phosphory- lation by phosphorylase kinase. Phosphorylase kinase can be st ~mulated by increased intracellular Ca++ via calmodulin, or by

From Mere Coincidences to Meaningful Discoveries

ERIC Educational Resources Information Center

Griffiths, Thomas L.; Tenenbaum, Joshua B.

2007-01-01

People's reactions to coincidences are often cited as an illustration of the irrationality of human reasoning about chance. We argue that coincidences may be better understood in terms of rational statistical inference, based on their functional role in processes of causal discovery and theory revision. We present a formal definition of…

Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Cong; Sekine, Shuichi, E-mail: ssekine@facult

Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and inmore » hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.« less

Three-dimensional hepatocyte culture system for the study of Echinococcus multilocularis larval development

PubMed Central

Chen, Bing; Yan, Hongbin; Zhao, Yannan; Lou, Zhongzi; Li, Jianqiu; Fu, Baoquan; Zhu, Xingquan; McManus, Donald P.; Dai, Jianwu; Jia, Wanzhong

2018-01-01

Background Hepatocyte-based metacestode culture is an attractive method to study alveolar echinococcosis (AE), but it is limited by the relatively short lifespan of cultured hepatocytes in maintaining their normal function. Methodology/principal findings We describe a three-dimensional (3D) hepatic culture system developed from co-cultured hepatocytes and mesenchymal stem cells using a collagen scaffold to study the development of Echinococcus multilocularis larvae. This 3D culture system preserved the function of hepatocytes for a longer period of time than their monolayer counterparts, with albumin secretion, 7-ethoxyresorufin O-deethylation activity, urea synthesis, CYP3A4 and CYP2D6 activity being highly preserved for 21–28 days. The expression levels of hepatocyte-specific genes including CLDN-3, Bsep, AFP, G6P, A1AT, CYP3A4 and NR1I3 were significantly higher in the 3D cultured system compared with their monolayer counterparts after 14-days in culture. Additionally, in the presence of 3D cultured hepatocytes, 81.2% of E. multilocularis protoscoleces rapidly de-differentiated into infective vesicles within eight weeks. Transcriptomic analyses revealed 807 differentially expressed genes between cultured vesicles and protoscoleces, including 119 genes uniquely expressed in protoscoleces, and 242 genes uniquely expressed in vesicles. These differentially expressed genes were mainly involved in parasite growth relating to the G-protein coupled receptor activity pathway, substrate-specific transmembrane transporter activity, cell-cell adhesion process, and potentially with neuroactive ligand-receptor interaction. Conclusions/significance This culture system provides a valuable advance in prolonging hepatocyte functionality, a foundation for future in-depth analysis of the host-parasite interaction in AE, and a useful model to evaluate potential therapeutic strategies to treat AE. PMID:29538424

[The polyploidization characteristics of the hepatocytes of the mouse-like hamster Calomyscus mystax].

PubMed

Anatskaia, O V; Malikov, V G; Meĭer, M N; Kudriavtsev, B N

1995-01-01

A cytophotometric measurement of DNA content in hepatocytes of maturing mouse-like hamsters was made. Cells belonging to ordinary mammalian ploidy classes 2c, 2c x 2, 4c, and 4c x 2 made about 90% of the hepatocyte population. The share of binucleated cells wa high (about 80%), the majority of these cells being 2c X 2 hepatocytes. Binucleated cells with tetraploid and diploid nuclei occur in almost every animal. An average hepatocyte ploidy level in mouse-like hamster is 4.6c. The main peculiarity of parenchymal liver cell populations is that up 5% of hepatocytes contain 3--11 nuclei of different ploidy classes. Multinucleated cells increase in number from 1.5% to 4% within the period from one year (the age of maturation) to two years. Later on their percentage does not change. It is found that in binucleated and multinucleated hepatocytes DNA synthesis can proceed asynchronously. Asynchrony in DNA synthesis elevates as the number of nuclei increases. Among the 2c x 2 and 2c x 3 cells an uneven distribution of 3H-thymidine label can occur, respectively, in 5 and in 50% cases, whereas all the cells with more than 3 nuclei display an uneven an uneven 3H-thymidin label distribution. The formation of multinucleated cells is supposed to be associated with asynchrony in DNA-synthesis in binucleated cells and with the restitution of mitosis.

Cell therapy from bench to bedside: Hepatocytes from fibroblasts - the truth and myth of transdifferentiation.

PubMed

Sanal, Madhusudana Girija

2015-06-07

Hepatocyte transplantation is an alternative to liver transplantation in certain disorders such as inherited liver diseases and liver failure. It is a relatively less complicated surgical procedure, and has the advantage that it can be repeated several times if unsuccessful. Another advantage is that hepatocytes can be isolated from partly damaged livers which are not suitable for liver transplantation. Despite these advantages hepatocyte transplantation is less popular. Important issues are poor engraftment of the transplanted cells and the scarcity of donor hepatocytes. Generation of "hepatocyte like cells"/iHeps from embryonic stem cells (ES) and induced pluripotent stem cells (iPSCs) by directed differentiation is an emerging solution to the latter issue. Direct conversation or trans-differentiation of fibroblasts to "hepatocyte like cells" is another way which is, being explored. However this method has several inherent and technical disadvantages compared to the directed differentiation from ES or iPSC. There are several methods claiming to be "highly efficient" for generating "highly functional" "hepatocyte like cells". Currently different groups are working independently and coming up with differentiation protocols and each group claiming an advantage for their protocol. Directed differentiation protocols need to be designed, compared, analyzed and tweaked systematically and logically than empirically. There is a need for a well-coordinated global initiative comparable to the Human Genome Project to achieve this goal in the near future.

Chronic alcohol feeding potentiates hormone‐induced calcium signalling in hepatocytes

PubMed Central

Bartlett, Paula J.; Antony, Anil Noronha; Agarwal, Amit; Hilly, Mauricette; Prince, Victoria L.; Combettes, Laurent; Hoek, Jan B.

2017-01-01

Key points Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined.We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca2+‐mobilizing hormones resulting in a leftward shift in the concentration–response relationship and the transition from oscillatory to more sustained and prolonged Ca2+ increases.Our data demonstrate that alcohol‐dependent adaptation in the Ca2+ signalling pathway occurs at the level of hormone‐induced inositol 1,4,5 trisphosphate (IP3) production and does not involve changes in the sensitivity of the IP3 receptor or size of internal Ca2+ stores.We suggest that prolonged and aberrant hormone‐evoked Ca2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol‐induced hepatocyte injury. Abstract ‘Adaptive’ responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide‐dependent cytosolic calcium ([Ca2+]i) increases, which can adversely affect mitochondrial Ca2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose–response for Ca2+‐mobilizing hormones resulting in more sustained and prolonged [Ca2+]i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone‐induced calcium increases in control livers, but not after chronic alcohol‐feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone‐induced inositol 1,4,5 trisphosphate (IP3) accumulation and

Chronic alcohol feeding potentiates hormone-induced calcium signalling in hepatocytes.

PubMed

Bartlett, Paula J; Antony, Anil Noronha; Agarwal, Amit; Hilly, Mauricette; Prince, Victoria L; Combettes, Laurent; Hoek, Jan B; Gaspers, Lawrence D

2017-05-15

Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca 2+ -mobilizing hormones resulting in a leftward shift in the concentration-response relationship and the transition from oscillatory to more sustained and prolonged Ca 2+ increases. Our data demonstrate that alcohol-dependent adaptation in the Ca 2+ signalling pathway occurs at the level of hormone-induced inositol 1,4,5 trisphosphate (IP 3 ) production and does not involve changes in the sensitivity of the IP 3 receptor or size of internal Ca 2+ stores. We suggest that prolonged and aberrant hormone-evoked Ca 2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol-induced hepatocyte injury. ABSTRACT: 'Adaptive' responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide-dependent cytosolic calcium ([Ca 2+ ] i ) increases, which can adversely affect mitochondrial Ca 2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose-response for Ca 2+ -mobilizing hormones resulting in more sustained and prolonged [Ca 2+ ] i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca 2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone-induced calcium increases in control livers, but not after chronic alcohol-feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone-induced inositol 1,4,5 trisphosphate (IP 3 ) accumulation and phospholipase C

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

PubMed Central

2011-01-01

Background The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies. Results The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells. Conclusion The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450. PMID:21961524

Characterization and Comprehensive Proteome Profiling of Exosomes Secreted by Hepatocytes

PubMed Central

Conde-Vancells, Javier; Rodriguez-Suarez, Eva; Embade, Nieves; Gil, David; Matthiesen, Rune; Valle, Mikel; Elortza, Felix; Lu, Shelly C.; Mato, Jose M.; Falcon-Perez, Juan M.

2009-01-01

Synopsis Exosomes constitute a discrete population of nanometer-sized (30-150 nm) vesicles formed in endocytic compartments and released to the extracellular environment by different cell types. In this work we demonstrated by electron microscopic, western blotting and proteomic analyses that primary hepatocytes secrete exosome-like vesicles containing proteins involved in metabolizing lipoproteins, endogenous compounds as well as xenobiotics. These new findings contribute to improve our knowledge about biology's hepatocyte and may have important diagnostic, prognosis and therapeutic implications in liver diseases Exosomes represent a discrete population of vesicles that are secreted from various cell types to the extracellular media. Their protein and lipid composition are a consequence of sorting events at the level of the multivesicular body, a central organelle which integrates endocytic and secretory pathways. Characterization of exosomes from different biological samples has shown the presence of common as well as cell-type specific proteins. Remarkably, the protein content of the exosomes is modified upon pathological or stress conditions. Hepatocytes play a central role in the body response to stress metabolizing potentially harmful endogenous substances as well as xenobiotics. In the present study we described and characterized for first time exosome secretion in non-tumoral hepatocytes, and using a systematic proteomic approach, we establish the first extensive proteome of a hepatocyte-derived exosome population which should be useful in furthering our understanding of the hepatic function and in the identification of components that may serve as biomarkers for hepatic alterations. Our analysis identifies a significant number of proteins previously described among exosomes derived from others cell types as well as proteins involved in metabolizing lipoproteins, endogenous compounds and xenobiotics, not previously described in exosomes. Furthermore, we

Hepatocytic differentiation of mesenchymal stem cells in cocultures with fetal liver cells.

PubMed

Lange, Claudia; Bruns, Helge; Kluth, Dietrich; Zander, Axel-R; Fiegel, Henning-C

2006-04-21

To investigate the hepatocytic differentiation of mesenchymal stem cells (MSCs) in co-cultures with fetal liver cells (FLC) and the possibility to expand differentiated hepatocytic cells. MSCs were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSCs were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with stem cell factor (SCF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), and fibroblast growth factor 4 (FGF-4) alone, or in presence of freshly isolated FLC. Cells in co-cultures were harvested, and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. Reverse transcription-polymerase chain reaction (RT-PCR) for the liver specific markers cytokeratin-18 (CK-18), albumin, and alpha-fetoprotein (AFP) was performed in different cell populations. Under the specified culture conditions, rat MSCs co-cultured with FLC expressed albumin, CK-18, and AFP-RNA over two weeks. At wk 3, MSCs lost hepatocytic gene expression, probably due to overgrowth of the cocultured FLC. FLC also showed a stable liver specific gene expression in the co-cultures and a very high growth potential. The rat MSCs from bone marrow can differentiate hepatocytic cells in the presence of FLC in vitro and the presence of MSCs in co-cultures also provides a beneficial environment for expansion and differentiation of FLC.

Deficiency of angiotensinogen in hepatocytes markedly decreases blood pressure in lean and obese male mice.

PubMed

Yiannikouris, Frederique; Wang, Yu; Shoemaker, Robin; Larian, Nika; Thompson, Joel; English, Victoria L; Charnigo, Richard; Su, Wen; Gong, Ming; Cassis, Lisa A

2015-10-01

We recently demonstrated that adipocyte deficiency of angiotensinogen (AGT) ablated high-fat diet-induced elevations in plasma angiotensin II (Ang II) concentrations and obesity-hypertension in male mice. Hepatocytes are the predominant source of systemic AGT. Therefore, in this study, we defined the contribution of hepatocyte-derived AGT to obesity-induced elevations in plasma AGT concentrations and hypertension. Male Agt(fl/fl) mice expressing albumin-driven Cre recombinase were bred to female Agt(fl/fl) mice to generate Agt(fl/fl) or hepatocyte AGT-deficient male mice (Agt(Alb)). Mice were fed a low-fat or high-fat diet for 16 weeks. Hepatocyte AGT deficiency had no significant effect on body weight. Plasma AGT concentrations were increased in obese Agt(fl/fl) mice. Hepatocyte AGT deficiency markedly reduced plasma AGT and Ang II concentrations in lean and obese mice. Moreover, hepatocyte AGT deficiency reduced the content and release of AGT from adipose explants. Systolic blood pressure was markedly decreased in lean (by 18 mm Hg) and obese Agt(Alb) mice (by 54 mm Hg) compared with Agt(fl/fl) controls. To define mechanisms, we quantified effects of Ang II on mRNA abundance of megalin, an AGT uptake transporter, in 3T3-L1 adipocytes. Ang II stimulated adipocyte megalin mRNA abundance and decreased media AGT concentrations. These results demonstrate that hepatocytes are the predominant source of systemic AGT in both lean and obese mice. Moreover, reductions in plasma angiotensin concentrations in obese hepatocyte AGT-deficient mice may have limited megalin-dependent uptake of AGT into adipocytes for the production of Ang II in the development of obesity-hypertension. © 2015 American Heart Association, Inc.

Intracellular pH regulation in hepatocytes isolated from three teleost species.

PubMed

Furimsky, M; Moon, T W; Perry, S F

1999-09-01

The mechanisms of intracellular pH (pH(i)) regulation were studied in hepatocytes isolated from three species of teleost: rainbow trout (Oncorhynchus mykiss), black bullhead (Ameiurus melas) and American eel (Anguilla rostrata). Intracellular pH was monitored over time using the pH-sensitive fluorescent dye BCECF in response to acid loading under control conditions and in different experimental media containing either low Na(+) or Cl(-) concentrations, the Na(+)-H(+) exchanger blocker amiloride or the blocker of the V-type H(+)-ATPase, bafilomycin A(1). In trout and bullhead hepatocytes, recovery to an intracellular acid load occurred principally by way of a Na(+)-dependent amiloride-sensitive Na(+)-H(+) exchanger. In eel hepatocytes, the Na(+)-H(+) exchanger did not contribute to recovery to an acid load though evidence suggests that it is present on the cell membrane and participates in the maintenance of steady-state pH(i). The V-type H(+)-ATPase did not participate in recovery to an acid load in any species. A Cl(-)-HCO(3)(-) exchanger may play a role in recovery to an acid load in eel hepatocytes by switching off and retaining base that would normally be tonically extruded. Thus, it is clear that hepatocytes isolated from the three species are capable of regulating pH(i), principally by way of a Na(+)-H(+) exchanger and a Cl(-)-HCO(3)(-) exchanger, but do not exploit identical mechanisms for pH(i) recovery. J. Exp. Zool. 284:361-367, 1999. Copyright 1999 Wiley-Liss, Inc.

Induced Mitogenic Activity in AML-12 Mouse Hepatocytes Exposed to Low-dose Ultra-Wideband Electromagnetic Radiation

PubMed Central

Dorsey, W. C.; Ford, B. D.; Roane, L.; Haynie, D. T.; Tchounwou, P. B.

2005-01-01

Ultra–wideband (UWB) technology has increased with the use of various civilian and military applications. In the present study, we hypothesized that low-dose UWB electromagnetic radiation (UWBR) could elicit a mitogenic effect in AML-12 mouse hepatocytes, in vitro. To test this hypothesis, we exposed AML-12 mouse hepatocytes, to UWBR in a specially constructed gigahertz transverse electromagnetic mode (GTEM) cell. Cells were exposed to UWBR for 2 h at a temperature of 23°C, a pulse width of 10 ns, a repetition rate of 1 kHz, and field strength of 5–20 kV/m. UWB pulses were triggered by an external pulse generator for UWBR exposure but were not triggered for the sham exposure. We performed an MTT Assay to assess cell viability for UWBR-treated and sham-exposed hepatocytes. Data from viability studies indicated a time-related increase in hepatocytes at time intervals from 8–24 h post exposure. UWBR exerted a statistically significant (p < 0.05) dose-dependent response in cell viability in both serum-treated and serum free medium (SFM) -treated hepatocytes. Western blot analysis of hepatocyte lysates demonstrated that cyclin A protein was induced in hepatocytes, suggesting that increased MTT activity after UWBR exposure was due to cell proliferation. This study indicates that UWBR has a mitogenic effect on AML-12 mouse hepatocytes and implicates a possible role for UWBR in hepatocarcinoma. PMID:16705798

Pro-apoptotic effect of fly ash leachates in hepatocytes of freshwater fish (Channa punctata Bloch).

PubMed

Ali, Mehboob; Rahman, Shakilur; Rehman, Hasibur; Bhatia, Kanchan; Ansari, Rizwan A; Raisuddin, Sheikh

2007-02-01

The pro-apoptotic effect of fly ash leachates (FAL) was studied in the hepatocytes of an Indian freshwater fish, Channa punctata Bloch. Hepatocytes were exposed to different concentrations of '7-day' FAL for 24 and 48h and various parameters of apoptosis were studied using standardized procedures. FAL-induced apoptosis in hepatocytes was indicated by cytological examination, DNA fragmentation and DNA laddering. The induction in cytochrome-c release, caspases 3, 7, 10 and 9 activities and lactate dehydrogenase level provide mechanistic platform for FAL-induced apoptosis. Cytological examination showed an unambiguous apoptotic effect of ash leachates in fish hepatocytes. Exposed hepatocytes also showed increased production of H(2)O(2), superoxide ions and an increase in lipid peroxidation (LPO). The present study suggests a possible role of reactive oxygen species (ROS) in FAL-induced apoptosis in hepatocytes. Lactate dehydrogenase, LPO and apoptosis as biomarkers of cytotoxicity have recently been used for assessment of ecotoxicological impact of environmental chemicals. Our findings show that these biomarkers may also be used for evaluation of ecotoxicological impact of complex chemical mixture such as fly ash and its leachates.

Isolation of GMP Grade Human Hepatocytes from Remnant Liver Tissue of Living Donor Liver Transplantation.

PubMed

Enosawa, Shin

2017-01-01

For the purpose of clinical research of hepatocyte transplantation, procedures for isolation, cryopreservation, thawing, and functional assessment of hepatocytes are described. Although demands for human hepatocytes are increasing in not only cell therapy but also drug development, it is highly difficult to obtain good lots of hepatocytes from human liver tissue. This chapter describes essential issues such as alleviation of warm ischemia, prevention of shear stress, optimization of cryopreservation, and functional assessment, along with securement of quality. All procedures described here are compliant with good manufacturing procedure (GMP) in cell processing facility, approved by the act on measures to ensure safety of regenerative medicine and ethical regulations in Japan.

Mesenchymal stem cells support hepatocyte function in engineered liver grafts.

PubMed

Kadota, Yoshie; Yagi, Hiroshi; Inomata, Kenta; Matsubara, Kentaro; Hibi, Taizo; Abe, Yuta; Kitago, Minoru; Shinoda, Masahiro; Obara, Hideaki; Itano, Osamu; Kitagawa, Yuko

2014-01-01

Recent studies suggest that organ decellularization is a promising approach to facilitate the clinical application of regenerative therapy by providing a platform for organ engineering. This unique strategy uses native matrices to act as a reservoir for the functional cells which may show therapeutic potential when implanted into the body. Appropriate cell sources for artificial livers have been debated for some time. The desired cell type in artificial livers is primary hepatocytes, but in addition, other supportive cells may facilitate this stem cell technology. In this context, the use of mesenchymal stem cells (MSC) is an option meeting the criteria for therapeutic organ engineering. Ideally, supportive cells are required to (1) reduce the hepatic cell mass needed in an engineered liver by enhancing hepatocyte function, (2) modulate hepatic regeneration in a paracrine fashion or by direct contact, and (3) enhance the preservability of parenchymal cells during storage. Here, we describe enhanced hepatic function achieved using a strategy of sequential infusion of cells and illustrate the advantages of co-cultivating bone marrow-derived MSCs with primary hepatocytes in the engineered whole-liver scaffold. These co-recellularized liver scaffolds colonized by MSCs and hepatocytes were transplanted into live animals. After blood flow was established, we show that expression of adhesion molecules and proangiogenic factors was upregulated in the graft.

Rodent-borne diseases and their public health importance in Iran.

PubMed

Rabiee, Mohammad Hasan; Mahmoudi, Ahmad; Siahsarvie, Roohollah; Kryštufek, Boris; Mostafavi, Ehsan

2018-04-01

Rodents are reservoirs and hosts for several zoonotic diseases such as plague, leptospirosis, and leishmaniasis. Rapid development of industry and agriculture, as well as climate change throughout the globe, has led to change or increase in occurrence of rodent-borne diseases. Considering the distribution of rodents throughout Iran, the aim of this review is to assess the risk of rodent-borne diseases in Iran. We searched Google Scholar, PubMed, Science Direct, Scientific Information Database (SID), and Magiran databases up to September 2016 to obtain articles reporting occurrence of rodent-borne diseases in Iran and extract information from them. Out of 70 known rodent-borne diseases, 34 were reported in Iran: 17 (50%) parasitic diseases, 13 (38%) bacterial diseases, and 4 (12%) viral diseases. Twenty-one out of 34 diseases were reported from both humans and rodents. Among the diseases reported in the rodents of Iran, plague, leishmaniasis, and hymenolepiasis were the most frequent. The most infected rodents were Rattus norvegicus (16 diseases), Mus musculus (14 diseases), Rattus rattus (13 diseases), Meriones persicus (7 diseases), Apodemus spp. (5 diseases), Tatera indica (4 diseases), Meriones libycus (3 diseases), Rhombomys opimus (3 diseases), Cricetulus migratorius (3 diseases), and Nesokia indica (2 diseases). The results of this review indicate the importance of rodent-borne diseases in Iran. Considering notable diversity of rodents and their extensive distribution throughout the country, it is crucial to pay more attention to their role in spreading infectious diseases for better control of the diseases.

The Fecal Viral Flora of Wild Rodents

PubMed Central

Phan, Tung G.; Kapusinszky, Beatrix; Wang, Chunlin; Rose, Robert K.; Lipton, Howard L.; Delwart, Eric L.

2011-01-01

The frequent interactions of rodents with humans make them a common source of zoonotic infections. To obtain an initial unbiased measure of the viral diversity in the enteric tract of wild rodents we sequenced partially purified, randomly amplified viral RNA and DNA in the feces of 105 wild rodents (mouse, vole, and rat) collected in California and Virginia. We identified in decreasing frequency sequences related to the mammalian viruses families Circoviridae, Picobirnaviridae, Picornaviridae, Astroviridae, Parvoviridae, Papillomaviridae, Adenoviridae, and Coronaviridae. Seventeen small circular DNA genomes containing one or two replicase genes distantly related to the Circoviridae representing several potentially new viral families were characterized. In the Picornaviridae family two new candidate genera as well as a close genetic relative of the human pathogen Aichi virus were characterized. Fragments of the first mouse sapelovirus and picobirnaviruses were identified and the first murine astrovirus genome was characterized. A mouse papillomavirus genome and fragments of a novel adenovirus and adenovirus-associated virus were also sequenced. The next largest fraction of the rodent fecal virome was related to insect viruses of the Densoviridae, Iridoviridae, Polydnaviridae, Dicistroviriade, Bromoviridae, and Virgaviridae families followed by plant virus-related sequences in the Nanoviridae, Geminiviridae, Phycodnaviridae, Secoviridae, Partitiviridae, Tymoviridae, Alphaflexiviridae, and Tombusviridae families reflecting the largely insect and plant rodent diet. Phylogenetic analyses of full and partial viral genomes therefore revealed many previously unreported viral species, genera, and families. The close genetic similarities noted between some rodent and human viruses might reflect past zoonoses. This study increases our understanding of the viral diversity in wild rodents and highlights the large number of still uncharacterized viruses in mammals. PMID:21909269

How many food additives are rodent carcinogens?

PubMed

Johnson, F M

2002-01-01

One generally assumes that chemical agents added to foods are reasonably free of risks to human health, and practically everyone consumes some additives in his or her food daily throughout life. In the United States, the 1958 Food Additives Amendment to the Federal Food, Drug and Cosmetic Act of 1938 requires food manufacturers to demonstrate the safety of food additives to the Food and Drug Administration (FDA). The Amendment contains a provision that prohibits approval of an additive if it is found to cause cancer in humans or animals. In the present study, data from the National Toxicology Program rodent bioassay (NTPRB) were used to identify a sample of approximately 50 rodent-tested additives and other chemicals added to food that had been evaluated independently of the FDA/food industry. Surprisingly, the sample shows more than 40% of these food chemicals to be carcinogenic in one or more rodent groups. If this percentage is extrapolated to all substances added to food in the United States, it would imply that more than 1000 of such substances are potential rodent carcinogens. The NTP and FDA test guidelines use similar, though not necessarily identical, rodent test procedures, including near lifetime exposures to the maximum tolerated dose. The FDA specifies that test chemicals should be administered by the oral route. However, the oral route includes three methods of delivering chemicals, that is, mixed in the food or water or delivered by stomach tube (gavage). The NTP data show only 1 of 18 food chemicals mixed in the food are rodent carcinogens, but 16 of 23 gavage-administered food chemicals are carcinogenic to rodents. The distribution suggests that among orally delivered chemicals, those administered in the feed will more likely prove to be noncarcinogens than chemicals given by gavage. The rodent data also reveal that effects may vary according to dose and genotype, as well as by route of administration, to further complicate extrapolation to humans

Optimization of upcyte® human hepatocytes for the in vitro micronucleus assay.

PubMed

Nörenberg, Astrid; Heinz, Stefan; Scheller, Katharina; Hewitt, Nicola J; Braspenning, Joris; Ott, Michael

2013-12-12

"Upcyte(®) human hepatocytes" have the unique property of combining proliferation with the expression of drug metabolising activities. In our current study, we evaluated whether these cells would be suitable for early in vitro micronucleus (MN) tests. A treatment period of 96 h without a recovery period was most reliable for detecting MN formation in upcyte(®) hepatocytes from Donor 740. The basal MN rate in upcyte(®) hepatocytes varied considerably between donors (7-28%); therefore, modifications to the assay medium were tested to determine whether they could decrease inherent MN formation. Optimal medium supplements were 10 ng/ml oncostatin M for the pre-culture and recovery periods and 25 ng/ml epidermal growth factor and 10 ng/ml oncostatin M for the treatment period. Using the optimised conditions and outcome criteria, the upcyte(®) hepatocyte MN assay could correctly identify directly acting (e.g. mitomycin C, etoposide) and metabolically activated genotoxins (e.g. benzo[a]pyrene, cyclophosphamide). "True negative" and "false positive" compounds were also correctly identified as negative. The basal %MN in upcyte(®) hepatocytes from Donor 740 treated with DMSO, cyclophosphamide or MMC, was essentially unaffected by the growth stage ranging from population doublings of 14-61, suggesting that billions of cells could be produced from a single donor for standardised drug toxicity testing. In conclusion, we have established and optimised an in vitro MN test by using upcyte(®) hepatocytes to correctly identify known direct and metabolically activated genotoxicants as well as "false positives" and true negative compounds. The almost unlimited supply of cells from a single donor and optimised test conditions increase reproducibility in early and more predictive in vitro MN tests. Copyright © 2013 Elsevier B.V. All rights reserved.

DNA content of hepatocyte and erythrocyte nuclei of the spined loach (Cobitis taenia L.) and its polyploid forms.

PubMed

Juchno, Dorota; Lackowska, Bozena; Boron, Alicja; Kilarski, Wincenty

2010-09-01

We analyzed the DNA content of hepatocyte and erythrocyte nuclei of the spined loach Cobitis taenia (diploid) and its allopolyploid forms. Twenty triploid females and one tetraploid were used. At least 20,000 hepatocyte and erythrocyte nuclei were acquired and analyzed by flow cytometry. C. taenia erythrocyte nuclei contain 3.15 +/- 0.21 pg of DNA and the hepatocyte nuclei 4.45 +/- 0.46 pg of DNA. Triploid Cobitis have 5.08 +/- 0.41 pg of DNA in erythrocyte nuclei and 6.11 +/- 0.40 pg of DNA in hepatocyte nuclei, whereas the tetraploid erythrocyte and hepatocyte nuclei contained 6.60 and 7.40 pg of DNA, respectively. In general, the DNA contents correlate positively with the ploidy level of the fish investigated. The DNA content variation in the hepatocyte and erythrocyte nuclei may be due to differences in extent of chromatin condensation, which is more pronounced in the erythrocyte than hepatocyte nuclei, or to the several orders of ploidy that occur in the parenchymal liver cells.

Nrf2 Knockdown Disrupts the Protective Effect of Curcumin on Alcohol-Induced Hepatocyte Necroptosis.

PubMed

Lu, Chunfeng; Xu, Wenxuan; Zhang, Feng; Shao, Jiangjuan; Zheng, Shizhong

2016-12-05

It has emerged that hepatocyte necroptosis plays a critical role in chronic alcoholic liver disease (ALD). Our previous study has identified that the beneficial therapeutic effect of curcumin on alcohol-caused liver injury might be attributed to activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), whereas the role of curcumin in regulating necroptosis and the underlying mechanism remain to be determined. We first found that chronic alcohol consumption triggered obvious hepatocyte necroptosis, leading to increased expression of receptor-interacting protein 1, receptor-interacting protein 3, high-mobility group box 1, and phosphorylated mixed lineage kinase domain-like in murine livers. Curcumin dose-dependently ameliorated hepatocyte necroptosis and alleviated alcohol-caused decrease in hepatic Nrf2 expression in alcoholic mice. Then Nrf2 shRNA lentivirus was introduced to generate Nrf2-knockdown mice. Our results indicated that Nrf2 knockdown aggravated the effects of alcohol on liver injury and necroptosis and even abrogated the inhibitory effect of curcumin on necroptosis. Further, activated Nrf2 by curcumin inhibited p53 expression in both livers and cultured hepatocytes under alcohol stimulation. The next in vitro experiments, similar to in vivo ones, revealed that although Nrf2 knockdown abolished the suppression of curcumin on necroptosis of hepatocytes exposed to ethanol, p53 siRNA could clearly rescued the relative effect of curcumin. In summary, for the first time, we concluded that curcumin attenuated alcohol-induced hepatocyte necroptosis in a Nrf2/p53-dependent mechanism. These findings make curcumin an excellent candidate for ALD treatment and advance the understanding of ALD mechanisms associated with hepatocyte necroptosis.

Cryopreservation of isolated primary rat hepatocytes: enhanced survival and long-term hepatospecific function.

PubMed

Sosef, Meindert N; Baust, John M; Sugimachi, Keishi; Fowler, Alex; Tompkins, Ronald G; Toner, Mehmet

2005-01-01

To investigate the long-term effect of cryopreservation on hepatocyte function, as well as attempt to improve cell viability and function through the utilization of the hypothermic preservation solution, HypoThermosol (HTS), as the carrier solution. Advances in the field of bioartificial liver support have led to an increasing demand for successful, efficient means of cryopreservation of hepatocytes. Fresh rat hepatocytes were cryopreserved in suspension in culture media (Media-cryo group) or HTS (HTS-cryo group), both supplemented with 10% DMSO. Following storage up to 2 months in liquid nitrogen, cells were thawed and maintained in a double collagen gel culture for 14 days. Hepatocyte yield and viability were assessed up to 14 days postthaw. Serial measurements of albumin secretion, urea synthesis, deethylation of ethoxyresorufin (CYT P450 activity), and responsiveness to stimulation with interleukin-6 (IL-6) were performed. Immediate postthaw viability was 60% in Media-cryo and 79% in HTS-cryo, in comparison with control (90%). Albumin secretion, urea synthesis and CYT P450 activity yielded 33%, 55%, and 59% in Media-cryo and 71%, 80%, and 88% in HTS-cryo, respectively, compared with control (100%). Assessment of cellular response to IL-6 following cryopreservation revealed a similar pattern of up-regulation in fibrinogen production and suppression of albumin secretion compared with nonfrozen controls. This study demonstrates that isolated rat hepatocytes cryopreserved using HTS showed high viability, long-term hepatospecific function, and response to cytokine challenge. These results may represent an important step forward to the utilization of cryopreserved isolated hepatocytes in bioartificial liver devices.

Generation of functional hepatocyte-like cells from human deciduous periodontal ligament stem cells.

PubMed

Vasanthan, Punitha; Jayaraman, Pukana; Kunasekaran, Wijenthiran; Lawrence, Anthony; Gnanasegaran, Nareshwaran; Govindasamy, Vijayendran; Musa, Sabri; Kasim, Noor Hayaty Abu

2016-08-01

Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases.

Generation of functional hepatocyte-like cells from human deciduous periodontal ligament stem cells

NASA Astrophysics Data System (ADS)

Vasanthan, Punitha; Jayaraman, Pukana; Kunasekaran, Wijenthiran; Lawrence, Anthony; Gnanasegaran, Nareshwaran; Govindasamy, Vijayendran; Musa, Sabri; Kasim, Noor Hayaty Abu

2016-08-01

Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases.

Depressed gluconeogenesis and ureogenesis in isolated hepatocytes after intermittent hypoxia in rats.

PubMed

Freminet, A; Megas, P; Puceat, M

1990-01-01

1. Rats were exposed to hypobaric hypoxia (equivalent altitude 4500 m), 2 x 2 hr per day, for 5 days. Isolated hepatocytes were prepared on day 6 after 18 hr of fast and also from control normoxic animals. The hepatocytes were incubated (120 min) with various substrates. 2. ATP contents were lower in hepatocytes from exposed as compared to control animals whether at the beginning (14%) or at the end (-6 to -33%) of incubation depending on the substrate. 3. Gluconeogenesis from all precursors (lactate, alanine, pyruvate, glutamine) was significantly reduced (40-50%) in exposed as compared to control animals. 4. Ureogenesis from alanine and from pyruvate + NH4Cl was also markedly depressed in exposed animals but no differences were noticed with glutamine or lactate + NH4Cl and alanine + NH4Cl. 5. Results are discussed in relation to known effects of acute and chronic hypoxia, interrelationship between gluconeogenesis and ureogenesis, taking into account the inhomogeneity of liver and the metabolic properties of periportal and perivenous hepatocytes.

Receptor-Mediated and Fluid-Phase Transcytosis of Horseradish Peroxidase across Rat Hepatocytes

PubMed Central

Ellinger, Isabella; Fuchs, Renate

2010-01-01

Horseradish peroxidase (HRP) is often used as a fluid-phase marker to characterize endocytic and transcytotic processes. Likewise, it has been applied to investigate the mechanisms of biliary secretion of fluid in rat liver hepatocytes. However, HRP contains mannose residues and thus binds to mannose receptors (MRs) on liver cells, including hepatocytes. To study the role of MR-mediated endocytosis of HRP transport in hepatocytes, we determined the influence of the oligosaccharid mannan on HRP biliary secretion in the isolated perfused rat liver. A 1-minute pulse of HRP was applied followed by marker-free perfusion. HRP appeared in bile with biphasic kinetics: a first peak at 7 minutes and a second peak at 15 minutes after labeling. Perfusion with 0.8 mg/mL HRP in the presence of a twofold excess of mannan reduced the first peak by 41% without effect on the second one. Together with recently published data on MR expression in rat hepatocytes this demonstrates two different mechanisms for HRP transcytosis: a rapid, receptor-mediated transport and a slower fluid-phase transport. PMID:20168981

IL-6 modulates hepatocyte proliferation via induction of HGF/p21{sup cip1}: Regulation by SOCS3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Rui; Jaruga, Barbara; Kulkarni, Shailin

2005-12-30

The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21{sup cip1} protein expression in primary mouse hepatocytes. Disruption of the p21{sup cip1} gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21{sup cip1} protein expression and a slightly strongermore » inhibition of cell proliferation in SOCS3{sup +/-} mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3{sup +/-} mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21{sup cip1}-dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration.« less

Molecular perturbations restrict potential for liver repopulation of hepatocytes isolated from non-heart-beating donor rats.

PubMed

Enami, Yuta; Joseph, Brigid; Bandi, Sriram; Lin, Juan; Gupta, Sanjeev

2012-04-01

Organs from non-heart-beating donors are attractive for use in cell therapy. Understanding the nature of molecular perturbations following reperfusion/reoxygenation will be highly significant for non-heart-beating donor cells. We studied non-heart-beating donor rats for global gene expression with Affymetrix microarrays, hepatic tissue integrity, viability of isolated hepatocytes, and engraftment and proliferation of transplanted cells in dipeptidyl peptidase IV-deficient rats. In non-heart-beating donors, liver tissue was morphologically intact for >24 hours with differential expression of 1, 95, or 372 genes, 4, 16, or 34 hours after death, respectively, compared with heart-beating donors. These differentially expressed genes constituted prominent groupings in ontological pathways of oxidative phosphorylation, adherence junctions, glycolysis/gluconeogenesis, and other discrete pathways. We successfully isolated viable hepatocytes from non-heart-beating donors, especially up to 4 hours after death, although the hepatocyte yield and viability were inferior to those of hepatocytes from heart-beating donors (P < 0.05). Similarly, although hepatocytes from non-heart-beating donors engrafted and proliferated after transplantation in recipient animals, this was inferior to hepatocytes from heart-beating donors (P < 0.05). Gene expression profiling in hepatocytes isolated from non-heart-beating donors showed far greater perturbations compared with corresponding liver tissue, including representation of pathways in focal adhesion, actin cytoskeleton, extracellular matrix-receptor interactions, multiple ligand-receptor interactions, and signaling in insulin, calcium, wnt, Jak-Stat, or other cascades. Liver tissue remained intact over prolonged periods after death in non-heart-beating donors, but extensive molecular perturbations following reperfusion/reoxygenation impaired the viability of isolated hepatocytes from these donors. Insights into molecular changes in

Quinoline-glycomimetic conjugates reducing lipogenesis and lipid accumulation in hepatocytes.

PubMed

Palit, Subhadeep; Mukherjee, Sanghamitra; Niyogi, Sougata; Banerjee, Anindyajit; Patra, Dipendu; Chakraborty, Amit; Chakrabarti, Saikat; Chakrabarti, Partha; Dutta, Sanjay

2018-06-13

Non-alcoholic fatty liver disease (NAFLD) characterized by excess accumulation of triglyceride in hepatocyte is the major cause of chronic liver disease worldwide and no approved drug is available. The mechanistic target of rapamycin complexes (mTORC) have been implicated to promote lipogenesis and fat accumulation in liver and thus serve as attractive drug targets. Generation of no or low cytotoxic mTOR inhibitors are required as the existing cytotoxic mTOR inhibitors are not useful for NAFLD therapy. We have synthesized novel compounds based on the privileged ATP site binder quinoline scaffold conjugated to glucose and galactosamine derivatives that have significantly low cytotoxicity yet having strong mTORC1 inhibitory activity at low micromolar concentrations. These compounds also effectively inhibit the rate of lipogenesis and lipid accumulation in cultured hepatocytes. This is the first report of glycomimetic-quinoline derivatives that reduce lipid load in hepatocytes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

PubMed

Brodsky, V Y; Malchenko, L A; Butorina, N N; Lazarev Konchenko, D S; Zvezdina, N D; Dubovaya, T K

2017-08-01

Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

Parent Perspectives on Decisions to Participate in a Phase I Hepatocyte Transplant Trial

PubMed Central

Dreyzin, Alexandra; Barnato, Amber; Soltys, Kyle; Farris, Coreen; Sada, Rachel; Haberman, Kimberly; Fox, Ira

2013-01-01

We examined factors that affect decision-making for families presented with a phase I clinical trial of hepatocyte transplant as a potential alternative to liver transplant for their children among two groups: 1) families who were actually offered enrollment in the hepatocyte trial and; 2) families whose children had liver transplants before the trial was available. We conducted semi-structured interviews about actual and hypothetical decision-making regarding trial participation and used grounded theory analysis to identify common themes. The most common motivator for participation was decline in the child's health. The most common deterrent was lack of data from prior hepatocyte transplants, particularly compared to data available about liver transplant. Interviewees' point of comparison for evaluating relative benefits and risks of hepatocyte transplant oscillated between the alternative of doing nothing while waiting for a liver (the relevant alternative) versus the alternative of getting a liver. These results suggest that families' reluctance to participate may result from misconceptions about severity of the child's disease, underestimating risks of liver transplant, or confusion about the role of hepatocyte transplant in the treatment pathway. Clarification of available treatment alternatives and associated risks as part of informed consent may improve the quality of decision-making regarding trial enrollment. PMID:24251638

Rodent-borne diseases and their public health importance in Iran

PubMed Central

Mahmoudi, Ahmad; Siahsarvie, Roohollah; Kryštufek, Boris; Mostafavi, Ehsan

2018-01-01

Background Rodents are reservoirs and hosts for several zoonotic diseases such as plague, leptospirosis, and leishmaniasis. Rapid development of industry and agriculture, as well as climate change throughout the globe, has led to change or increase in occurrence of rodent-borne diseases. Considering the distribution of rodents throughout Iran, the aim of this review is to assess the risk of rodent-borne diseases in Iran. Methodology/Principal finding We searched Google Scholar, PubMed, Science Direct, Scientific Information Database (SID), and Magiran databases up to September 2016 to obtain articles reporting occurrence of rodent-borne diseases in Iran and extract information from them. Out of 70 known rodent-borne diseases, 34 were reported in Iran: 17 (50%) parasitic diseases, 13 (38%) bacterial diseases, and 4 (12%) viral diseases. Twenty-one out of 34 diseases were reported from both humans and rodents. Among the diseases reported in the rodents of Iran, plague, leishmaniasis, and hymenolepiasis were the most frequent. The most infected rodents were Rattus norvegicus (16 diseases), Mus musculus (14 diseases), Rattus rattus (13 diseases), Meriones persicus (7 diseases), Apodemus spp. (5 diseases), Tatera indica (4 diseases), Meriones libycus (3 diseases), Rhombomys opimus (3 diseases), Cricetulus migratorius (3 diseases), and Nesokia indica (2 diseases). Conclusions/Significance The results of this review indicate the importance of rodent-borne diseases in Iran. Considering notable diversity of rodents and their extensive distribution throughout the country, it is crucial to pay more attention to their role in spreading infectious diseases for better control of the diseases. PMID:29672510

Rodents as potential couriers for bioterrorism agents.

PubMed

Lõhmus, Mare; Janse, Ingmar; van de Goot, Frank; van Rotterdam, Bart J

2013-09-01

Many pathogens that can cause major public health, economic, and social damage are relatively easily accessible and could be used as biological weapons. Wildlife is a natural reservoir for many potential bioterrorism agents, and, as history has shown, eliminating a pathogen that has dispersed among wild fauna can be extremely challenging. Since a number of wild rodent species live close to humans, rodents constitute a vector for pathogens to circulate among wildlife, domestic animals, and humans. This article reviews the possible consequences of a deliberate spread of rodentborne pathogens. It is relatively easy to infect wild rodents with certain pathogens or to release infected rodents, and the action would be difficult to trace. Rodents can also function as reservoirs for diseases that have been spread during a bioterrorism attack and cause recurring disease outbreaks. As rats and mice are common in both urban and rural settlements, deliberately released rodentborne infections have the capacity to spread very rapidly. The majority of pathogens that are listed as potential agents of bioterrorism by the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases exploit rodents as vectors or reservoirs. In addition to zoonotic diseases, deliberately released rodentborne epizootics can have serious economic consequences for society, for example, in the area of international trade restrictions. The ability to rapidly detect introduced diseases and effectively communicate with the public in crisis situations enables a quick response and is essential for successful and cost-effective disease control.

Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes

PubMed Central

Yanhong, Fan; Chenghua, He; Guofang, Liu

2008-01-01

The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion, and pancreatin digestion. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll gradient centrifugation). The harvested hepatocytes were suspended in DMEM, M199 (cultured in 5% CO2), or L-15 (cultured without 5% CO2) medium then cultured at 17, 27, or 37 °C. Cell yield was counted by use of a hemocytometer, and the viability of the cells was assessed by use of the Trypan blue exclusion test. Results from these studies showed that the best method of isolation was pancreatin digestion (the cell yield was 2.7 × 108 per g (liver weight) and the viability was 98.4%) and the best medium was M199 (cultured in 5% CO2) or L-15 (cultured without 5% CO2). The optimum culture temperature was 27 °C. The primary hepatocytes culture of Cyprimus carpio grew well and satisfied requirements for most toxicological experiments in this condition. PMID:19002769

Independent, parallel pathways to CXCL10 induction in HCV-infected hepatocytes.

PubMed

Brownell, Jessica; Wagoner, Jessica; Lovelace, Erica S; Thirstrup, Derek; Mohar, Isaac; Smith, Wesley; Giugliano, Silvia; Li, Kui; Crispe, I Nicholas; Rosen, Hugo R; Polyak, Stephen J

2013-10-01

The pro-inflammatory chemokine CXCL10 is induced by HCV infection in vitro and in vivo, and is associated with outcome of IFN (interferon)-based therapy. We studied how hepatocyte sensing of early HCV infection via TLR3 (Toll-like receptor 3) and RIG-I (retinoic acid inducible gene I) led to expression of CXCL10. CXCL10, type I IFN, and type III IFN mRNAs and proteins were measured in PHH (primary human hepatocytes) and hepatocyte lines harboring functional or non-functional TLR3 and RIG-I pathways following HCV infection or exposure to receptor-specific stimuli. HuH7 human hepatoma cells expressing both TLR3 and RIG-I produced maximal CXCL10 during early HCV infection. Neutralization of type I and type III IFNs had no impact on virus-induced CXCL10 expression in TLR3+/RIG-I+ HuH7 cells, but reduced CXCL10 expression in PHH. PHH cultures were positive for monocyte, macrophage, and dendritic cell mRNAs. Immunodepletion of non-parenchymal cells (NPCs) eliminated marker expression in PHH cultures, which then showed no IFN requirement for CXCL10 induction during HCV infection. Immunofluorescence studies also revealed a positive correlation between intracellular HCV Core and CXCL10 protein expression (r(2) = 0.88, p ≤ 0.001). While CXCL10 induction in hepatocytes during the initial phase of HCV infection is independent of hepatocyte-derived type I and type III IFNs, NPC-derived IFNs contribute to CXCL10 induction during HCV infection in PHH cultures. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Inflammation-induced synthesis of proteoheparan sulfate: a novel acute-phase reactant in rat hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Djovkar, A.; Gressner, A.M.

1987-03-01

The synthesis of proteoheparan sulfate in hepatocytes is positively regulated under acute-phase conditions produced either by turpentine or deep back incision. In both cases the incorporation of (/sup 35/S)sulfate and (/sup 14/C)glucosamine is doubled during a 4-h incubation period if compared with control rat hepatocytes. Neither the fractional secretion rate of heparan sulfate into the medium (less than 0.1 of cell-associated glycosaminoglycans) nor the composition of newly formed proteoglycans in hepatocytes are affected during acute phase reaction.

Preventing hepatocyte oxidative stress cytotoxicity with Mangifera indica L. extract (Vimang).

PubMed

Remirez, Diadelis; Tafazoli, Shahrzad; Delgado, Rene; Harandi, Asghar A; O'Brien, Peter J

2005-01-01

Vimang is an aqueous extract of Mangifera indica used in Cuba to improve the quality of life in patients suffering from inflammatory diseases. In the present study we evaluated the effects of Vimang at preventing reactive oxygen species (ROS) formation and lipid peroxidation in intact isolated rat hepatocytes. Vimang at 20, 50 and 100 microg/ml inhibited hepatocyte ROS formation induced by glucose-glucose oxidase. Hepatocyte cytotoxicity and lipid peroxidation induced by cumene hydroperoxide was also inhibited by Vimang in a dose and time dependent manner at the same concentration. Vimang also inhibited superoxide radical formation by xanthine oxidase and hypoxanthine. The superoxide radical scavenging and antioxidant activity of the Vimang extract was likely related to its gallates, catechins and mangiferin content. To our knowledge, this is the first report of cytoprotective antioxidant effects of Vimang in cellular oxidative stress models.

Domestic Rodent Control Training Manual: A Training Aid for the Rodent Control Category for Certification of Pesticide Applicators.

ERIC Educational Resources Information Center

Childress, William R., Jr.; And Others

This training manual, designed for training applicants who wish to obtain certification in pesticide application relative to rodent control, covers the following topics: economic factors, public health factors, biological characteristics of domestic rodents, rat and mouse signs, trapping, repellents, poisons, baits, poisoned water, dumps, sewers,…

Protective effects of ACLF sera on metabolic functions and proliferation of hepatocytes co-cultured with bone marrow MSCs in vitro

PubMed Central

Shi, Xiao-Lei; Gu, Jin-Yang; Zhang, Yue; Han, Bing; Xiao, Jiang-Qiang; Yuan, Xian-Wen; Zhang, Ning; Ding, Yi-Tao

2011-01-01

AIM: To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells (MSCs) could be maintained in serum from acute-on-chronic liver failure (ACLF) patients. METHODS: Hepatocyte supportive functions and cytotoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evaluated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemokine profile was also examined for the normal serum and liver failure serum. RESULTS: Hepatocyte growth factor (HGF) and Tumor necrosis factor; tumor necrosis factor (TNF)-α were remarkably elevated in response to ACLF while epidermal growth factor (EGF) and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver support functions in the homo-hepatocyte culture. Hepatocytes co-cultured with MSCs could tolerate the cytotoxicity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cultured with healthy human serum in vitro. In addition, co-cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum. CONCLUSION: ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro. PMID:21633639

Comparative Gene Expression Profiles Induced by PPARγ and PPARα/γ Agonists in Human Hepatocytes

PubMed Central

Rogue, Alexandra; Lambert, Carine; Jossé, Rozenn; Antherieu, Sebastien; Spire, Catherine; Claude, Nancy; Guillouzo, André

2011-01-01

Background Several glitazones (PPARγ agonists) and glitazars (dual PPARα/γ agonists) have been developed to treat hyperglycemia and, simultaneously, hyperglycemia and dyslipidemia, respectively. However, most have caused idiosyncratic hepatic or extrahepatic toxicities through mechanisms that remain largely unknown. Since the liver plays a key role in lipid metabolism, we analyzed changes in gene expression profiles induced by these two types of PPAR agonists in human hepatocytes. Methodology/Principal Findings Primary human hepatocytes and the well-differentiated human hepatoma HepaRG cells were exposed to different concentrations of two PPARγ (troglitazone and rosiglitazone) and two PPARα/γ (muraglitazar and tesaglitazar) agonists for 24 h and their transcriptomes were analyzed using human pangenomic Agilent microarrays. Principal Component Analysis, hierarchical clustering and Ingenuity Pathway Analysis® revealed large inter-individual variability in the response of the human hepatocyte populations to the different compounds. Many genes involved in lipid, carbohydrate, xenobiotic and cholesterol metabolism, as well as inflammation and immunity, were regulated by both PPARγ and PPARα/γ agonists in at least a number of human hepatocyte populations and/or HepaRG cells. Only a few genes were selectively deregulated by glitazars when compared to glitazones, indicating that PPARγ and PPARα/γ agonists share most of their target genes. Moreover, some target genes thought to be regulated only in mouse or to be expressed in Kupffer cells were also found to be responsive in human hepatocytes and HepaRG cells. Conclusions/Significance This first comprehensive analysis of gene regulation by PPARγ and PPARα/γ agonists favor the conclusion that glitazones and glitazars share most of their target genes and induce large differential changes in gene profiles in human hepatocytes depending on hepatocyte donor, the compound class and/or individual compound, thereby

Nature and mechanisms of hepatocyte apoptosis induced by D-galactosamine/lipopolysaccharide challenge in mice.

PubMed

Wu, Yi-Hang; Hu, Shao-Qing; Liu, Jun; Cao, Hong-Cui; Xu, Wei; Li, Yong-Jun; Li, Lan-Juan

2014-06-01

Apoptosis plays a role in the normal development of liver. However, overactivation thereof may lead to hepatocellular damage. The aim of this study was to assess D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced hepatocyte apoptotic changes in mice and clarify the mechanisms involved in this process. DNA ladder detection was employed to determine the induction condition of hepatic apoptosis. An initial test indicated that typical hepatocyte apoptosis was observed at 6-10 h after the intraperitoneal injection of D-GalN (700 mg/kg) and LPS (10 µg/kg). Subsequently, we evaluated hepatocyte apoptosis at 8 h after administering D-GalN/LPS by histopathological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end‑labeling (TUNEL) detection, flow cytometry and electron microscopy analysis. To clarify the apoptosis-related gene expression, the expression levels of tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), caspase-3, and Fas/Fas ligand (FasL) were determined by serum enzyme immunoassay, immunohistochemistry and western blot analysis. Strong apoptotic positive signals following D-GalN/LPS injection were observed from the results of the serum analysis, histopathological and immunohistochemical analyses, DNA ladder detection, TUNEL detection, flow cytometry and electron microscopy analysis. Additionally, apoptotic hepatocytes were mainly at the late stage of cell apoptosis. The expression of TNF-α, TGF-β1, caspase-3 and Fas/FasL was significantly increased. In conclusion, this study evaluated the D-GalN/LPS-induced hepatocyte apoptotic changes and clarified the apoptosis-related gene expression in mice. The hepatocyte apoptosis induced by D-GalN/LPS may be mainly regulated by the death receptor pathway. TGF-β signaling pathway may also play a vital role in this process of hepatocyte apoptosis.

Differentiation of embryonic stem cells into hepatocytes that coexpress coagulation factors VIII and IX.

PubMed

Cao, Jun; Shang, Chang-zhen; Lü, Li-hong; Qiu, De-chuan; Ren, Meng; Chen, Ya-jin; Min, Jun

2010-11-01

To establish an efficient culture system to support embryonic stem (ES) cell differentiation into hepatocytes that coexpress F-VIII and F-IX. Mouse E14 ES cells were cultured in differentiation medium containing sodium butyrate (SB), basic fibroblast growth factor (bFGF), and/or bone morphogenetic protein 4 (BMP4) to induce the differentiation of endoderm cells and hepatic progenitor cells. Hepatocyte growth factor, oncostatin M, and dexamethasone were then used to induce the maturation of ES cell-derived hepatocytes. The mRNA expression levels of endoderm-specific genes and hepatocyte-specific genes, including the levels of F-VIII and F-IX, were detected by RT-PCR and real-time PCR during various stages of differentiation. Protein expression was examined by immunofluorescence and Western blot. At the final stage of differentiation, flow cytometry was performed to determine the percentage of cells coexpressing F-VIII and F-IX, and ELISA was used to detect the levels of F-VIII and F-IX protein secreted into the culture medium. The expression of endoderm-specific and hepatocyte-specific markers was upregulated to highest level in response to the combination of SB, bFGF, and BMP4. Treatment with the three inducers during hepatic progenitor differentiation significantly enhanced the mRNA and protein levels of F-VIII and F-IX in ES cell-derived hepatocytes. More importantly, F-VIII and F-IX were coexpressed with high efficiency at the final stage of differentiation, and they were also secreted into the culture medium. We have established a novel in vitro differentiation protocol for ES-derived hepatocytes that coexpress F-VIII and F-IX that may provide a foundation for stem cell replacement therapy for hemophilia.

[Current status and future perspectives of hepatocyte transplantation].

PubMed

Pareja, Eugenia; Cortés, Miriam; Gómez-Lechón, M José; Maupoey, Javier; San Juan, Fernando; López, Rafael; Mir, Jose

2014-02-01

The imbalance between the number of potential beneficiaries and available organs, originates the search for new therapeutic alternatives, such as Hepatocyte transplantation (HT).Even though this is a treatment option for these patients, the lack of unanimity of criteria regarding indications and technique, different cryopreservation protocols, as well as the different methodology to assess the response to this therapy, highlights the need of a Consensus Conference to standardize criteria and consider future strategies to improve the technique and optimize the results.Our aim is to review and update the current state of hepatocyte transplantation, emphasizing the future research attempting to solve the problems and improve the results of this treatment. Copyright © 2013 AEC. Published by Elsevier Espana. All rights reserved.

[Hepatocyte apoptosis and mitochondrial permeability transition pore opening in rats with nonalcoholic fatty liver].

PubMed

Kang, Min; Li, Sen; Zhong, Dejun; Yang, Zhimin; Li, Peng

2013-07-01

To investigate the role of hepatocyte apoptosis and mitochondrial permeability transition pore (MPTP) opening in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Thirty male SD rats were randomized into normal diet group and high-fat diet group. At 4, 8 and 12 week of feeding. The hepatocyte apoptosis index (AI) was measured using flow cytometry, and MPTP opening was evaluated with ultraviolet spectrophotometry. Immunohistochemistry was employed to detect hepatic expressions of Bcl-2 and Bax, and Western blotting was used to detect Bax protein expression changes. High-fat feeding resulted in significantly increased hepatocyte AI at 4-12 weeks and gradually increased MPTP opening. In the high-fat diet group, hepatic Bcl-2 expression was detected but the positive cell number remained stable, whereas Bax-positive cell number increased steadily with time with progressively increased intensity of Bax protein expression, resulting in gradually decreased Bcl-2/Bax ratio. Hepatocyte apoptosis occurs in the rat model of NAFLD in close correlation with mitochondrial damage. Increased MPTP opening as the result of increased Bax expression and aberrant Bcl-2/Bax ratio is an important mechanism of hepatocyte mitochondrial damage in NAFLD.

Comparative Proteomic Analysis of Human Liver Tissue and Isolated Hepatocytes with a Focus on Proteins Determining Drug Exposure.

PubMed

Vildhede, Anna; Wiśniewski, Jacek R; Norén, Agneta; Karlgren, Maria; Artursson, Per

2015-08-07

Freshly isolated human hepatocytes are considered the gold standard for in vitro studies of liver functions, including drug transport, metabolism, and toxicity. For accurate predictions of the in vivo outcome, the isolated hepatocytes should reflect the phenotype of their in vivo counterpart, i.e., hepatocytes in human liver tissue. Here, we quantified and compared the membrane proteomes of freshly isolated hepatocytes and human liver tissue using a label-free shotgun proteomics approach. A total of 5144 unique proteins were identified, spanning over 6 orders of magnitude in abundance. There was a good global correlation in protein abundance. However, the expression of many plasma membrane proteins was lower in the isolated hepatocytes than in the liver tissue. This included transport proteins that determine hepatocyte exposure to many drugs and endogenous compounds. Pathway analysis of the differentially expressed proteins confirmed that hepatocytes are exposed to oxidative stress during isolation and suggested that plasma membrane proteins were degraded via the protein ubiquitination pathway. Finally, using pitavastatin as an example, we show how protein quantifications can improve in vitro predictions of in vivo liver clearance. We tentatively conclude that our data set will be a useful resource for improved hepatocyte predictions of the in vivo outcome.

[The miR-21 attenuates hepatocyte hypoxia/reoxygenation injury via inhibiting PTEN/PI3K/AKT signaling pathway].

PubMed

Lu, Xiuxian; Sun, Chao; Zheng, Daofeng; Liu, Rui; Wei, Xufu; Wu, Zhongjun

2017-04-01

Objective To study the effect of microRNA-21 (miR-21) on hypoxia/reoxygenation (H/R)-treated primary hepatocytes from C57BL/6J mice and analyze its possible molecular mechanism. Methods The H/R model of primary hepatocytes was established and the expression of miR-21 was detected by the quantitative real-time PCR. Western blotting was used to detect protein expression levels of phosphatase and tension homology deleted on chromosome 10 (PTEN), phosphorylated AKT (p-AKT), Bcl-2 and Bax. Flow cytometry was performed to observe the hepatocyte apoptosis. Results The expression of miR-21 in primary hepatocytes decreased after H/R injury. After transfected with exogenous miR-21 mimics, the expression of PTEN decreased, while the expressions of p-AKT and Bcl-2 and the ratio of Bcl-2/Bax increased in hepatocytes; the apoptotic level of hepatocytes was downregulated. The inhibition of AKT phosphorylation could downregulate the expression of Bcl-2 and the ratio of Bcl-2/Bax, and upregulate the level of hepatocyte apoptosis. Conclusion The miR-21 can alleviate the hepatocyte apoptosis by inhibiting the PTEN/PI3K/AKT signaling pathway in the process of H/R.

Regeneration of hepatocyte 'buds' in cirrhosis from intrabiliary stem cells.

PubMed

Falkowski, Olga; An, Hee Jung; Ianus, I Andreea; Chiriboga, Luis; Yee, Herman; West, A Brian; Theise, Neil D

2003-09-01

In massive hepatic necrosis, hepatic stem cells constitute a canal of Hering derived, cytokeratin 19 (CK19) positive 'ductular reaction' (DR). Whether DRs in cirrhosis are activated stem cells (so called 'buds') or biliary metaplasia of cholestatic, injured hepatocytes is still debated. We investigate derivation of intraseptal hepatocytes (ISHs) from DRs and from the biliary tree in cirrhosis. Explants of hepatitis B and C, alcohol, primary biliary cirrhosis and primary sclerosing cholangitis-related cirrhosis were examined. ISHs were quantified and their associations with DRs and cholestasis recorded. 3D-reconstruction of ISHs and nearby bile ducts was performed in blocks from hepatitis C and primary sclerosing cholangitis cirrhosis. Seven hundred seventy five/830 (94%) ISHs were associated with CK19 positive DRs. ISHs without ductular reactions were more likely to show cholestatic features (P<0.0001). In 3D, ISHs were seen to bud directly from the biliary tree. In summary: ISHs: (1) are usually associated with stem cell-like DRs; (2) are rarely cholestatic, leaving the associated DRs unexplained; and (3) are linked to the biliary tree in 3D. Dynamic proliferation rates in hepatitis C over time suggest that hepatocyte replication diminishes in late stages, with an associated activation of the biliary stem cell compartment. We therefore suggest that the biliary tree, from at least its smaller branches up to the canals of Hering, are composed of or at least harbor facultative hepatic stem cells, and that ISH largely represent 'buds' of newly formed hepatocytes.

Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes.

PubMed

Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

2016-11-30

The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N -acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

PubMed Central

Wang, Yue; Gao, Hong; Na, Xiao-Lin; Dong, Shu-Ying; Dong, Hong-Wei; Yu, Jia; Jia, Li; Wu, Yong-Hui

2016-01-01

The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL) for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC). Levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), activities of superoxide dismutase (SOD) and catalase (CAT), mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes. PMID:27916916

EXPERIMENTAL SUBSTANTIATION OF PERMEABILIZED HEPATOCYTES MODEL FOR INVESTIGATION OF MITOCHONDRIA IN SITU RESPIRATION.

PubMed

Merlavsky, V M; Manko, B O; Ikkert, O V; Manko, V V

2015-01-01

To verify experimentally the model of permeabilized hepatocytes, the degree of cell permeability was assessed using trypan blue and polarographycally determined cell respiration rate upon succinate (0.35 mM) and a-ketoglutarate (1 mM) oxidation. Oxidative phosphorylation was stimulated by ADP (750 μM). Hepatocyte permeabilization depends on digitonin concentraion in medium and on the number of cells in suspension. Thus, the permeabilization of 0.9-1.7 million cells/ml was completed by 25 μg/ml of digitonin, permeabilization of 2.0-3.0 million cells/ml--by 50 μg/ml of digitonin and permeabilization of 4.0-5.6 million cells/ml--by 100 μg/ml. Thus, the higher is the suspension density, the higher digitonin concentration is required. Treatment of hepatocytes with digitonin resulted in a decrease of endogenous respiration rate to a minimum upon 20-22 μg of digitonin per 1 million cells. Supplementation of permeabilized hepatocytes with α-ketoglutarate maintained stable respiration rate, on the level higher than endogenous respiration at the corresponding digitonin concentration, unlike the intact cells. Respiration rate of permeabilized hepatocytes at the simultaneous addition of α-ketoglutarate and ADP increased to the level of intact cell respiration, irrespective of digitonin concentration. Addition of solely succinate and especially succinate plus ADP markedly intensified the respiration of permeabilized hepatocytes to the level higher than that of intact cells. The dependence of succinate-stimulated respiration on digitonin concentration reached maximum at 20-22 αg of digitonin per 1 million cells. Optimal ratio of digitonin amount and the cell number in suspension is expected to be different in various tissues.

Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Zhichao; Yu, Xuemei; Wu, Weibin

2012-07-13

Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated thatmore » the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.« less

Entrapment of hepatocyte spheroids in a hollow fiber bioreactor as a potential bioartificial liver.

PubMed

Wu, F J; Peshwa, M V; Cerra, F B; Hu, W S

1995-01-01

A bioartificial liver (BAL) employing xenogeneic hepatocytes has been developed as a potential interim support for patients in hepatic failure. For application in human therapy, the BAL requires a substantial increase in liver-specific functions. Cultivation of hepatocytes as spheroids leads to enhanced liver specific functions. We explored the possibility of entrapping spheroids into the BAL in order to improve device performance. Rat hepatocyte spheroids were entrapped in collagen gel within the lumen fibers of the BAL. The morphology and ultrastructure of collagen-entrapped spheroids resembled those of suspended spheroids formed on petri dishes. Albumin synthesis and P-450 enzyme activity were measured as markers of liver specific functions of spheroids entrapped in the BAL. At least a 4-fold improvement in these functions was observed compared to BAL devices entrapped with dispersed hepatocytes in collagen gels.

Generation of Hepatocytes from Pluripotent Stem Cells for Drug Screening and Developmental Modeling.

PubMed

Gieseck, Richard L; Vallier, Ludovic; Hannan, Nicholas R F

2015-01-01

Hepatocytes produced from the differentiation of human pluripotent stem cells can be used to study human development and liver disease, to investigate the toxicological response of novel drug candidates, and as an alternative source of primary cells for transplantation therapies. Here, we describe a method to produce hepatocytes by differentiating human pluripotent stem cells into definitive endoderm, patterning definitive endoderm into anterior definitive endoderm, specifying anterior definitive endoderm into hepatic endoderm, and differentiating hepatic endoderm into immature hepatocytes. These cells are further matured in either two-dimensional or three-dimensional culture conditions to produce cells capable of metabolizing xenobiotics and generating liver-specific proteins, such as albumin and alpha 1 antitrypsin.

Microencapsulation of Hepatocytes and Mesenchymal Stem Cells for Therapeutic Applications.

PubMed

Meier, Raphael P H; Montanari, Elisa; Morel, Philippe; Pimenta, Joël; Schuurman, Henk-Jan; Wandrey, Christine; Gerber-Lemaire, Sandrine; Mahou, Redouan; Bühler, Leo H

2017-01-01

Encapsulated hepatocyte transplantation and encapsulated mesenchymal stem cell transplantation are newly developed potential treatments for acute and chronic liver diseases, respectively. Cells are microencapsulated in biocompatible semipermeable alginate-based hydrogels. Microspheres protect cells against antibodies and immune cells, while allowing nutrients, small/medium size proteins and drugs to diffuse inside and outside the polymer matrix. Microencapsulated cells are assessed in vitro and designed for experimental transplantation and for future clinical applications.Here, we describe the protocol for microencapsulation of hepatocytes and mesenchymal stem cells within hybrid poly(ethylene glycol)-alginate hydrogels.

Hantavirus Prevention: Cleanup of Rodent Contamination

DTIC Science & Technology

2008-09-01

Hantaviruses in the Americas may cause human disease involving the lungs, hence the name " hantavirus pulmonary syndrome" (HPS). Since May 1993, a...humans are also found in other rodents, but the number of cases stemming from these hantaviruses is small when compared to SNV. Hantavirus is shed in... HANTAVIRUS PREVENTION: CLEANUP OF RODENT CONTAMINATION Technical Information Paper 18-001-0306

7 CFR 58.147 - Insect and rodent control program.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Insect and rodent control program. 58.147 Section 58... Service 1 Operations and Operating Procedures § 58.147 Insect and rodent control program. In addition to... made responsible for the performance of a regularly scheduled insect and rodent control program...

7 CFR 58.247 - Insect and rodent control program.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Insect and rodent control program. 58.247 Section 58... Service 1 Operations and Operating Procedures § 58.247 Insect and rodent control program. In addition to... made responsible for the performance of a regularly scheduled insect and rodent control program as...

7 CFR 58.147 - Insect and rodent control program.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 3 2011-01-01 2011-01-01 false Insect and rodent control program. 58.147 Section 58... Service 1 Operations and Operating Procedures § 58.147 Insect and rodent control program. In addition to... made responsible for the performance of a regularly scheduled insect and rodent control program...

7 CFR 58.247 - Insect and rodent control program.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 3 2011-01-01 2011-01-01 false Insect and rodent control program. 58.247 Section 58... Service 1 Operations and Operating Procedures § 58.247 Insect and rodent control program. In addition to... made responsible for the performance of a regularly scheduled insect and rodent control program as...

Roles for Coincidence Detection in Coding Amplitude-Modulated Sounds

PubMed Central

Ashida, Go; Kretzberg, Jutta; Tollin, Daniel J.

2016-01-01

Many sensory neurons encode temporal information by detecting coincident arrivals of synaptic inputs. In the mammalian auditory brainstem, binaural neurons of the medial superior olive (MSO) are known to act as coincidence detectors, whereas in the lateral superior olive (LSO) roles of coincidence detection have remained unclear. LSO neurons receive excitatory and inhibitory inputs driven by ipsilateral and contralateral acoustic stimuli, respectively, and vary their output spike rates according to interaural level differences. In addition, LSO neurons are also sensitive to binaural phase differences of low-frequency tones and envelopes of amplitude-modulated (AM) sounds. Previous physiological recordings in vivo found considerable variations in monaural AM-tuning across neurons. To investigate the underlying mechanisms of the observed temporal tuning properties of LSO and their sources of variability, we used a simple coincidence counting model and examined how specific parameters of coincidence detection affect monaural and binaural AM coding. Spike rates and phase-locking of evoked excitatory and spontaneous inhibitory inputs had only minor effects on LSO output to monaural AM inputs. In contrast, the coincidence threshold of the model neuron affected both the overall spike rates and the half-peak positions of the AM-tuning curve, whereas the width of the coincidence window merely influenced the output spike rates. The duration of the refractory period affected only the low-frequency portion of the monaural AM-tuning curve. Unlike monaural AM coding, temporal factors, such as the coincidence window and the effective duration of inhibition, played a major role in determining the trough positions of simulated binaural phase-response curves. In addition, empirically-observed level-dependence of binaural phase-coding was reproduced in the framework of our minimalistic coincidence counting model. These modeling results suggest that coincidence detection of excitatory

The MAM rodent model of schizophrenia

PubMed Central

Lodge, Daniel J.

2013-01-01

Rodent models of human disease are essential to obtain a better understanding of disease pathology, the mechanism of action underlying conventional treatments, as well as for the generation of novel therapeutic approaches. There are a number of rodent models of schizophrenia based on either genetic manipulations, acute or sub-chronic drug administration, or developmental disturbances. The prenatal methylazoxymethanol acetate (MAM) rodent model is a developmental disruption model gaining increased attention because it displays a number of histological, neurophysiological and behavioral deficits analogous to those observed in schizophrenia patients. This unit describes the procedures required to safely induce the MAM phenotype in rats. In addition, we describe a simple behavioral procedure, amphetamine-induced hyper-locomotion, which can be utilized to verify the MAM phenotype. PMID:23559309

Wheat extracts as an efficient cryoprotective agent for primary cultures of rat hepatocytes.

PubMed

Hamel, Francine; Grondin, Mélanie; Denizeau, Francine; Averill-Bates, Diana A; Sarhan, Fathey

2006-11-05

Hepatocytes are an important physiological model for evaluation of metabolic and biological effects of xenobiotics. They do not proliferate in culture and are extremely sensitive to damage during freezing and thawing, even after the addition of classical cryoprotectants. Thus improved cryopreservation techniques are needed to reduce cell injury and functional impairment. Here, we describe a new and efficient cryopreservation method, which permits long-term storage and recovery of large quantities of healthy cells that maintain high hepatospecific functions. In culture, the morphology of hepatocytes cryopreserved with wheat protein extracts (WPE) was similar to that of fresh cells. Furthermore, hepatospecific functions such as albumin secretion and biotransformation of ammonium to urea were well maintained during 4 days in culture. Inductions of CYP1A1 and CYP2B in hepatocytes cryopreserved with WPEs were similar to those in fresh hepatocytes. These findings clearly show that WPEs are an excellent cryopreservant for primary hepatocytes. The extract was also found to cryopreserve other human and animal cell types such as lung carcinoma, colorectal adenocarcinoma, Chinese hamster ovary transfected with TGF-b1 cDNA, cervical cancer taken from Henrietta Lacks, intestinal epithelium, and T cell leukemia. WPEs have potential as a universal cryopreservant agent of mammalian cells. It is an economic, efficient and non-toxic agent. (c) 2006 Wiley Periodicals, Inc.

Hunting, Food Preparation, and Consumption of Rodents in Lao PDR

PubMed Central

Suwannarong, Kanokwan; Chapman, Robert S.; Lantican, Cecile; Michaelides, Tula; Zimicki, Susan

2015-01-01

A cross-sectional study was conducted in 29 villages of Khamkeuth District in Bolikhamxay Province in the Lao PDR during March to May 2013. The study aimed to determine the characteristics associated with rodent consumption and related behaviors among different ethnic groups, ages, and genders. Five-hundred-eighty-four (584) males and females from 18-50 years of age participated in this study. Half of them were Hmong (292, 50%) while 152 respondents were Lao-Tai (26%) or other ethnic groups (140, 24%). Most of the respondents (79.5%) had farming as their main occupation. Prevalences of the studied outcomes were high: 39.9 for hunting or capturing rodents in the previous year, 77.7% for preparing rodents as food, and 86.3% for rodent consumption. Multivariable logistic regression analysis showed that likelihood of these types of rodent contact was more consistently associated with behavioral factors (gathering things from the forest and elsewhere, cultivation-related activities, and taking measures to prevent rodent-borne disease) than with socio-demographic, environmental, or cultural factors. The strongest associations were observed for gathering things; these associations were consistently positive and statistically significant. Although this study did not directly assess rodent-borne zoonosis risk, we believe that study findings raise concern that such risk may be substantial in the study area and other similar areas. Further epidemiological studies on the association between rodent-borne disease infection and rodent hunting, preparation for food, and consumption are recommended. Moreover, further studies are needed on the association between these potential exposure factors (i.e., rodent hunting, preparation for food, and consumption) and rodent-borne infections, especially among ethnic groups like the Hmong in Lao PDR and those in neighboring countries with similar socio-demographic, environmental, behavioral and cultural contexts. PMID:26196134

Hunting, Food Preparation, and Consumption of Rodents in Lao PDR.

PubMed

Suwannarong, Kanokwan; Chapman, Robert S; Lantican, Cecile; Michaelides, Tula; Zimicki, Susan

2015-01-01

A cross-sectional study was conducted in 29 villages of Khamkeuth District in Bolikhamxay Province in the Lao PDR during March to May 2013. The study aimed to determine the characteristics associated with rodent consumption and related behaviors among different ethnic groups, ages, and genders. Five-hundred-eighty-four (584) males and females from 18-50 years of age participated in this study. Half of them were Hmong (292, 50%) while 152 respondents were Lao-Tai (26%) or other ethnic groups (140, 24%). Most of the respondents (79.5%) had farming as their main occupation. Prevalences of the studied outcomes were high: 39.9 for hunting or capturing rodents in the previous year, 77.7% for preparing rodents as food, and 86.3% for rodent consumption. Multivariable logistic regression analysis showed that likelihood of these types of rodent contact was more consistently associated with behavioral factors (gathering things from the forest and elsewhere, cultivation-related activities, and taking measures to prevent rodent-borne disease) than with socio-demographic, environmental, or cultural factors. The strongest associations were observed for gathering things; these associations were consistently positive and statistically significant. Although this study did not directly assess rodent-borne zoonosis risk, we believe that study findings raise concern that such risk may be substantial in the study area and other similar areas. Further epidemiological studies on the association between rodent-borne disease infection and rodent hunting, preparation for food, and consumption are recommended. Moreover, further studies are needed on the association between these potential exposure factors (i.e., rodent hunting, preparation for food, and consumption) and rodent-borne infections, especially among ethnic groups like the Hmong in Lao PDR and those in neighboring countries with similar socio-demographic, environmental, behavioral and cultural contexts.

Effects of silver nanoparticles on the liver and hepatocytes in vitro.

PubMed

Gaiser, Birgit K; Hirn, Stephanie; Kermanizadeh, Ali; Kanase, Nilesh; Fytianos, Kleanthis; Wenk, Alexander; Haberl, Nadine; Brunelli, Andrea; Kreyling, Wolfgang G; Stone, Vicki

2013-02-01

With the increasing use and incorporation of nanoparticles (NPs) into consumer products, screening for potential toxicity is necessary to ensure customer safety. NPs have been shown to translocate to the bloodstream following inhalation and ingestion, and such studies demonstrate that the liver is an important organ for accumulation. Silver (Ag) NPs are highly relevant for human exposure due to their use in food contact materials, dietary supplements, and antibacterial wound treatments. Due to the large number of different NPs already used in various products and being developed for new applications, it is essential that relevant, quick, and cheap methods of in vitro risk assessment suitable for these new materials are established. Therefore, this study used a simple hepatocytes model combined with an in vivo injection model to simulate the passage of a small amount of NPs into the bloodstream following exposure, e.g., via ingestion or inhalation, and examined the potential of Ag NPs of 20 nm diameter to cause toxicity, inflammation, and oxidative stress in the liver following in vivo exposures of female Wistar rats via iv injection to 50 μg of NPs and in vitro exposures using the human hepatocyte cell line C3A. We found that Ag NPs were highly cytotoxic to hepatocytes (LC(50) lactate dehydrogenase: 2.5 μg/cm(2)) and affected hepatocyte homeostasis by reducing albumin release. At sublethal concentrations with normal cell or tissue morphology, Ag NPs were detected in cytoplasm and nuclei of hepatocytes. We observed similar effects of Ag NPs on inflammatory mediator expression in vitro and in vivo with increase of interleukin-8 (IL-8)/macrophage inflammatory protein 2, IL-1RI, and tumor necrosis factor-α expression in both models and increased IL-8 protein release in vitro. This article presents evidence of the potential toxicity and inflammogenic potential of Ag NPs in the liver following ingestion. In addition, the similarities between in vitro and in vivo

Hepatocyte spheroid arrays inside microwells connected with microchannels

PubMed Central

Fukuda, Junji; Nakazawa, Kohji

2011-01-01

Spheroid culture is a preferable cell culture approach for some cell types, including hepatocytes, as this type of culture often allows maintenance of organ-specific functions. In this study, we describe a spheroid microarray chip (SM chip) that allows stable immobilization of hepatocyte spheroids in microwells and that can be used to evaluate drug metabolism with high efficiency. The SM chip consists of 300-μm-diameter cylindrical wells with chemically modified bottom faces that form a 100-μm-diameter cell adhesion region surrounded by a nonadhesion region. Primary hepatocytes seeded onto this chip spontaneously formed spheroids of uniform diameter on the cell adhesion region in each microwell and these could be used for cytochrome P-450 fluorescence assays. A row of microwells could also be connected to a microchannel for simultaneous detection of different cytochrome P-450 enzyme activities on a single chip. The miniaturized features of this SM chip reduce the numbers of cells and the amounts of reagents required for assays. The detection of four cytochrome P-450 enzyme activities was demonstrated following induction by 3-methylcholantlene, with a sensitivity significantly higher than that in conventional monolayer culture. This microfabricated chip could therefore serve as a novel culture platform for various cell-based assays, including those used in drug screening, basic biological studies, and tissue engineering applications. PMID:21799712

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology

PubMed Central

Dong, Jia; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K.N.; Knobeloch, Daniel; Gerlach, Jörg C.; Zeilinger, Katrin

2008-01-01

Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes. PMID:19003182

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology.

PubMed

Dong, Jia; Mandenius, Carl-Fredrik; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K N; Knobeloch, Daniel; Gerlach, Jörg C; Zeilinger, Katrin

2008-07-01

Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes.

Arenavirus Diversity and Phylogeography of Mastomys natalensis Rodents, Nigeria

PubMed Central

Obadare, Adeoba; Oyeyiola, Akinlabi; Igbokwe, Joseph; Fasogbon, Ayobami; Igbahenah, Felix; Ortsega, Daniel; Asogun, Danny; Umeh, Prince; Vakkai, Innocent; Abejegah, Chukwuyem; Pahlman, Meike; Becker-Ziaja, Beate; Günther, Stephan; Fichet-Calvet, Elisabeth

2016-01-01

Mastomys natalensis rodents are natural hosts for Lassa virus (LASV). Detection of LASV in 2 mitochondrial phylogroups of the rodent near the Niger and Benue Rivers in Nigeria underlines the potential for LASV emergence in fresh phylogroups of this rodent. A Mobala-like sequence was also detected in eastern Nigeria. PMID:26982388

Investigation of metabolic objectives in cultured hepatocytes.

PubMed

Uygun, Korkut; Matthew, Howard W T; Huang, Yinlun

2007-06-15

Using optimization based methods to predict fluxes in metabolic flux balance models has been a successful approach for some microorganisms, enabling construction of in silico models and even inference of some regulatory motifs. However, this success has not been translated to mammalian cells. The lack of knowledge about metabolic objectives in mammalian cells is a major obstacle that prevents utilization of various metabolic engineering tools and methods for tissue engineering and biomedical purposes. In this work, we investigate and identify possible metabolic objectives for hepatocytes cultured in vitro. To achieve this goal, we present a special data-mining procedure for identifying metabolic objective functions in mammalian cells. This multi-level optimization based algorithm enables identifying the major fluxes in the metabolic objective from MFA data in the absence of information about critical active constraints of the system. Further, once the objective is determined, active flux constraints can also be identified and analyzed. This information can be potentially used in a predictive manner to improve cell culture results or clinical metabolic outcomes. As a result of the application of this method, it was found that in vitro cultured hepatocytes maximize oxygen uptake, coupling of urea and TCA cycles, and synthesis of serine and urea. Selection of these fluxes as the metabolic objective enables accurate prediction of the flux distribution in the system given a limited amount of flux data; thus presenting a workable in silico model for cultured hepatocytes. It is observed that an overall homeostasis picture is also emergent in the findings.

Development of an Apparatus for High-Resolution Auger Photoelectron Coincidence Spectroscopy (APECS) and Electron Ion Coincidence (EICO) Spectroscopy

NASA Astrophysics Data System (ADS)

Kakiuchi, Takuhiro; Hashimoto, Shogo; Fujita, Narihiko; Mase, Kazuhiko; Tanaka, Masatoshi; Okusawa, Makoto

We have developed an electron electron ion coincidence (EEICO) apparatus for high-resolution Auger photoelectron coincidence spectroscopy (APECS) and electron ion coincidence (EICO) spectroscopy. It consists of a coaxially symmetric mirror electron energy analyzer (ASMA), a miniature double-pass cylindrical mirror electron energy analyzer (DP-CMA), a miniature time-of-flight ion mass spectrometer (TOF-MS), a magnetic shield, an xyz stage, a tilt-adjustment mechanism, and a conflat flange with an outer diameter of 203 mm. A sample surface was irradiated by synchrotron radiation, and emitted electrons were energy-analyzed and detected by the ASMA and the DP-CMA, while desorbed ions were mass-analyzed and detected by the TOF-MS. The performance of the new EEICO analyzer was evaluated by measuring Si 2p photoelectron spectra of clean Si(001)-2×1 and Si(111)-7×7, and by measuring Si-L23VV-Si-2p Auger photoelectron coincidence spectra (Si-L23VV-Si-2p APECS) of clean Si(001)-2×1.

A Field Study of Plague and Tularemia in Rodents, Western Iran.

PubMed

Mostafavi, Ehsan; Shahraki, Abdolrazagh Hashemi; Japoni-Nejad, Alireza; Esmaeili, Saber; Darvish, Jamshid; Sedaghat, Mohammad Mehdi; Mohammadi, Ali; Mohammadi, Zeinolabedin; Mahmoudi, Ahmad; Pourhossein, Behzad; Ghasemi, Ahmad; Gyuranecz, Miklós; Carniel, Elisabeth

2017-04-01

Kurdistan Province in Iran is a historical focus for plague and tularemia. This study aimed at assessing the current status of these two foci by studying their rodent reservoirs. Rodents were trapped and their ectoparasites were collected. The genus and species of both rodents and ectoparasites were determined. Serological analyses of rodent blood samples were done by enzyme-linked immunosorbent assay for plague and by standard tube agglutination assay for tularemia. Rodent spleen samples were subjected to bacterial culture, microscopic examination, and real-time PCR to search for active plague or tularemia infection. During this study, 245 rodents were trapped, of which the most abundant genera were Apodemus (40%), Mus (24.49%), and Meriones (12.65%). One hundred fifty-three fleas, 37 mites, and 54 ticks were collected on these rodents. The results of all direct and indirect tests were negative for plague. Serological tests were positive for tularemia in 4.8% of trapped rodents. This study is the first report on the presence of tularemia infection in rodents in Western Iran. Since Meriones persicus is a known reservoir for plague and tularemia, and this rodent carried plague and tularemia vectors in Marivan and Sanandaj districts, there is a real potential for the occurrence of these two diseases in this region.

The differentiation and isolation of mouse embryonic stem cells toward hepatocytes using galactose-carrying substrata.

PubMed

Meng, Qingyuan; Haque, Amranul; Hexig, Bayar; Akaike, Toshihiro

2012-02-01

A simple culture system to achieve the differentiation of embryonic stem (ES) cells toward hepatocytes with high efficiency is crucial in providing a cell source for the medical application. In this study, we report the effect of a matrix-dependent enrichment of ES cell-derived hepatocytes using immobilized poly(N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide) (PVLA) with E-cadherin-IgG Fc (E-cad-Fc) as a galactose-carrying substratum. PVLA and E-cad-Fc were confirmed to be stably co-adsorbed onto polystyrene surface by quartz crystal microbalance (QCM). We showed that the E-cad-Fc/PVLA hybrid substratum was efficient in culturing primary hepatocytes and maintaining liver functions, on which the undifferentiated ES cells also maintained high proliferative capability. Furthermore, ES cell-derived hepatocytes on this hybrid matrix expressed elevated level of liver specific genes and functions together with early expression of definitive hepatocyte marker, asialoglycoprotein receptor (ASGPR). Finally, we isolated a high percentage of cells (about 60%) with ASGPR expression after re-seeding onto PVLA-coated surface, and observed the elimination of the poorly differentiated cells (Gata6(+) and Sox17(+)) and the ones toward another cell lineage (brachyury(+) and Pdx1(+)). The system uses a glycopolymer as an extracellular substratum for isolation and enrichment of ES cell-derived hepatocytes with adequate homogeneity and functionality. Copyright © 2011 Elsevier Ltd. All rights reserved.

Endothelial cell-derived matrix promotes the metabolic functional maturation of hepatocyte via integrin-Src signalling.

PubMed

Guo, Xinyue; Li, Weihong; Ma, Minghui; Lu, Xin; Zhang, Haiyan

2017-11-01

The extracellular matrix (ECM) microenvironment is involved in the regulation of hepatocyte phenotype and function. Recently, the cell-derived extracellular matrix has been proposed to represent the bioactive and biocompatible materials of the native ECM. Here, we show that the endothelial cell-derived matrix (EC matrix) promotes the metabolic maturation of human adipose stem cell-derived hepatocyte-like cells (hASC-HLCs) through the activation of the transcription factor forkhead box protein A2 (FOXA2) and the nuclear receptors hepatocyte nuclear factor 4 alpha (HNF4α) and pregnane X receptor (PXR). Reducing the fibronectin content in the EC matrix or silencing the expression of α5 integrin in the hASC-HLCs inhibited the effect of the EC matrix on Src phosphorylation and hepatocyte maturation. The inhibition of Src phosphorylation using the inhibitor PP2 or silencing the expression of Src in hASC-HLCs also attenuated the up-regulation of the metabolic function of hASC-HLCs in a nuclear receptor-dependent manner. These data elucidate integrin-Src signalling linking the extrinsic EC matrix signals and metabolic functional maturation of hepatocyte. This study provides a model for studying the interaction between hepatocytes and non-parenchymal cell-derived matrix. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Monoacylglycerol O-acyltransferase 1 is regulated by peroxisome proliferator-activated receptor γ in human hepatocytes and increases lipid accumulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Jung Hwan; Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul 120-752; Lee, Yoo Jeong

2015-05-08

Monoacylglycerol O-acyltransferase (MGAT) is an enzyme that is involved in triglyceride synthesis by catalyzing the formation of diacylglycerol from monoacylglycerol and fatty acyl CoAs. Recently, we reported that MGAT1 has a critical role in hepatic TG accumulation and that its suppression ameliorates hepatic steatosis in a mouse model. However, the function of MGAT enzymes in hepatic lipid accumulation has not been investigated in humans. Unlike in rodents, MGAT3 as well as MGAT1 and MGAT2 are present in humans. In this study, we evaluated the differences between MGAT subtypes and their association with peroxisome proliferator-activated receptor γ (PPARγ), a regulator ofmore » mouse MGAT1 expression. In human primary hepatocytes, basal expression of MGAT1 was lower than that of MGAT2 or MGAT3, but was strongly induced by PPARγ overexpression. A luciferase assay as well as an electromobility shift assay revealed that human MGAT1 promoter activity is driven by PPARγ by direct binding to at least two regions of the promoter in 293T and HepG2 cells. Moreover, siRNA-mediated suppression of MGAT1 expression significantly attenuated lipid accumulation by PPARγ overexpression in HepG2 cells, as evidenced by oil-red-O staining. These results suggest that human MGAT1 has an important role in fatty liver formation as a target gene of PPARγ, and blocking MGAT1 activity could be an efficient therapeutic way to reduce nonalcoholic fatty liver diseases in humans. - Highlights: • PPARγ promotes MGAT1 expression in human primary hepatocytes. • PPARγ directly regulates MGAT1 promoter activity. • Human MGAT1 promoter has at least two PPARγ-binding elements. • Inhibition of MGAT1 expression attenuates hepatic lipid accumulation in humans.« less

Comparison of trout hepatocytes and liver S9 fractions as in ...

EPA Pesticide Factsheets

Isolated hepatocytes and liver S9 fractions have been used to collect in vitro biotransformation data for fish as a means of improving modeled estimates of chemical bioaccumulation. To date, however, there have been few direct comparisons of these two methods. In the present study, cryopreserved trout hepatocytes were used to measure in vitro intrinsic clearance rates for 6 polycyclic aromatic hydrocarbons (PAHs). These rates were extrapolated to estimates of in vivo intrinsic clearance and used as inputs to a well-stirred liver model to predict hepatic clearance. Predicted rates of hepatic clearance were then evaluated by comparison to measured rates determined previously using isolated perfused livers. Hepatic clearance rates predicted using hepatocytes were in good agreement with measured values (< 2.1 fold difference for 5 of 6 compounds) under two competing binding assumptions. These findings, which may be attributed in part to high rates of PAH metabolism, are similar to those obtained previously using data from liver S9 fractions. For one compound (benzo[a]pyrene), the in vivo intrinsic clearance rate calculated using S9 data was 10-fold higher than that determined using hepatocytes, possibly due to a diffusion limitation on cellular uptake. Generally, however, there was good agreement between calculated in vivo intrinsic clearance rates obtained using either in vitro test system. These results suggest that both systems can be used to improve

The ultrastructural characteristics of porcine hepatocytes donated after cardiac death and preserved with warm machine perfusion preservation.

PubMed

Bochimoto, Hiroki; Matsuno, Naoto; Ishihara, Yo; Shonaka, Tatsuya; Koga, Daisuke; Hira, Yoshiki; Nishikawa, Yuji; Furukawa, Hiroyuki; Watanabe, Tsuyoshi

2017-01-01

The effects of warm machine perfusion preservation of liver grafts donated after cardiac death on the intracellular three-dimensional ultrastructure of the organelles in hepatocytes remain unclear. Here we analyzed comparatively the ultrastructure of the endomembrane systems in porcine hepatocytes under warm ischemia and successive hypothermic and midthermic machine perfusion preservation, a type of the warm machine perfusion. Porcine liver grafts which had a warm ischemia time of 60 minutes were perfused for 4 hours with modified University of Wisconsin gluconate solution. Group A grafts were preserved with hypothermic machine perfusion preservation at 8°C constantly for 4 hours. Group B grafts were preserved with rewarming up to 22°C by warm machine perfusion preservation for 4 hours. An analysis of hepatocytes after 60 minutes of warm ischemia by scanning electron microscope revealed the appearance of abnormal vacuoles and invagination of mitochondria. In the hepatocytes preserved by subsequent hypothermic machine perfusion preservation, strongly swollen mitochondria were observed. In contrast, the warm machine perfusion preservation could preserve the functional appearance of mitochondria in hepatocytes. Furthermore, abundant vacuoles and membranous structures sequestrating cellular organelles like autophagic vacuoles were frequently observed in hepatocytes after warm machine perfusion preservation. In conclusion, the ultrastructure of the endomembrane systems in the hepatocytes of liver grafts changed in accordance with the temperature conditions of machine perfusion preservation. In addition, temperature condition of the machine perfusion preservation may also affect the condition of the hepatic graft attributed to autophagy systems, and consequently alleviate the damage of the hepatocytes.

Micropatterned coculture of hepatocytes on electrospun fibers as a potential in vitro model for predictive drug metabolism.

PubMed

Liu, Yaowen; Wei, Jiaojun; Lu, Jinfu; Lei, Dongmei; Yan, Shili; Li, Xiaohong

2016-06-01

The liver is the major organ of importance to determine drug dispositions in the body, thus the development of hepatocyte culture systems is of great scientific and practical interests to provide reliable and predictable models for in vitro drug screening. In the current study, to address the challenges of a rapid function loss of primary hepatocytes, the coculture of hepatocytes with fibroblasts and endothelial cells (Hep-Fib-EC) was established on micropatterned fibrous scaffolds. Liver-specific functions, such as the albumin secretion and urea synthesis, were well maintained in the coculture system, accompanied by a rapid formation of multicellular hepatocyte spheroids. The activities of phase I (CYP3A11 and CYP2C9) and phase II enzymes indicated a gradual increase for cocultured hepatocytes, and a maximum level was achieved after 5 days and maintained throughout 15 days of culture. The metabolism testing on model drugs indicated that the scaled clearance rates for hepatocytes in the Hep-Fib-EC coculture system were significantly higher than those of other culture methods, and a linear regression analysis indicated good correlations between the observed data of rats and in vitro predicted values during 15 days of culture. In addition, the enzyme activities and drug clearance rates of hepatocytes in the Hep-Fib-EC coculture model experienced sensitive responsiveness to the inducers and inhibitors of metabolizing enzymes. These results demonstrated the feasibility of micropatterned coculture of hepatocytes as a potential in vitro testing model for the prediction of in vivo drug metabolism. Copyright © 2016 Elsevier B.V. All rights reserved.

Parasite-induced ER stress response in hepatocytes facilitates Plasmodium liver stage infection.

PubMed

Inácio, Patricia; Zuzarte-Luís, Vanessa; Ruivo, Margarida T G; Falkard, Brie; Nagaraj, Nagarjuna; Rooijers, Koos; Mann, Matthias; Mair, Gunnar; Fidock, David A; Mota, Maria M

2015-08-01

Upon infection of a mammalian host, Plasmodium parasites first replicate inside hepatocytes, generating thousands of new parasites. Although Plasmodium intra-hepatic development represents a substantial metabolic challenge to the host hepatocyte, how infected cells respond to and integrate this stress remains poorly understood. Here, we present proteomic and transcriptomic analyses, revealing that the endoplasmic reticulum (ER)-resident unfolded protein response (UPR) is activated in host hepatocytes upon Plasmodium berghei infection. The expression of XBP1s--the active form of the UPR mediator XBP1--and the liver-specific UPR mediator CREBH is induced by P. berghei infection in vivo. Furthermore, this UPR induction increases parasite liver burden. Altogether, our data suggest that ER stress is a central feature of P. berghei intra-hepatic development, contributing to the success of infection. © 2015 The Authors.

Hepatotoxin N-nitrosomorpholine-induced carcinogenesis in rat liver: ex vivo exploration of preneoplastic and neoplastic hepatocytes.

PubMed

Jeong, Jin Sook; Lee, Sang Hyeung; Jung, Kap Joong; Choi, Yong C; Park, Woong Yang; Kim, In Hoo; Kim, Sang Soon

2003-02-01

N-nitrosomorpholine (NNM) is a hepatotoxic and hepatocarcinogenic agent. This agent was administered in the form of drinking water which contained 200 mg of NNM/liter. Its time-dependent intake profile showed four phases over 20 weeks, followed by a fifth phase where only water was supplied. Most frequently, hepatocellular carcinoma appeared between the end of phase IV and the beginning of phase V. At 5 weeks of NNM administration, foci of altered hepatocytes (FAH) containing 100-1000 hepatocytes could be isolated together with free hepatocytes by the collagenase perfusion method. When these foci were grown on the William's Medium E containing hormonally defined medium, they were able to survive approximately twice as long as normal hepatocytes At 10 weeks of NNM administration, few FAH were isolated together with free hepatocytes. The hepatocytes which had been placed under extended chemical stress showed increased heat tolerance (7 to 8 h) at 43 degrees C, while normal hepatocytes could survive 3 to 4 h. At the neoplastic phase spanning the end of the 20 weeks of the NNM administration and water phase, the rats bearing hepatocellular carcinoma entered the terminal stage, where observable tumor masses could be isolated from the tumor bearing liver and tested for ex vivo growth in tissue culture. After stabilization of the isolated primary hepatoma cells through 10 passages of propagation on William's Medium E or minimal Eagle's medium containing 10% FBS, their gene expression profile was analyzed by DNA microarray and compared with the profile of normal hepatocytes. The comparison revealed that upregulation involved ribosome-dependent protein synthesis, including 40S ribosomal proteins (S4, S7, S18, S20), 60S ribosomal proteins (L6, L21, L32, L37, P1), initiation factor 4A, and elongation factor 1alpha.

[Changes in the chromatin structure of hepatocyte nuclei of rats trained to hypoxia].

PubMed

Domkina, L K; Bresler, V M; Simanovskiĭ, L N

1976-03-01

Structure of chromatin in the nuclei of the isolated surviving hepatocytes and in the isolated nuclei of hepatocytes were studied by fluorochroming with acridine orange and by microfluorimetry of fluorescenc connected with the stain chromatin at 530 and 590 nm in intact rats and in the animals trained to hypoxia in a pressure chamber for 60 days. The nuclei of hepatocytes of intact rats were distributed by fluorescence at 530 nm into three classes with the intensity ratio of 1:2:4; as to the nuclei of hepatocytes of the rats trained to hypoxia - they formed a single class corresponding to the second class of control. In intact rats the ratio of the fluorescence intensity at 590 nm to such at 530 nm (alpha coefficient) formed normal distribution; in trained rats - a bimodal distribution with a shift of the maximum in the direction of reduction and increase of alpha in comparison with control. It is supposed that in hypoxia there is a repression of one and depression of other genes in the chromatine of the nuclei of the liver.

Quantifying radionuclide signatures from a γ-γ coincidence system.

PubMed

Britton, Richard; Jackson, Mark J; Davies, Ashley V

2015-11-01

A method for quantifying gamma coincidence signatures has been developed, and tested in conjunction with a high-efficiency multi-detector system to quickly identify trace amounts of radioactive material. The γ-γ system utilises fully digital electronics and list-mode acquisition to time-stamp each event, allowing coincidence matrices to be easily produced alongside typical 'singles' spectra. To quantify the coincidence signatures a software package has been developed to calculate efficiency and cascade summing corrected branching ratios. This utilises ENSDF records as an input, and can be fully automated, allowing the user to quickly and easily create/update a coincidence library that contains all possible γ and conversion electron cascades, associated cascade emission probabilities, and true-coincidence summing corrected γ cascade detection probabilities. It is also fully searchable by energy, nuclide, coincidence pair, γ multiplicity, cascade probability and half-life of the cascade. The probabilities calculated were tested using measurements performed on the γ-γ system, and found to provide accurate results for the nuclides investigated. Given the flexibility of the method, (it only relies on evaluated nuclear data, and accurate efficiency characterisations), the software can now be utilised for a variety of systems, quickly and easily calculating coincidence signature probabilities. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Attenuation of alcohol-induced apoptosis of hepatocytes in rat livers by polyenylphosphatidylcholine (PPC).

PubMed

Mi, L J; Mak, K M; Lieber, C S

2000-02-01

Alcohol consumption increases apoptosis of hepatocytes. This effect appears to be mediated by the induction of hepatic cytochrome P-4502E1(CYP2E1) and its generation of free radicals, which results in an enhanced lipid peroxidation that initiates apoptosis. Because polyenylphosphatidylcholine (PPC), a soybean extract rich in polyunsaturated phosphatidylcholines, decreases the induction of ethanol-specific CYP2E1 and opposes oxidative stress, we hypothesized that PPC supplementation may attenuate hepatocyte apoptosis caused by ethanol ingestion. Twenty-eight male Sprague Dawley rats were pair-fed Lieber-DeCarli liquid diets containing 36% of energy as alcohol or an isocaloric amount of carbohydrate for 28 days. Half of the rats were given PPC (3 g/liter), whereas the other half received the same amount of linoleate (as safflower oil) and of choline as the bitartrate. An additional dose of alcohol (3 g/kg) was given intragastrically 90 min before the livers were removed. We assessed apoptosis in formalin-fixed, paraffin-embedded liver sections by using the TUNEL (terminal transferase dUTP nick end labeling) assay. Apoptotic hepatocytes were identified by positive TUNEL staining in conjunction with condensation of nucleoplasm or margination of chromatin. In each rat, 20,000 to 60,000 hepatocytes were counted by light microscopy by using Image-Pro Plus computer software, and the incidence of apoptosis was expressed as the percentage of total hepatocytes. Alcohol feeding resulted in a 4.5-fold increase in apoptosis of hepatocytes compared to pair-fed control rats; PPC supplementation decreased the alcohol-induced apoptosis to less than half. No difference in the incidence of apoptosis between the control and PPC-supplemented rats was found in the absence of alcohol. Apoptosis was distributed randomly in the liver lobules of the rats fed the control diet, whereas the alcohol-induced apoptosis was significantly increased in the perivenular area. PPC supplementation

Identification of Novel Rosavirus Species That Infects Diverse Rodent Species and Causes Multisystemic Dissemination in Mouse Model

PubMed Central

Fan, Rachel Y. Y.; Zhang, Anna J. X.; Chan, Brandon C. C.; Lam, Carol S. F.; Yip, Cyril C. Y.; Chan, Kwok-Hung; Chen, Zhi-Wei; Yuen, Kwok-Yung

2016-01-01

While novel picornaviruses are being discovered in rodents, their host range and pathogenicity are largely unknown. We identified two novel picornaviruses, rosavirus B from the street rat, Norway rat, and rosavirus C from five different wild rat species (chestnut spiny rat, greater bandicoot rat, Indochinese forest rat, roof rat and Coxing's white-bellied rat) in China. Analysis of 13 complete genome sequences showed that “Rosavirus B” and “Rosavirus C” represent two potentially novel picornavirus species infecting different rodents. Though being most closely related to rosavirus A, rosavirus B and C possessed distinct protease cleavage sites and variations in Yn-Xm-AUG sequence in 5’UTR and myristylation site in VP4. Anti-rosavirus B VP1 antibodies were detected in Norway rats, whereas anti-rosavirus C VP1 and neutralizing antibodies were detected in Indochinese forest rats and Coxing's white-bellied rats. While the highest prevalence was observed in Coxing's white-bellied rats by RT-PCR, the detection of rosavirus C from different rat species suggests potential interspecies transmission. Rosavirus C isolated from 3T3 cells causes multisystemic diseases in a mouse model, with high viral loads and positive viral antigen expression in organs of infected mice after oral or intracerebral inoculation. Histological examination revealed alveolar fluid exudation, interstitial infiltration, alveolar fluid exudate and wall thickening in lungs, and hepatocyte degeneration and lymphocytic/monocytic inflammatory infiltrates with giant cell formation in liver sections of sacrificed mice. Since rosavirus A2 has been detected in fecal samples of children, further studies should elucidate the pathogenicity and emergence potential of different rosaviruses. PMID:27737017

Construction of the Database of Rat Repeated-dose Toxicity Tests of Pesticides for the Toxicological Characterization of Hepatocyte Hypertrophy.

PubMed

Masuda, Akane; Masuda, Miyabi; Kawano, Takuya; Kitsunai, Yoko; Nakayama, Haruka; Nakajima, Hiroyuki; Kojima, Hiroyuki; Kitamura, Shigeyuki; Uramaru, Naoto; Hosaka, Takuomi; Sasaki, Takamitsu; Yoshinari, Kouichi

2017-01-01

Liver and hepatocyte hypertrophy can be induced by exposure to chemical compounds, but the mechanisms and toxicological characteristics of these phenomena have not yet been investigated extensively. In particular, it remains unclear whether the hepatocyte hypertrophy induced by chemical compounds should be judged as an adaptive response or an adverse effect. Thus, understanding of the toxicological characteristics of hepatocyte hypertrophy is of great importance to the safety evaluation of pesticides and other chemical compounds. To this end, we have constructed a database of potentially toxic pesticides. Using risk assessment reports of pesticides that are publicly available from the Food Safety Commission of Japan, we extracted all observations/findings that were based on 90-day subacute toxicity tests and 2-year chronic toxicity and carcinogenicity tests in rats. Analysis of the database revealed that hepatocyte hypertrophy was observed for 37-47% of the pesticides investigated (varying depending on sex and testing period), and that centrilobular hepatocyte hypertrophy was the most frequent among the various types of hepatocyte hypertrophy in both the 90-day and 2-year studies. The database constructed in this study enables us to investigate the relationships between hepatocyte hypertrophy and other toxicological observations/findings, and thus will be useful for characterizing hepatocyte hypertrophy.

ASGPR-Mediated Uptake of Multivalent Glycoconjugates for Drug Delivery in Hepatocytes.

PubMed

Monestier, Marie; Charbonnier, Peggy; Gateau, Christelle; Cuillel, Martine; Robert, Faustine; Lebrun, Colette; Mintz, Elisabeth; Renaudet, Olivier; Delangle, Pascale

2016-04-01

Liver cells are an essential target for drug delivery in many diseases. The hepatocytes express the asialoglycoprotein receptor (ASGPR), which promotes specific uptake by means of N-acetylgalactosamine (GalNAc) recognition. In this work, we designed two different chemical architectures to treat Wilson's disease by intracellular copper chelation. Two glycoconjugates functionalized with three or four GalNAc units each were shown to enter hepatic cells and chelate copper. Here, we studied two series of compounds derived from these glycoconjugates to find key parameters for the targeting of human hepatocytes. Efficient cellular uptake was demonstrated by flow cytometry using HepG2 human heptic cells that express the human oligomeric ASGPR. Dissociation constants in the nanomolar range showed efficient multivalent interactions with the receptor. Both architectures were therefore concluded to be able to compete with endogeneous asialoglycoproteins and serve as good vehicles for drug delivery in hepatocytes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The effect of phenformin on insulin-stimulated isoaminobutyric acid (AIB) transport by isolated hepatocytes.

PubMed Central

Woods, R. J.; Dandona, P.

1984-01-01

Hepatocytes isolated from adult rat livers by collagenase perfusion were used to investigate the effect of phenformin on amino acid transport by measuring the uptake of aminoisobutyric acid (AIB). At concentrations between 0.01 and 100 micrograms/ml, phenformin hydrochloride did not affect AIB uptake, but concentrations of 500 and 1000 micrograms/ml were inhibitory. Incubating hepatocytes with insulin increased their accumulation of AIB. Inclusion of up to 100 micrograms/ml, phenformin hydrochloride during maximal and submaximal stimulation by insulin did not affect AIB uptake, but inhibition occurred with 500 and 1000 micrograms/ml. The continued presence of phenformin after addition of AIB was not required in order to produce inhibition. Furthermore, increasing the duration of exposure of hepatocytes to phenformin increased the degree of inhibition, suggesting that it is a non-competitive inhibitor. We conclude that phenformin does not enhance the sensitivity of hepatocytes to insulin and that at higher concentrations it may exert an inhibitory effect on basal and insulin stimulated aminoacid transport. PMID:6370292

Measurement of the low energy spectral contribution in coincidence with valence band (VB) energy levels of Ag(100) using VB-VB coincidence spectroscopy

NASA Astrophysics Data System (ADS)

Gladen, R. W.; Joglekar, P. V.; Lim, Z. H.; Shastry, K.; Hulbert, S. L.; Weiss, A. H.

A set of coincidence measurements were obtained for the study and measurement of the electron contribution arising from the inter-valence band (VB) transitions along with the inelastically scattered VB electron contribution. These Auger-unrelated contributions arise in the Auger spectrum (Ag 4p NVV) obtained using Auger Photoelectron Coincidence Spectroscopy (APECS). The measured Auger-unrelated contribution can be eliminated from Auger spectrum to obtain the spectrum related to Auger. In our VB-VB coincidence measurement, a photon beam of energy 180eV was used to probe the Ag(100) sample. The coincidence spectrum was obtained using two Cylindrical Mirror Analyzers (CMA's). The scan CMA measured the low energy electron contribution in the energy range 0-70eV in coincidence with VB electrons measured by the fixed CMA. In this talk, we present the data obtained for VB-VB coincidence at the valence band energy of 171eV along with the coincidence measurements in the energy range of 4p core and valence band. NSF DMR 0907679, NSF Award Number: 1213727. Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the U.S. DOE, Office of Science, Office of Basic Energy Sciences, under Contract No. DEAC02-98CH10886.

Measurement of the low energy spectral contribution in coincidence with valence band (VB) energy levels of Ag(100) using VB-VB coincidence spectroscopy

NASA Astrophysics Data System (ADS)

Joglekar, P. V.; Gladen, R.; Lim, Z. H.; Shastry, K.; Hulbert, S. L.; Weiss, A. H.

2015-03-01

A set of coincidence measurements were obtained for the study and measurement of the electron contribution arising from the inter-valence band (VB) transitions along with the inelastically scattered VB electron contribution. These Auger-unrelated contributions arise in the Auger spectrum (Ag 4p NVV) obtained using Auger Photoelectron Coincidence Spectroscopy (APECS). The measured Auger-unrelated contribution can be eliminated from Auger spectrum to obtain the spectrum related to Auger. In our VB-VB coincidence measurement, a photon beam of energy 180eV was used to probe the Ag(100) sample. The coincidence spectrum was obtained using two Cylindrical Mirror Analyzers (CMA's). The scan CMA measured the low energy electron contribution in the energy range 0-70eV in coincidence with VB electrons measured by the fixed CMA. In this talk, we present the data obtained for VB-VB coincidence at the valence band energy of 171eV along with the coincidence measurements in the energy range of 4p core and valence band. NSF DMR 0907679, NSF Award Number: 1213727. Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the U.S. DOE, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886.

Human hepatocytes derived from pluripotent stem cells: a promising cell model for drug hepatotoxicity screening.

PubMed

Gómez-Lechón, María José; Tolosa, Laia

2016-09-01

Drug-induced liver injury (DILI) is a frequent cause of failure in both clinical and post-approval stages of drug development, and poses a key challenge to the pharmaceutical industry. Current animal models offer poor prediction of human DILI. Although several human cell-based models have been proposed for the detection of human DILI, human primary hepatocytes remain the gold standard for preclinical toxicological screening. However, their use is hindered by their limited availability, variability and phenotypic instability. In contrast, pluripotent stem cells, which include embryonic and induced pluripotent stem cells (iPSCs), proliferate extensively in vitro and can be differentiated into hepatocytes by the addition of soluble factors. This provides a stable source of hepatocytes for multiple applications, including early preclinical hepatotoxicity screening. In addition, iPSCs also have the potential to establish genotype-specific cells from different individuals, which would increase the predictivity of toxicity assays allowing more successful clinical trials. Therefore, the generation of human hepatocyte-like cells derived from pluripotent stem cells seems to be promising for overcoming limitations of hepatocyte preparations, and it is expected to have a substantial repercussion in preclinical hepatotoxicity risk assessment in early drug development stages.

Menstrual blood-derived mesenchymal stem cells differentiate into functional hepatocyte-like cells*

PubMed Central

Mou, Xiao-zhou; Lin, Jian; Chen, Jin-yang; Li, Yi-fei; Wu, Xiao-xing; Xiang, Bing-yu; Li, Cai-yun; Ma, Ju-ming; Xiang, Charlie

2013-01-01

Orthotopic liver transplantation (OLT) is the only proven effective treatment for both end-stage and metabolic liver diseases. Hepatocyte transplantation is a promising alternative for OLT, but the lack of available donor livers has hampered its clinical application. Hepatocyte-like cells (HLCs) differentiated from many multi-potential stem cells can help repair damaged liver tissue. Yet almost suitable cells currently identified for human use are difficult to harvest and involve invasive procedures. Recently, a novel mesenchymal stem cell derived from human menstrual blood (MenSC) has been discovered and obtained easily and repeatedly. In this study, we examined whether the MenSCs are able to differentiate into functional HLCs in vitro. After three weeks of incubation in hepatogenic differentiation medium containing hepatocyte growth factor (HGF), fibroblast growth factor-4 (FGF-4), and oncostain M (OSM), cuboidal HLCs were observed, and cells also expressed hepatocyte-specific marker genes including albumin (ALB), α-fetoprotein (AFP), cytokeratin 18/19 (CK18/19), and cytochrome P450 1A1/3A4 (CYP1A1/3A4). Differentiated cells further demonstrated in vitro mature hepatocyte functions such as urea synthesis, glycogen storage, and indocyanine green (ICG) uptake. After intrasplenic transplantation into mice with 2/3 partial hepatectomy, the MenSC-derived HLCs were detected in recipient livers and expressed human ALB protein. We also showed that MenSC-derived HLC transplantation could restore the serum ALB level and significantly suppressed transaminase activity of liver injury animals. In conclusion, MenSCs may serve as an ideal, easily accessible source of material for tissue engineering and cell therapy of liver tissues. PMID:24190442

Selective Toxicity of Apigenin on Cancerous Hepatocytes by Directly Targeting their Mitochondria.

PubMed

Seydi, Enayatollah; Rasekh, Hamid R; Salimi, Ahmad; Mohsenifar, Zhaleh; Pourahmad, Jalal

2016-01-01

hepatocellular carcinoma (HCC) is the third cause of mortality due to cancer throughout the world. The main goal of the current research was to evaluate the selective toxicity of apigenin (APG) on hepatocytes and mitochondria obtained from the liver of HCC rats). In this research, HCC induced by a single dose of diethylnitrosamine (DEN); 200 mg/kg, i.p, and 2-acetylaminofluorene (2-AAF) (0.02%, through dietary) for 14 days. For confirmation of HCC, histopathological evaluations and determination of serum concentrations of liver toxicity enzymes and specific liver cancer marker; alpha-fetoprotein (AFP) were performed. Then, cancerous and non- cancerous hepatocytes were isolated by using the collagen perfusion method. Eventually, mitochondria isolated from HCC and normal hepatocytes were tested for every eventual toxic effects of APG. After confirmation of HCC, the results of this research showed that APG (10, 20 and 40 μM) increased mitochondrial parameters such as, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) level, mitochondrial swelling and cytochrome c expulsion only in cancerous hepatocytes. Apoptotic effect of APG on HCC cells was confirmed by caspase-3 activation and Annexin V-FITC and PI double staining analysis. These results propose the eligibility of the flavonoid APG as a complementary therapeutic agent for patients with hepatocellular carcinoma.

Ferritin expression in rat hepatocytes and Kupffer cells after lead nitrate treatment.

PubMed

Fan, Yang; Yamada, Toshiyuki; Shimizu, Takeshi; Nanashima, Naoki; Akita, Miki; Suto, Kohji; Tsuchida, Shigeki

2009-02-01

Lead nitrate induces hepatocyte proliferation and subsequent apoptosis in rat livers. Iron is a constituent of heme and is also required for cell proliferation. In this study, the expression of ferritin light-chain (FTL), the major iron storage protein, was investigated in rat livers after a single intravenous injection of lead nitrate. Western blotting and immunohistochemistry revealed that FTL was increased in hepatocytes around the central veins and strongly expressed in nonparenchymal cells. Some FTL-positive nonparenchymal cells were identified as Kupffer cells that were positive for CD68. FTL-positive Kupffer cells occupied about 60% of CD68-positive cells in the periportal and perivenous areas. The relationships between FTL expression and apoptosis induction or the engulfment of apoptotic cells were examined. TUNEL-positive cells were increased in the treatment group, and enhanced expression of milk fat globule EGF-like 8 was demonstrated in some Kupffer cells and hepatocytes, indicating enhanced apoptosis induction and phagocytosis of apoptotic cells. FTL-positive Kupffer cells were not detected without lead nitrate treatment or in rat livers treated with clofibrate, which induces hepatocyte proliferation but not apoptosis. These results suggest that FTL expression in Kupffer cells after lead treatment is dependent on phagocytosis of apoptotic cells.

Experimental encephalomyocarditis virus infection in small laboratory rodents.

PubMed

Doi, K

2011-01-01

Encephalomyocarditis virus (EMCV) is a cardiovirus that belongs to the family Picornaviridae. EMCV is an important cause of acute myocarditis in piglets and of fetal death or abortion in pregnant sows. Small rodents, especially rats, have been suspected to be reservoir hosts or carriers. This virus also induces type 1 diabetes mellitus, encephalomyelitis, myocarditis, orchitis and/or sialodacryoadenitis in small laboratory rodents. This paper reviews the pathology and pathogenesis of experimental infection with EMCV in small laboratory rodents. Copyright © 2010 Elsevier Ltd. All rights reserved.

Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from Kupffer cells.

PubMed

Prill, Jan-Michael; Espenlaub, Sigrid; Samen, Ulrike; Engler, Tatjana; Schmidt, Erika; Vetrini, Francesco; Rosewell, Amanda; Grove, Nathan; Palmer, Donna; Ng, Philip; Kochanek, Stefan; Kreppel, Florian

2011-01-01

In vivo gene transfer with adenovirus vectors would significantly benefit from a tight control of the adenovirus-inherent liver tropism. For efficient hepatocyte transduction, adenovirus vectors need to evade from Kupffer cell scavenging while delivery to peripheral tissues or tumors could be improved if both scavenging by Kupffer cells and uptake by hepatocytes were blocked. Here, we provide evidence that a single point mutation in the hexon capsomere designed to enable defined chemical capsid modifications may permit both detargeting from and targeting to hepatocytes with evasion from Kupffer cell scavenging. Vector particles modified with small polyethylene glycol (PEG) moieties specifically on hexon exhibited decreased transduction of hepatocytes by shielding from blood coagulation factor binding. Vector particles modified with transferrin or, surprisingly, 5,000 Da PEG or dextran increased hepatocyte transduction up to 18-fold independent of the presence of Kupffer cells. We further show that our strategy can be used to target high-capacity adenovirus vectors to hepatocytes emphasizing the potential for therapeutic liver-directed gene transfer. Our approach may lead to a detailed understanding of the interactions between adenovirus vectors and Kupffer cells, one of the most important barriers for adenovirus-mediated gene delivery.

MicroRNA-21 accelerates hepatocyte proliferation in vitro via PI3K/Akt signaling by targeting PTEN

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan-nan, Bai; Department of Hepatobiliary Surgery, Fujian Provincial Hospital, Provincial Clinical College of Fujian Medical University, Fuzhou 350001, Fujian Province; Zhao-yan, Yu

2014-01-17

Highlights: •miRNAs-expression patterns of primary hepatocytes under proliferative status. •miR-21 expression level peaked at 12 h after stimulated by EGF. •miR-21 drive rapid S phase entry of primary hepatocytes. •PI3K/Akt signaling was modulated via targeting PTEN by miR-21. -- Abstract: MicroRNAs (miRNAs) are involved in controlling hepatocyte proliferation during liver regeneration. In this study, we established the miRNAs-expression patterns of primary hepatocytes in vitro under stimulation of epidermal growth factor (EGF), and found that microRNA-21 (miR-21) was appreciably up-regulated and peaked at 12 h. In addition, we further presented evidences indicating that miR-21 promotes primary hepatocyte proliferation through in vitromore » transfecting with miR-21 mimics or inhibitor. We further demonstrated that phosphatidylinositol 3′-OH kinase (PI3K)/Akt signaling was altered accordingly, it is, by targeting phosphatase and tensin homologue deleted on chromosome 10, PI3K/Akt signaling is activated by miR-21 to accelerate hepatocyte rapid S-phase entry and proliferation in vitro.« less

Parasite-induced ER stress response in hepatocytes facilitates Plasmodium liver stage infection

PubMed Central

Inácio, Patricia; Zuzarte-Luís, Vanessa; Ruivo, Margarida TG; Falkard, Brie; Nagaraj, Nagarjuna; Rooijers, Koos; Mann, Matthias; Mair, Gunnar; Fidock, David A; Mota, Maria M

2015-01-01

Upon infection of a mammalian host, Plasmodium parasites first replicate inside hepatocytes, generating thousands of new parasites. Although Plasmodium intra-hepatic development represents a substantial metabolic challenge to the host hepatocyte, how infected cells respond to and integrate this stress remains poorly understood. Here, we present proteomic and transcriptomic analyses, revealing that the endoplasmic reticulum (ER)-resident unfolded protein response (UPR) is activated in host hepatocytes upon Plasmodium berghei infection. The expression of XBP1s—the active form of the UPR mediator XBP1—and the liver-specific UPR mediator CREBH is induced by P. berghei infection in vivo. Furthermore, this UPR induction increases parasite liver burden. Altogether, our data suggest that ER stress is a central feature of P. berghei intra-hepatic development, contributing to the success of infection. PMID:26113366

T lymphocytes from mice immunized with irradiated sporozoites eliminated malaria from hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoffman, S.L.; Isenbarger, D.; Long, G.W.

When mice are immunized with radiation-attenuated sporozoites they are solidly protected against sporozoite challenge by an immune response that has been shown to require CD8+ lymphocytes in several strains of mice. The target of this CD8+ T-cell-dependent immunity has not been established. Immune BALB/c mice were shown to develop malaria-specific. CD8+ T-cell-dependent inflammatory infiltrates in their livers after challenge with Plasmodium berghei sporozoites. Spleen cells from immune BALB/c and C57BL/6 mice eliminated hepatocytes infected with the liver stage of P. berghei in vitro. The activity against infected hepatocytes is not inhibited by antibodies to interferon-y and is not present inmore » culture supernatants. It is generally restricted, an indication that malaria antigens on the hepatocyte surface are recognized by immune T-effector cells.« less

Emmonsiosis of rodents in an agroecosystem.

PubMed

Hubálek, Z; Nesvadbová, J; Halouzka, J

1998-12-01

Adiaspores of Emmonsia crescens were found in the lungs of 62.1% of 87 adult rodents from 10 windbreaks compared to only 8.2% of 184 adult rodents caught in 10 adjacent arable fields in South Moravia, Czechland. A significantly higher mean weight proportion of plant remnants (predominantly small roots) was present in the soil from windbreaks (0.74%) than from fields (0.24%). In addition, mean relative abundance of rodents was significantly higher in windbreaks than in fields, and the windbreak soil was more acidic (pH 6.2 vs. 6.9, respectively). On the other hand, there was no significant difference in the water content (17.4% vs. 18.5%, respectively). The difference in the infection rate between the two habitats of the agroecosystem could be caused or affected by land use and farming technology.

Enamel mineral concentration in diabetic rodents.

PubMed

Atar, M; Davis, G R; Verry, P; Wong, F S L

2007-12-01

This was to use x-ray microtomography (XMT), to assess the mineral composition and 3-D structure of enamel and bone in the teeth and skulls of diabetic rodents. Three-dimensional images of the skull were reconstructed using computer generated false colour to highlight different levels of mineralization in bone and enamel. These showed that diabetic rodents exhibited more wear in their teeth. Deformities were observed in the alveolar process of the mandible and maxilla. Regions of extensive hypomineralization were found in the calvarial bone of skulls. The maximum mineral concentrations and the time constants for diabetic rodents were similar to the controls. The diabetic mice appeared to have random regions of hypomineralization and one diabetic rat had areas of hypoplasia in the mandibular incisors. Diabetes may have a detrimental influence on the function of ameloblasts in laying down enamel.

Resveratrol Differentially Regulates NAMPT and SIRT1 in Hepatocarcinoma Cells and Primary Human Hepatocytes

PubMed Central

Schuster, Susanne; Penke, Melanie; Gorski, Theresa; Petzold-Quinque, Stefanie; Damm, Georg; Gebhardt, Rolf; Kiess, Wieland; Garten, Antje

2014-01-01

Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells. PMID:24603648

Can a native rodent species limit the invasive potential of a non-native rodent species in tropical agroforest habitats?

PubMed

Stuart, Alexander M; Prescott, Colin V; Singleton, Grant R

2016-06-01

Little is known about native and non-native rodent species interactions in complex tropical agroecosystems. We hypothesised that the native non-pest rodent Rattus everetti may be competitively dominant over the invasive pest rodent Rattus tanezumi within agroforests. We tested this experimentally by using pulse removal for three consecutive months to reduce populations of R. everetti in agroforest habitat, and assessed over 6 months the response of R. tanezumi and other rodent species. Following removal, R. everetti individuals rapidly immigrated into removal sites. At the end of the study period, R. tanezumi were larger and there was a significant shift in their microhabitat use with respect to the use of ground vegetation cover following the perturbation of R. everetti. Irrespective of treatment, R. tanezumi selected microhabitat with less tree canopy cover, indicative of severely disturbed habitat, whereas R. everetti selected microhabitat with a dense canopy. Our results suggest that sustained habitat disturbance in agroforests favours R. tanezumi, while the regeneration of agroforests towards a more natural state would favour native species and may reduce pest pressure in adjacent crops. In addition, the rapid recolonisation of R. everetti suggests this species would be able to recover from non-target impacts of short-term rodent pest control. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Mir-24 regulates hepatocyte apoptosis via BIM during acute liver failure.

PubMed

Feng, Zhiwen; Li, Zhi; Zhu, Deming; Ling, Wei; Zheng, Lei; Pu, Liyong; Kong, Lianbao

2017-01-01

Acuteliver failure (ALF) has a high mortality rate and is characterized by massive hepatocyte destruction. Although microRNAs (miRNAs) play an important role in manyliver diseases, the role of miRNAs in ALF development is unknown. In this study, the murine ALF model was induced by intraperitoneal injection of D-galactosamine/lipopolysaccharide (D-GalN/LPS). Compared with saline-treated mice, miR-24 was distinctly down-regulated post D-GalN/LPS challenge in vivo and D-galactosamine/tumor necrosis factor (D-GalN/TNF) challenge in vitro , which was confirmed by quantitative real-time polymerase chain reaction. Meanwhile, the mRNA and protein levels of the BH3-only-domain-containing protein BIM were upregulated after challenge both in vivo and in vitro . Previous studies have demonstrated that hepatocyte apoptosis is a distinguishing feature of D-GalN/LPS-associated liver failure. In this study, D-GalN/LPS-challenged mice showed higher alanine aminotransferase and aspartate aminotransferase levels, more severe liver damage, increased numbers of apoptotic hepatocytes and higher levels of caspase-3 compared with saline-treated mice. In D-GalN/TNF-treated BNLCL2 cells, miR-24 overexpression attenuated apoptosis.Furthermore, miR-24 overexpression reduced BIM mRNA and protein levels in vitro . Taken together, these findings demonstrate that miR-24 regulates hepatocyte apoptosis via BIM during ALF development, suggesting that miR-24 is a novel onco-miRNA that may provide potential therapeutic targets for ALF.

Magentic Cell labeling of primary and stem cell-derived pig hepatocytes for MRI-based cell tracking of heptocytes transplantation

USDA-ARS?s Scientific Manuscript database

Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this wo...

Methionine sulfoxide reductase A protects hepatocytes against acetaminophen-induced toxicity via regulation of thioredoxin reductase 1 expression.

PubMed

Singh, Mahendra Pratap; Kwak, Geun-Hee; Kim, Ki Young; Kim, Hwa-Young

2017-06-03

Thioredoxin reductase 1 (TXNRD1) is associated with susceptibility to acetaminophen (APAP)-induced liver damage. Methionine sulfoxide reductase A (MsrA) is an antioxidant and protein repair enzyme that specifically catalyzes the reduction of methionine S-sulfoxide residues. We have previously shown that MsrA deficiency exacerbates acute liver injury induced by APAP. In this study, we used primary hepatocytes to investigate the underlying mechanism of the protective effect of MsrA against APAP-induced hepatotoxicity. MsrA gene-deleted (MsrA -/- ) hepatocytes showed higher susceptibility to APAP-induced cytotoxicity than wild-type (MsrA +/+ ) cells, consistent with our previous in vivo results. MsrA deficiency increased APAP-induced glutathione depletion and reactive oxygen species production. APAP treatment increased Nrf2 activation more profoundly in MsrA -/- than in MsrA +/+ hepatocytes. Basal TXNRD1 levels were significantly higher in MsrA -/- than in MsrA +/+ hepatocytes, while TXNRD1 depletion in both MsrA -/- and MsrA +/+ cells resulted in increased resistance to APAP-induced cytotoxicity. In addition, APAP treatment significantly increased TXNRD1 expression in MsrA -/- hepatocytes, while no significant change was observed in MsrA +/+ cells. Overexpression of MsrA reduced APAP-induced cytotoxicity and TXNRD1 expression levels in APAP-treated MsrA -/- hepatocytes. Collectively, our results suggest that MsrA protects hepatocytes from APAP-induced cytotoxicity through the modulation of TXNRD1 expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Testing the limits of Rodent Sperm Analysis: azoospermia in an otherwise healthy wild rodent population.

PubMed

Tannenbaum, Lawrence V; Thran, Brandolyn H; Willams, Keith J

2009-01-01

By comparing the sperm parameters of small rodents trapped at contaminated terrestrial sites and nearby habitat-matched noncontaminated locations, the patent-pending Rodent Sperm Analysis (RSA) method provides a direct health status appraisal for the maximally chemical-exposed mammalian ecological receptor in the wild. RSA outcomes have consistently allowed for as definitive determinations of receptor health as are possible at the present time, thereby streamlining the ecological risk assessment (ERA) process. Here, we describe the unanticipated discovery, at a contaminated US EPA Superfund National Priorities List site, of a population of Hispid cotton rats (Sigmodon hispidus), with a high percentage of adult males lacking sperm entirely (azoospermia). In light of the RSA method's role in streamlining ERAs and in bringing contaminated Superfund-type site investigations to closure, we consider the consequences of the discovery. The two matters specifically discussed are (1) the computation of a population's average sperm count where azoospermia is present and (2) the merits of the RSA method and its sperm parameter thresholds-for-effect when azoospermia is masked in an otherwise apparently healthy rodent population.

Stimulation of fibrinogen synthesis in cultured rat hepatocytes by fibrinogen degradation product fragment D

DOE Office of Scientific and Technical Information (OSTI.GOV)

LaDuca, F.M.; Tinsley, L.A.; Dang, C.V.

The direct stimulation of fibrinogen biosynthesis by fibrinogen degradation produces (FDPs) was studied in rat hepatocyte cultures. Pure rat FDP fragment D (FDP-D) (Mr 90,000) and FDP fragment E (FDP-E) (Mr 40,000) and mixtures of the two (FDP-DE) were added to rat hepatocytes cultured in serum-free hormonally defined medium. Hydrocortisone (20 microM) significantly increased synthesis of fibrinogen, as determined by incorporation of (35S)methionine. FDP-D and FDP-E did not increase fibrinogen synthesis in the presence of hydrocortisone. However, hepatocytes cultured without hydrocortisone displayed increased fibrinogen synthesis (2.0- to 2.8-fold) with FDP-D (2.6-6.7 microM) but not with FDP-E (5.7 microM). At thesemore » FDP concentrations the synthesis of albumin, haptoglobin, and transferrin was not increased. FDP-D-induced fibrinogen synthesis was inhibited (greater than 90%) by actinomycin D and cycloheximide, indicating that the increase in (35S)methionine incorporation was from de novo protein synthesis. The role of FDP-D was further substantiated by showing that FDP-D, but not FDP-E, bound to the hepatocytes. These data indicate that FDP-D, but not FDP-E, directly and specifically stimulates fibrinogen synthesis in rat hepatocytes; this stimulation does not require any additional serum or protein cofactors.« less

Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures.

PubMed

Kim, Yohan; Kang, Kyojin; Yoon, Sangtae; Kim, Ji Sook; Park, Su A; Kim, Wan Doo; Lee, Seung Bum; Ryu, Ki-Young; Jeong, Jaemin; Choi, Dongho

2018-01-02

Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.

Competition between harvester ants and rodents in the cold desert

DOE Office of Scientific and Technical Information (OSTI.GOV)

Landeen, D.S.; Jorgensen, C.D.; Smith, H.D.

1979-09-30

Local distribution patterns of three rodent species (Perognathus parvus, Peromyscus maniculatus, Reithrodontomys megalotis) were studied in areas of high and low densities of harvester ants (Pogonomyrmex owyheei) in Raft River Valley, Idaho. Numbers of rodents were greatest in areas of high ant-density during May, but partially reduced in August; whereas, the trend was reversed in areas of low ant-density. Seed abundance was probably not the factor limiting changes in rodent populations, because seed densities of annual plants were always greater in areas of high ant-density. Differences in seasonal population distributions of rodents between areas of high and low ant-densities weremore » probably due to interactions of seed availability, rodent energetics, and predation.« less

Old World Hantaviruses in Rodents in New Orleans, Louisiana

PubMed Central

Cross, Robert W.; Waffa, Bradley; Freeman, Ashley; Riegel, Claudia; Moses, Lina M.; Bennett, Andrew; Safronetz, David; Fischer, Elizabeth R.; Feldmann, Heinz; Voss, Thomas G.; Bausch, Daniel G.

2014-01-01

Seoul virus, an Old World hantavirus, is maintained in brown rats and causes a mild form of hemorrhagic fever with renal syndrome (HFRS) in humans. We captured rodents in New Orleans, Louisiana and tested them for the presence of Old World hantaviruses by reverse transcription polymerase chain reaction (RT-PCR) with sequencing, cell culture, and electron microscopy; 6 (3.4%) of 178 rodents captured—all brown rats—were positive for a Seoul virus variant previously coined Tchoupitoulas virus, which was noted in rodents in New Orleans in the 1980s. The finding of Tchoupitoulas virus in New Orleans over 25 years since its first discovery suggests stable endemicity in the city. Although the degree to which this virus causes human infection and disease remains unknown, repeated demonstration of Seoul virus in rodent populations, recent cases of laboratory-confirmed HFRS in some US cities, and a possible link with hypertensive renal disease warrant additional investigation in both rodents and humans. PMID:24639295

Old World hantaviruses in rodents in New Orleans, Louisiana.

PubMed

Cross, Robert W; Waffa, Bradley; Freeman, Ashley; Riegel, Claudia; Moses, Lina M; Bennett, Andrew; Safronetz, David; Fischer, Elizabeth R; Feldmann, Heinz; Voss, Thomas G; Bausch, Daniel G

2014-05-01

Seoul virus, an Old World hantavirus, is maintained in brown rats and causes a mild form of hemorrhagic fever with renal syndrome (HFRS) in humans. We captured rodents in New Orleans, Louisiana and tested them for the presence of Old World hantaviruses by reverse transcription polymerase chain reaction (RT-PCR) with sequencing, cell culture, and electron microscopy; 6 (3.4%) of 178 rodents captured--all brown rats--were positive for a Seoul virus variant previously coined Tchoupitoulas virus, which was noted in rodents in New Orleans in the 1980s. The finding of Tchoupitoulas virus in New Orleans over 25 years since its first discovery suggests stable endemicity in the city. Although the degree to which this virus causes human infection and disease remains unknown, repeated demonstration of Seoul virus in rodent populations, recent cases of laboratory-confirmed HFRS in some US cities, and a possible link with hypertensive renal disease warrant additional investigation in both rodents and humans.

Autocrine stimulation of human hepatocytes triggers late DNA synthesis and stabilizes long-term differentiation in vitro.

PubMed

Leckel, Kerstin; Strey, Christoph; Bechstein, Wolf O; Boost, Kim A; Auth, Marcus K H; El Makhfi, Amal; Juengel, Eva; Wedel, Steffen; Jones, Jon; Jonas, Dietger; Blaheta, Roman A

2008-05-01

Isolated human hepatocytes are of great value in investigating cell transplantation, liver physiology, pathology, and drug metabolism. Though hepatocytes possess a tremendous proliferative capacity in vivo, their ability to grow in culture is severely limited. We postulated that repeated medium change, common to most in vitro systems, may prevent long-term maintenance of hepato-specific functions and growth capacity. To verify our hypotheses we compared the DNA synthesis and differentiation status of isolated human hepatocytes, cultured in medium which was renewed every day or was not changed for 3 weeks ('autocrine' setting). Daily medium change led to rapid hepatocellular de-differentiation without any signs of DNA replication. In contrast, the autocrine setting allowed hepatocytes to become highly differentiated, demonstrated by an elevated ASGPr expression level, and increased albumin and fibrinogen synthesis and release. Cytokeratin 18 filaments were stably expressed, whereas cytokeratin 19 remained undetectable. Hepatocytes growing in an autocrine fashion were activated in the presence of hepatocyte growth factor (HGF), evidenced by c-Met phosphorylation. However, HGF response was not achieved when the culture medium was renewed daily. Furthermore, the autocrine setting evoked a late but strong interleukin 6 release into the culture supernatant, reaching maximum values after a 10-day cultivation period, and intense BrdU incorporation after a further 5-day period. Our data suggest that preservation of the same medium creates environmental conditions which allow hepatocytes to control their differentiation status and DNA synthesis in an autocrine fashion. Further studies are necessary to identify the key mediators involved in autocrine communication and to design the optimal culture configuration for clinical application.

Ground-based rodent control in a remote Hawaiian rainforest on Maui

USGS Publications Warehouse

Malcolm, T.R.; Swinnerton, K.J.; Groombridge, J.J.; Sparklin, B.D.; Brosius, C.N.; Vetter, J.P.; Foster, J.T.

2008-01-01

Effective control of introduced mammalian predators is essential to the recovery of native bird species in Hawai'i. Between August 1996 and December 2004, introduced rodents were controlled within three home ranges of the Po'ouli Melamprosops phaeosoma, a critically endangered Hawaiian honeycreeper. Rats were controlled using a combination of ground-based rodenticide (0.005% diphacinone) application and snap traps. Beginning in August 2001, we monitored the effectiveness of these rodent control efforts. Relative abundances of Black Rats Rattus rattus and Polynesian Rats R. exulans were measured in each of five snap-trapping grids seven times over a 35-month period. Rat populations decreased inside of the rodent control areas, but control effectiveness differed between rat species. During the first year of monitoring, target control levels for R. rattus were consistently achieved in only one of the rodent control areas. Control techniques were refined in areas failing to meet targets. Subsequently, we achieved target control levels for R. rattus more consistently in all three rodent control areas. However, relative abundances of R. exulans did not differ between rodent control and reference areas, indicating that our rodent control techniques were insufficient to reduce population levels of this species. These findings signify a need for further improvement of rodent control methods in Hawai'i, especially for Polynesian Rats, and demonstrate the critical importance of periodic monitoring of the response of rodent populations to management. In the future, managers may need to design rodent control operations targeting R. rattus and R. exulans independently to achieve best results.

Hemodynamic flow improves rat hepatocyte morphology, function, and metabolic activity in vitro.

PubMed

Dash, A; Simmers, M B; Deering, T G; Berry, D J; Feaver, R E; Hastings, N E; Pruett, T L; LeCluyse, E L; Blackman, B R; Wamhoff, B R

2013-06-01

In vitro primary hepatocyte systems typically elicit drug induction and toxicity responses at concentrations much higher than corresponding in vivo or clinical plasma C(max) levels, contributing to poor in vitro-in vivo correlations. This may be partly due to the absence of physiological parameters that maintain metabolic phenotype in vivo. We hypothesized that restoring hemodynamics and media transport would improve hepatocyte architecture and metabolic function in vitro compared with nonflow cultures. Rat hepatocytes were cultured for 2 wk either in nonflow collagen gel sandwiches with 48-h media changes or under controlled hemodynamics mimicking sinusoidal circulation within a perfused Transwell device. Phenotypic, functional, and metabolic parameters were assessed at multiple times. Hepatocytes in the devices exhibited polarized morphology, retention of differentiation markers [E-cadherin and hepatocyte nuclear factor-4α (HNF-4α)], the canalicular transporter [multidrug-resistant protein-2 (Mrp-2)], and significantly higher levels of liver function compared with nonflow cultures over 2 wk (albumin ~4-fold and urea ~5-fold). Gene expression of cytochrome P450 (CYP) enzymes was significantly higher (fold increase over nonflow: CYP1A1: 53.5 ± 10.3; CYP1A2: 64.0 ± 15.1; CYP2B1: 15.2 ± 2.9; CYP2B2: 2.7 ± 0.8; CYP3A2: 4.0 ± 1.4) and translated to significantly higher basal enzyme activity (device vs. nonflow: CYP1A: 6.26 ± 2.41 vs. 0.42 ± 0.015; CYP1B: 3.47 ± 1.66 vs. 0.4 ± 0.09; CYP3A: 11.65 ± 4.70 vs. 2.43 ± 0.56) while retaining inducibility by 3-methylcholanthrene and dexamethasone (fold increase over DMSO: CYP1A = 27.33 and CYP3A = 4.94). These responses were observed at concentrations closer to plasma levels documented in vivo in rats. The retention of in vivo-like hepatocyte phenotype and metabolic function coupled with drug response at more physiological concentrations emphasizes the importance of restoring in vivo physiological transport

Protective effects of melittin on transforming growth factor-{beta}1 injury to hepatocytes via anti-apoptotic mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Woo-Ram; Park, Ji-Hyun; Kim, Kyung-Hyun

Melittin is a cationic, hemolytic peptide that is the main toxic component in the venom of the honey bee (Apis mellifera). Melittin has multiple effects, including anti-bacterial, anti-viral and anti-inflammatory, in various cell types. However, the anti-apoptotic mechanisms of melittin have not been fully elucidated in hepatocytes. Apoptosis contributes to liver inflammation and fibrosis. Knowledge of the apoptotic mechanisms is important to develop new and effective therapies for treatment of cirrhosis, portal hypertension, liver cancer, and other liver diseases. In the present study, we investigated the anti-apoptotic effect of melittin on transforming growth factor (TGF)-{beta}1-induced apoptosis in hepatocytes. TGF-{beta}1-treated hepatocytesmore » were exposed to low doses (0.5 and 1 {mu}g/mL) and high dose (2 {mu}g/mL) of melittin. The low doses significantly protected these cells from DNA damage in TGF-{beta}1-induced apoptosis compared to the high dose. Also, melittin suppressed TGF-{beta}1-induced apoptotic activation of the Bcl-2 family and caspase family of proteins, which resulted in the inhibition of poly-ADP-ribose polymerase (PARP) cleavage. These results demonstrate that TGF-{beta}1 induces hepatocyte apoptosis and that an optimal dose of melittin exerts anti-apoptotic effects against TGF-{beta}1-induced injury to hepatocytes via the mitochondrial pathway. These results suggest that an optimal dose of melittin can serve to protect cells against TGF-{beta}1-mediated injury. - Highlights: > We investigated the anti-apoptotic effect of melittin on TGF-{beta}1-induced hepatocyte. > TGF-{beta}1 induces hepatocyte apoptosis. > TGF-{beta}1-treated hepatocytes were exposed to low doses and high dose of melittin. > Optimal dose of melittin exerts anti-apoptotic effects to hepatocytes.« less

Stathmin Mediates Hepatocyte Resistance to Death from Oxidative Stress by down Regulating JNK

PubMed Central

Zhao, Enpeng; Amir, Muhammad; Lin, Yu; Czaja, Mark J.

2014-01-01

Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK). The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth. PMID:25285524

Energetic costs of pyrene metabolism in isolated hepatocytes of rainbow trout, Oncorhynchus mykiss.

PubMed

Bains, Onkar S; Kennedy, Christopher J

2004-04-28

The respiratory costs of pyrene exposure and biotransformation were examined in isolated hepatocytes of adult rainbow trout, Oncorhynchus mykiss. Baseline oxygen consumption rates measured at an acclimation temperature of 7.5 degrees C and during an acute temperature increase to 15 degrees C were 10.1 +/- 0.1 and 22.6 +/- 0.4 ng O(2)/min/mg cells, respectively. Hepatocytes exposed to pyrene at 1, 5 and 10 microg/ml exhibited concentration-dependent increases in oxygen consumption. Respiration rates of cells exposed to these concentrations at their acclimation temperature were 12.5 +/- 0.1, 14.7 +/- 0.1 and 17.1 +/- 0.2 ng O(2)/min/mg cells, respectively. Exposure of cells to pyrene at 15 degrees C also elevated oxygen consumption to a maximum of 34.4 +/- 0.3 ng O(2)/min/mg cells, however, the relationship with pyrene concentration was biphasic. The major metabolite identified through a series of solvent extractions, acid hydrolysis, and synchronous fluorometric spectroscopy was conjugated 1-hydroxypyrene. At 7.5 degrees C, increased pyrene metabolism correlated with increased hepatocyte respiration rates. At 15 degrees C, however, pyrene metabolism reached a maximum at 5 microg/ml, suggesting saturation of detoxification enzymes, which correlated with maximum respiration rates at this concentration. Measures of respiration by isolated mitochondria indicated that changes in hepatocyte oxygen consumption were not through direct effects of pyrene on mitochondria. This study indicates that significant respiratory costs may be accrued by teleost hepatocytes actively metabolizing and secreting xenobiotic compounds.

Hepatocyte-based in vitro model for assessment of drug-induced cholestasis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chatterjee, Sagnik, E-mail: Sagnik.Chatterjee@pharm.kuleuven.be; Richert, Lysiane, E-mail: l.richert@kaly-cell.com; Augustijns, Patrick, E-mail: Patrick.Augustijns@pharm.kuleuven.be

Early detection of drug-induced cholestasis remains a challenge during drug development. We have developed and validated a biorelevant sandwich-cultured hepatocytes- (SCH) based model that can identify compounds causing cholestasis by altering bile acid disposition. Human and rat SCH were exposed (24–48 h) to known cholestatic and/or hepatotoxic compounds, in the presence or in the absence of a concentrated mixture of bile acids (BAs). Urea assay was used to assess (compromised) hepatocyte functionality at the end of the incubations. The cholestatic potential of the compounds was expressed by calculating a drug-induced cholestasis index (DICI), reflecting the relative residual urea formation bymore » hepatocytes co-incubated with BAs and test compound as compared to hepatocytes treated with test compound alone. Compounds with clinical reports of cholestasis, including cyclosporin A, troglitazone, chlorpromazine, bosentan, ticlopidine, ritonavir, and midecamycin showed enhanced toxicity in the presence of BAs (DICI ≤ 0.8) for at least one of the tested concentrations. In contrast, the in vitro toxicity of compounds causing hepatotoxicity by other mechanisms (including diclofenac, valproic acid, amiodarone and acetaminophen), remained unchanged in the presence of BAs. A safety margin (SM) for drug-induced cholestasis was calculated as the ratio of lowest in vitro concentration for which was DICI ≤ 0.8, to the reported mean peak therapeutic plasma concentration. SM values obtained in human SCH correlated well with reported % incidence of clinical drug-induced cholestasis, while no correlation was observed in rat SCH. This in vitro model enables early identification of drug candidates causing cholestasis by disturbed BA handling. - Highlights: • Novel in vitro assay to detect drug-induced cholestasis • Rat and human sandwich-cultured hepatocytes (SCH) as in vitro models • Cholestatic compounds sensitize SCH to toxic effects of accumulating bile acids �

[Pathological changes in hepatocytes of mice with obesity-induced type 2 diabetes by monosodium glutamate].

PubMed

Nakadate, Kazuhiko; Motojima, Kento; Kamata, Sumito; Yoshida, Testuro; Hikita, Masaaki; Wakamatsu, Hisanori

2014-01-01

Type 2 diabetes caused by chronic obesity is a major lifestyle-related disease. The present study aimed to determine the pathological changes in hepatocytes in chronic obesity. To develop our type 2 diabetes mouse model, we induced chronic obesity to mice by monosodium glutamate. By overeating, the mice significantly increased their body weight compared with age-matched healthy animals. To analyze the pathological changes in hepatocytes of chronic obesity before preclinical stage of type 2 diabetes, the mice were analyzed by hematoxylin-eosin staining of tissue sections at 15 w of age. In these mice, we observed eosin-negative accumulations of hepatocytes around central veins in the hepatic lobule. By Oil-Red O staining, the eosin-negative granules were identified in the lipid droplets. We then ascertained whether these lipid droplets of hepatocytes in the obese mice could be modified by diet. After 24 h of diet restriction, the lipid droplets of hepatocytes in the obese mice were swollen. Furthermore, after 48 h of the diet restriction, the lipid droplets continued swelling and the autophagy-like structures that were found in the healthy mice under the same condition in the obese mice were not observed. These results suggest that the obese mice might have delayed energy metabolism, which might have influenced the mechanisms of hepatocytes. These findings provide new insight into the functional changes in chronic obesity-induced type 2 diabetes and it is possible that the pathological feature make a contribution to promise the target of pharmacological therapy.

AMPKα1 controls hepatocyte proliferation independently of energy balance by regulating Cyclin A2 expression.

PubMed

Merlen, Grégory; Gentric, Géraldine; Celton-Morizur, Séverine; Foretz, Marc; Guidotti, Jacques-Emmanuel; Fauveau, Véronique; Leclerc, Jocelyne; Viollet, Benoit; Desdouets, Chantal

2014-01-01

AMP-activated protein kinase (AMPK) is an evolutionarily conserved sensor of cellular energy status that contributes to restoration of energy homeostasis by slowing down ATP-consuming pathways and activating ATP-producing pathways. Unexpectedly, in different systems, AMPK is also required for proper cell division. In the current study, we evaluated the potential effect of the AMPK catalytic subunit, AMPKα1, on hepatocyte proliferation. Hepatocyte proliferation was determined in AMPKα1 knockout and wild-type mice in vivo after two thirds partial hepatectomy, and in vitro in primary hepatocyte cultures. The activities of metabolic and cell cycle-related signaling pathways were measured. After partial hepatectomy, hepatocytes proliferated rapidly, correlating with increased AMPK phosphorylation. Deletion of AMPKα1 delayed liver regeneration by impacting on G1/S transition phase. The proliferative defect of AMPKα1-deficient hepatocytes was cell autonomous, and independent of energy balance. The priming phase, lipid droplet accumulation, protein anabolic responses and growth factor activation after partial hepatectomy occurred normally in the absence of AMPKα1 activity. By contrast, mRNA and protein expression of cyclin A2, a key driver of S phase progression, were compromised in the absence of AMPK activity. Importantly, AMPKα1 controlled cyclin A2 transcription mainly through the ATF/CREB element. Our study highlights a novel role for AMPKα1 as a positive regulator of hepatocyte division occurring independently of energy balance. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Lassa fever or lassa hemorrhagic fever risk to humans from rodent-borne zoonoses.

PubMed

El-Bahnasawy, Mamdouh M; Megahed, Laila Abdel-Mawla; Abdalla Saleh, Hala Ahmed; Morsy, Tosson A

2015-04-01

Viral hemorrhagic fevers (VHFs) typically manifest as rapidly progressing acute febrile syndromes with profound hemorrhagic manifestations and very high fatality rates. Lassa fever, an acute hemorrhagic fever characterized by fever, muscle aches, sore throat, nausea, vomiting, diarrhea and chest and abdominal pain. Rodents are important reservoirs of rodent-borne zoonosis worldwide. Transmission rodents to humans occur by aerosol spread, either from the genus Mastomys rodents' excreta (multimammate rat) or through the close contact with infected patients (nosocomial infection). Other rodents of the genera Rattus, Mus, Lemniscomys, and Praomys are incriminated rodents hosts. Now one may ask do the rodents' ectoparasites play a role in Lassa virus zoonotic transmission. This paper summarized the update knowledge on LHV; hopping it might be useful to the clinicians, nursing staff, laboratories' personals as well as those concerned zoonoses from rodents and rodent control.

New fossils from the Paleogene of central Libya illuminate the evolutionary history of endemic African anomaluroid rodents

NASA Astrophysics Data System (ADS)

Coster, Pauline; Beard, K. Christopher; Salem, Mustafa; Chaimanee, Yaowalak; Jaeger, Jean-Jacques

2015-10-01

Anomaluroid rodents show interesting biogeographic and macroevolutionary patterns, although their fossil record is meager and knowledge of the natural history of extant members of the clade remains inadequate. Living anomaluroids (Anomaluridae) are confined to equatorial parts of western and central Africa, but the oldest known fossil anomaluroid (Pondaungimys) comes from the late middle Eocene of Myanmar. The first appearance of anomaluroids in the African fossil record coincides with the first appearances of hystricognathous rodents and anthropoid primates there. Both of the latter taxa are widely acknowledged to have originated in Asia, suggesting that anomaluroids may show a concordant biogeographic pattern. Here we describe two new taxa of African Paleogene anomaluroids from sites in the Sirt Basin of central Libya. These include a new Eocene species of the nementchamyid genus Kabirmys, which ranks among the oldest African anomaluroids recovered to date, and a new genus and species of Anomaluridae from the early Oligocene, which appears to be closely related to extant Zenkerella, the only living non-volant anomalurid. Phylogenetic analyses incorporating the new Libyan fossils suggest that anomaluroids are not specially related to Zegdoumyidae, which are the only African rodents known to antedate the first appearance of anomaluroids there. The evolution of gliding locomotion in Anomaluridae appears to conflict with traditional assessments of relationships among living anomalurid taxa. If the historically accepted division of Anomaluridae into Anomalurinae (extant and Miocene Anomalurus and Miocene Paranomalurus) and Zenkerellinae (extant and Miocene Zenkerella and extant Idiurus) is correct, then either gliding locomotion evolved independently in Anomalurinae and Idiurus or non-volant Zenkerella evolved from a gliding ancestor. Anatomical data related to gliding in Anomaluridae are more consistent with a nontraditional systematic arrangement, whereby non

Superoxide dismutase and catalase protect cultured hepatocytes from the cytotoxicity of acetaminophen.

PubMed

Kyle, M E; Miccadei, S; Nakae, D; Farber, J L

1987-12-31

Superoxide dismutase, catalase and mannitol prevent the killing of cultured hepatocytes by acetaminophen in the presence of an inhibitor of glutathione reductase, BCNU. Under these conditions, the cytotoxicity of acetaminophen depends upon its metabolism, since beta-naphthoflavone, an inhibitor of mixed function oxidation, prevents the cell killing. In hepatocytes made resistant to acetaminophen by pretreatment with the ferric iron chelator, deferoxamine, addition of ferric or ferrous iron restores the sensitivity to acetaminophen. In such a situation, both superoxide dismutase and catalase prevent the killing by acetaminophen in the presence of ferric iron. By contrast, catalase, but not superoxide dismutase, prevents the cell killing dependent upon addition of ferrous iron. These results document the participation of both superoxide anion and hydrogen peroxide in the killing of cultured hepatocytes by acetaminophen and suggest that hydroxyl radicals generated by an iron catalyzed Haber-Weiss reaction mediate the cell injury.

Transversal inducing differentiation of human amniotic epithelial cells into hepatocyte-like cells.

PubMed

Luo, Hongwu; Huang, Xiangjun; Huang, Feizhou; Liu, Xunyang

2011-06-01

To evaluate the in vitro differentiation of human amniotic epithelial cells (hAECs ) into hepatocyte-like cells. Combined approach of dexamethasone, HGF, IGF and other cytokines were used to induce the differentiation of hAECs into hepatocyte-like cells. The induction lasted 2 weeks. During the induction, the expression of albumin ALB, CYP1A1, CYP1A2, IGFR, c-met and key functional genes related to liver cells as well as transcription factors HNF3, HNF4 and C/EBPa were monitored by RT-PCR. Time dependent changes of the surface marker colony ALB, AFP and CK18 were analyzed by cell flow cytometry. After the 2 week induction, the expressions of liver hepatocyte-like cell functional genes such as albumin, CYP1A1, CYP1A2, c-met, and transcription factors such as HNF3, HNF4, C/EBPa and HNF1 were observed. Six days after the induction, hAECs mainly were stained AFP+, and the positive rate was (15.1 ± 2.1)%. While 10 days after the induction, part of the hAECs showed AFP+/ALB+ (6.5 ± 1.4)%; and on 14th day, hAECs only showed ALB+, and the rate was (13.9 ± 2.3)%. ALB+ cell increase indicated a gradual functional maturation from the hAECs to hepatocyte-like cells. Similaritly, the number of CK18+ cells in the whole population was also increased: On 10th day, the rate was (16.1 ± 1.2)%; on 14th day, that was (21.3 ± 4.6)%, which proved the above hypothesis of the trandifferentiation. By extending the induction time, the expression of functional genes increased gradually, and a maturing process of hAECs was detected by cell surface markers. The differentiation of hAECs induced in vitro has the characteristics of hepatocyte-like cells.

Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mohammad, Mohammad K.; Alcohol Research Center, University of Louisville; Avila, Diana

2012-11-15

Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. Also, it is produced endogenously via lipid peroxidation and cellular metabolism of certain amino acids and drugs. Acrolein is cytotoxic to many cell types including hepatocytes; however the mechanisms are not fully understood. We examined the molecular mechanisms underlying acrolein hepatotoxicity in primary human hepatocytes and hepatoma cells. Acrolein, at pathophysiological concentrations, caused a dose-dependent loss of viability of hepatocytes. The death was apoptotic at moderate and necrotic at high concentrations of acrolein. Acrolein exposure rapidly and dramatically decreased intracellular glutathione and overall antioxidantmore » capacity, and activated the stress-signaling MAP-kinases JNK, p42/44 and p38. Our data demonstrate for the first time in human hepatocytes, that acrolein triggered endoplasmic reticulum (ER) stress and activated eIF2α, ATF-3 and -4, and Gadd153/CHOP, resulting in cell death. Notably, the protective/adaptive component of ER stress was not activated, and acrolein failed to up-regulate the protective ER-chaperones, GRP78 and GRP94. Additionally, exposure to acrolein disrupted mitochondrial integrity/function, and led to the release of pro-apoptotic proteins and ATP depletion. Acrolein-induced cell death was attenuated by N-acetyl cysteine, phenyl-butyric acid, and caspase and JNK inhibitors. Our data demonstrate that exposure to acrolein induces a variety of stress responses in hepatocytes, including GSH depletion, oxidative stress, mitochondrial dysfunction and ER stress (without ER-protective responses) which together contribute to acrolein toxicity. Our study defines basic mechanisms underlying liver injury caused by reactive aldehyde pollutants such as acrolein. -- Highlights: ► Human primary hepatocytes and cultured cell lines are used. ► Multiple cell death signaling pathways are activated by acrolein. ► Novel

Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes*

PubMed Central

Itoh, Yumi; Sanosaka, Masato; Fuchino, Hiroyuki; Yahara, Yasuhito; Kumagai, Ayako; Takemoto, Daisaku; Kagawa, Mai; Doi, Junko; Ohta, Miho; Tsumaki, Noriyuki; Kawahara, Nobuo; Takemori, Hiroshi

2015-01-01

Salt-inducible kinases (SIKs), members of the 5′-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes. PMID:26048985

The mitochondrial import gene tomm22 is specifically required for hepatocyte survival and provides a liver regeneration model

PubMed Central

Curado, Silvia; Ober, Elke A.; Walsh, Susan; Cortes-Hernandez, Paulina; Verkade, Heather; Koehler, Carla M.; Stainier, Didier Y. R.

2010-01-01

SUMMARY Understanding liver development should lead to greater insights into liver diseases and improve therapeutic strategies. In a forward genetic screen for genes regulating liver development in zebrafish, we identified a mutant – oliver – that exhibits liver-specific defects. In oliver mutants, the liver is specified, bile ducts form and hepatocytes differentiate. However, the hepatocytes die shortly after their differentiation, and thus the resulting mutant liver consists mainly of biliary tissue. We identified a mutation in the gene encoding translocase of the outer mitochondrial membrane 22 (Tomm22) as responsible for this phenotype. Mutations in tomm genes have been associated with mitochondrial dysfunction, but most studies on the effect of defective mitochondrial protein translocation have been carried out in cultured cells or unicellular organisms. Therefore, the tomm22 mutant represents an important vertebrate genetic model to study mitochondrial biology and hepatic mitochondrial diseases. We further found that the temporary knockdown of Tomm22 levels by morpholino antisense oligonucleotides causes a specific hepatocyte degeneration phenotype that is reversible: new hepatocytes repopulate the liver as Tomm22 recovers to wild-type levels. The specificity and reversibility of hepatocyte ablation after temporary knockdown of Tomm22 provides an additional model to study liver regeneration, under conditions where most hepatocytes have died. We used this regeneration model to analyze the signaling commonalities between hepatocyte development and regeneration. PMID:20483998

Rodent-associated Bartonella Febrile Illness, Southwestern United States

PubMed Central

Iralu, Jonathan; Bai, Ying; Crook, Larry; Tempest, Bruce; Simpson, Gary; McKenzie, Taylor

2006-01-01

Serum specimens from 114 patients hospitalized with a febrile illness were tested with an indirect immunofluorescence assay (IFA) using Bartonella antigens prepared from 6 species of sigmodontine rodents and 3 known human Bartonella pathogens: B. henselae, B. quintana, and B. elizabethae. Acute- and convalescent-phase serum samples from 5 of these patients showed seroconversion with an IFA titer >512 to rodent-associated Bartonella antigens. The highest titer was against antigen derived from the white-throated woodrat (Neotoma albigula), although this rodent is not necessarily implicated as the source of infection. Three of the 5 who seroconverted showed no cross-reaction to the 3 Bartonella human pathogens. Common clinical characteristics were fever, chills, myalgias, leukopenia, thrombocytopenia, and transaminasemia. Although antibodies to Bartonella are cross-reactive, high-titer seroconversions to rodent-associated Bartonella antigens in adults with common clinical characteristics should stimulate the search for additional Bartonella human pathogens. PMID:16836824

Problems associated with the control of rodents in tropical Africa

PubMed Central

Gratz, N. G.; Arata, A. A.

1975-01-01

As elsewhere in the world, rodents are responsible for very considerable economic losses in tropical Africa because of their depredations on both growing crops and stored food products. Unfortunately, few accurate data are available on the extent of these losses but there is evidence that they are considerable. The public health importance of rodents, both as reservoirs and vectors of disease in tropical Africa, is also great; plague, leptospirosis, murine typhus, and Lassa fever are among the diseases associated with rodent hosts. Scientifically based rodent control programmes have been carried out in very few areas of Africa and there is urgent need for studies and demonstrations on rodent control in both urban and rural areas. The problems likely to be encountered are reviewed and methods of control proposed. PMID:1085224

Claudin-2 Promotes Breast Cancer Liver Metastasis by Facilitating Tumor Cell Interactions with Hepatocytes

PubMed Central

Tabariès, Sébastien; Dupuy, Fanny; Dong, Zhifeng; Monast, Anie; Annis, Matthew G.; Spicer, Jonathan; Ferri, Lorenzo E.; Omeroglu, Atilla; Basik, Mark; Amir, Eitan; Clemons, Mark

2012-01-01

We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes. PMID:22645303

The energetic cost of protein synthesis in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss).

PubMed

Pannevis, M C; Houlihan, D F

1992-01-01

To establish the energetic cost of protein synthesis, isolated trout hepatocytes were used to measure protein synthesis and respiration simultaneously at a variety of temperatures. The presence of bovine serum albumin was essential for the viability of isolated hepatocytes during isolation, but, in order to measure protein synthesis rates, oxygen consumption rates and RNA-to-protein ratios, BSA had to be washed from the cells. Isolated hepatocytes were found to be capable of protein synthesis and oxygen consumption at constant rates over a wide range of oxygen tension. Cycloheximide was used to inhibit protein synthesis. Isolated hepatocytes used on average 79.7 +/- 9.5% of their total oxygen consumption on cycloheximide-sensitive protein synthesis and 2.8 +/- 2.8% on maintaining ouabain-sensitive Na+/K(+)-ATPase activity. The energetic cost of protein synthesis in terms of moles of adenosine triphosphate per gram of protein synthesis decreased with increasing rates of protein synthesis at higher temperatures. It is suggested that the energetic cost consists of a fixed (independent of synthesis rate) and a variable component (dependent on synthesis rate).

Gene-specific alterations of hepatic nuclear receptor regulated gene expression by ligand activation or hepatocyte-selective knockout inhibition of RXRα signaling during inflammation

PubMed Central

Kosters, Astrid; Tian, Feng; Wan, Yvonne Yu-Jie; Karpen, Saul J.

2013-01-01

Background Inflammation leads to transcriptional downregulation of many hepatic genes, particulary those activated by RXRα-heterodimers. Inflammation-mediated reduction of nuclear RXRα levels is a main factor in reduced nuclear receptor (NR)–regulated hepatic gene expression, eventually leading to cholestasis and liver damage. Aim To investigate roles for RXRα in hepatic gene expression during inflammation, using two complementary mouse models: ligand–activation of RXRα, and in mice expressing hepatocyte-specific expression of RXRα missing its DNA-binding-domain (DBD; hs-RxrαΔex4−/−) Methods To activate RXRα, mice were gavage-fed with LG268 or vehicle for 5 days. To inhibit RXRα function, hs-RxrαΔex4−/− were used. All mice were IP-injected with LPS or saline for 16 hrs prior to analysis of hepatic RNA, protein and NR-DNA binding. Results LG268-treatment attenuated the LPS-mediated reductions of several RXRα-regulated genes, coinciding with maintained RXRα occupancy in both Bsep and Ostβ promoters. Lacking full hepatocyte-RXRα function (hs-RxrαΔex4−/− mice) led to enhancement of LPS-mediated changes in gene expression, but surprisingly, maintenance of RNA levels of some RXRα-regulated genes. Investigations revealed that Hs-Rxrα−/− hepatocytes expressed an internally-truncated, ~44 kDa, RXRα-form. DNA-binding capacity of NR-heterodimers was equivalent in wt and hs-RxrαΔex4−/− livers, but reduced by LPS in both. ChIP-QPCR revealed reduced RXRα occupancy to the Bsep RXRα:FXR site was reduced, but not absent, in hs-RxrαΔex4−/− livers. Conclusions There are differential regulatory roles for hepatic RXRα, both in basal and inflammatory states, suggesting new and complex multi-domain roles for RXRα in regulating hepatic gene expression. Moreover, there is an unexpected non-obligate role for the DBD of RXRα. PMID:22098603

Malaria parasites target the hepatocyte receptor EphA2 for successful host infection.

PubMed

Kaushansky, Alexis; Douglass, Alyse N; Arang, Nadia; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Kain, Heather S; Austin, Laura S; Sather, D Noah; Kappe, Stefan H I

2015-11-27

The invasion of a suitable host hepatocyte by mosquito-transmitted Plasmodium sporozoites is an essential early step in successful malaria parasite infection. Yet precisely how sporozoites target their host cell and facilitate productive infection remains largely unknown. We found that the hepatocyte EphA2 receptor was critical for establishing a permissive intracellular replication compartment, the parasitophorous vacuole. Sporozoites productively infected hepatocytes with high EphA2 expression, and the deletion of EphA2 protected mice from liver infection. Lack of host EphA2 phenocopied the lack of the sporozoite proteins P52 and P36. Our data suggest that P36 engages EphA2, which is likely to be a key step in establishing the permissive replication compartment. Copyright © 2015, American Association for the Advancement of Science.

Purification, characterisation and protective effects of polysaccharides from alfalfa on hepatocytes.

PubMed

Wang, Shaopu; Dong, Xiaofang; Ma, Hao; Cui, Yaoming; Tong, Jianming

2014-11-04

The objective of this study was to determine the preliminary characteristics and protective effects of alfalfa polysaccharides (APS) on hepatocytes in vitro. The crude APS was purified by DEAE-cellulose and Sephadex G-100 chromatography, resulting in the four purified fractions: APS-1, APS-2, APS-3 and APS-4. The results indicated that APS-3 had higher carbohydrate and uronic acid contents and that APS-4 had a more complicated monosaccharide composition compared to the other purified fractions. The average molecular weights of APS-1, APS-2, APS-3 and APS-4 were 48,536, 6,221, 66,559 and 13,076 Da, respectively. Furthermore, APS (crude and its purified fractions) restored the activities of antioxidant enzymes and increased the total antioxidant capacity of hepatocytes subjected to H2O2-induced oxidative stress. Furthermore, APS treatment counteracted the increases in lactic dehydrogenase and malonaldehyde in the culture supernatant. These results clearly demonstrate that APS possesses a protective effect against oxidative injury in hepatocytes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells

PubMed Central

Fagoonee, Sharmila; Famulari, Elvira Smeralda; Silengo, Lorenzo; Tolosano, Emanuela; Altruda, Fiorella

2015-01-01

One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine. PMID:26323094

[The influence of hepatoprotector 2-ethylthiobenzimidazole hydrobromide (bemithyl) on the content of glycogen in cirrhotic rat liver hepatocytes located in various microenvironments].

PubMed

Kudriavtseva, M V; Bezborodkina, N N; Okovityĭ, S V; Kudriavtsev, B N

2004-01-01

Using absorption and fluorescent cytophotometry methods, glycogen contents were studied in hepatocytes located in liver lobules and in hepatocytes, which make the general population of these cells in normal and cirrhotic rat liver. In cirrhosis, the content of glycogen in hepatocytes located in lobules obviously rises in comparison with the norm, but to a lesser degree, than in hepatocytes making the general population of these cells in cirrhotic liver. The content of glycogen in hepatocytes, located in lobules of pathologically changed liver in bemithyl treated rats, did not differ from the norm. At the same time, the glycogen content in hepatocytes, representing the general population of these cells in cirrhotically altered bemithyl injected rat liver, remained higher than in the norm. The data obtained indicate that distinctions in particular cell microinvironment, obviously present in cirrhotic liver, render essential influence on hepatocyte functional activity.

Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes

PubMed Central

Casciano, Jessica C.; Duchemin, Nicholas J.; Lamontagne, R. Jason; Steel, Laura F.; Bouchard, Michael J.

2017-01-01

Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases

Nor-ursodeoxycholic acid reverses hepatocyte-specific nemo-dependent steatohepatitis.

PubMed

Beraza, Naiara; Ofner-Ziegenfuss, Lisa; Ehedego, Haksier; Boekschoten, Mark; Bischoff, Stephan C; Mueller, Michael; Trauner, Michael; Trautwein, Christian

2011-03-01

Hepatocyte-specific NEMO/NF-κB deleted mice (NEMO(Δhepa)) develop spontaneous non-alcoholic steatohepatitis (NASH). Free fatty acids and bile acids promote DR5 expression. TRAIL/NK cell-mediated activation of TRAIL-R2/DR5 plays an important role during acute injury in NEMO(Δhepa) mice. To inhibit the progression of NASH in the absence of hepatocyte-NEMO/NF-kB signaling. NEMOf/f and NEMO(Δhepa) mice were fed with a low-fat diet, and with two anticholestatic diets; UDCA and NorUDCA. The impact of these treatments on the progression of NASH was evaluated. We show that high expression of DR5 in livers from NEMO(Δhepa) mice is accompanied by an abundant presence of bile acids (BAs), misregulation of BA transporters and significant alteration of lipid metabolism-related genes. Additionally, mice lacking NEMO in hepatocytes spontaneously showed ductular response at young age. Unexpectedly, feeding of NEMO(Δhepa) mice with low-fat diet failed to improve chronic liver injury. Conversely, anti-cholestatic treatment with nor-ursodeoxycholic acid (NorUDCA), but not with ursodeoxycholic acid (UDCA), led to a significant attenuation of liver damage in NEMO(Δhepa) mice. The strong therapeutic effect of NorUDCA relied on a significant downregulation of LXR-dependent lipogenesis and the normalisation of BA metabolism through mechanisms involving cross-talk between Cyp7a1 and SHP. This was associated with the significant improvement of liver histology, NEMO(Δhepa)/NorUDCA-treated mice showed lower apoptosis and reduced CyclinD1 expression, indicating attenuation of the compensatory proliferative response to hepatocellular damage. Finally, fibrosis and ductular reaction markers were significantly reduced in NorUDCA-treated NEMO(Δhepa) mice. Overall, our work demonstrates the contribution of bile acids metabolism to the progression of NASH in the absence of hepatocyte-NF-kB through mechanisms involving DR5-apoptosis, inflammation and fibrosis. Our work suggests a potential

Microbial-derived lithocholic acid and vitamin K2 drive the metabolic maturation of pluripotent stem cells-derived and fetal hepatocytes.

PubMed

Avior, Yishai; Levy, Gahl; Zimerman, Michal; Kitsberg, Daniel; Schwartz, Robert; Sadeh, Ronen; Moussaieff, Arieh; Cohen, Merav; Itskovitz-Eldor, Joseph; Nahmias, Yaakov

2015-07-01

The liver is the main organ responsible for the modification, clearance, and transformational toxicity of most xenobiotics owing to its abundance in cytochrome P450 (CYP450) enzymes. However, the scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSCs) represent an excellent source of differentiated hepatocytes; however, current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly, fetal hepatocytes acquire mature CYP450 expression only postpartum, suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K2 and lithocholic acid, a by-product of intestinal flora, activate pregnane X receptor (PXR) and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produce albumin and apolipoprotein B100 at levels equivalent to primary human hepatocytes, while demonstrating an 8-fold induction of CYP450 activity in response to aryl hydrocarbon receptor (AhR) agonist omeprazole and a 10-fold induction in response to PXR agonist rifampicin. Flow cytometry showed that over 83% of cells were albumin and hepatocyte nuclear factor 4 alpha (HNF4α) positive, permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R(2) correlation of 0.94 between TC50 values obtained in stem cell-derived hepatocytes and primary cells, compared to 0.62 for HepG2 cells. Finally, stem cell-derived hepatocytes demonstrate all toxicological endpoints examined, including steatosis, apoptosis, and cholestasis, when exposed to nine known hepatotoxins. Our work provides fresh insights into liver development, suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes postpartum. Addition of these cues results in the first functional, inducible, hPSC-derived hepatocyte for predictive toxicology. © 2015 by the American Association for the Study of Liver Diseases.

Rodent analgesia: Assessment and therapeutics.

PubMed

Flecknell, Paul

2018-02-01

Current use of analgesics to control procedure-related pain in laboratory rodents is unacceptably low. Almost all currently available analgesics were developed in small rodents, prior to use in man, so that safety and efficacy data in laboratory assays are available. Greater use of analgesics would be encouraged by critical evaluation of the potential interactions of these compounds with the outcomes of specific research studies. As in other species, effective post-procedural analgesia requires reliable 'cage-side' methods of assessing pain. Recent advances in pain assessment should lead to both more extensive and more effective use of analgesics in these species. Copyright © 2017 Elsevier Ltd. All rights reserved.

Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

PubMed

Takayama, Kazuo; Mizuguchi, Hiroyuki

2017-02-01

Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Immunogenicity and immunomodulatory properties of hepatocyte-like cells derived from human amniotic epithelial cells.

PubMed

Tee, Jing Yang; Vaghjiani, Vijesh; Liu, Yu Han; Murthi, Padma; Chan, James; Manuelpillai, Ursula

2013-01-01

Hepatocyte transplantation is being trialled as an alternative to whole organ transplant for patients with acute liver failure and liver specific metabolic diseases. Due to the scarcity of human hepatocytes, hepatocyte-like cells (HLC) generated from stem cells may become a viable alternative to hepatocyte transplantation. Human amniotic epithelial cells (hAEC) from the placenta have stem cell-like properties and can be differentiated into HLC. Naïve hAEC have low immunogenicity and exert immunomodulatory effects that may facilitate allogeneic transplantation. However, whether the immunogenicity and immunomodulatory properties alter with differentiation into HLC are unknown. We further characterized HLC generated from hAEC, examined changes in human leucocyte antigens (HLA) and co-stimulatory molecules and effects exerted by the HLC on human peripheral blood mononuclear cells (PBMC). HLC derived from hAEC expressed proteins found in hepatocytes, had CYP3A4 drug metabolizing enzyme activity and secreted urea. IFN-γ treatment increased HLA Class IA, Class II and co-stimulatory molecule CD40 expression in the HLC. IFN-γ treated HLC stimulated proliferation of PBMC in one-way mixed lymphocyte reactions and were more immunogenic than undifferentiated hAEC. However, the HLC showed immunomodulatory properties and inhibited mitogen induced PBMC proliferation in vitro. PBMC proliferation may have been inhibited by IL-6, TGF-β1, PGE2 and HLA-G secreted by the HLC. The retention of immunomodulatory properties may enable HLC grafts to survive for longer periods despite the immunogenicity of the HLC.

Generation, characterization and potential therapeutic applications of mature and functional hepatocytes from stem cells.

PubMed

Zhang, Zhenzhen; Liu, Jianfang; Liu, Yang; Li, Zheng; Gao, Wei-Qiang; He, Zuping

2013-02-01

Liver cancer is the sixth most common tumor in the world and the majority of patients with this disease usually die within 1 year. The effective treatment for end-stage liver disease (also known as liver failure), including liver cancer or cirrhosis, is liver transplantation. However, there is a severe shortage of liver donors worldwide, which is the major handicap for the treatment of patients with liver failure. Scarcity of liver donors underscores the urgent need of using stem cell therapy to the end-stage liver disease. Notably, hepatocytes have recently been generated from hepatic and extra-hepatic stem cells. We have obtained mature and functional hepatocytes from rat hepatic stem cells. Here, we review the advancements on hepatic differentiation from various stem cells, including hepatic stem cells, embryonic stem cells, the induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, and probably spermatogonial stem cells. The advantages, disadvantages, and concerns on differentiation of these stem cells into hepatic cells are highlighted. We further address the methodologies, phenotypes, and functional characterization on the differentiation of numerous stem cells into hepatic cells. Differentiation of stem cells into mature and functional hepatocytes, especially from an extra-hepatic stem cell source, would circumvent the scarcity of liver donors and human hepatocytes, and most importantly it would offer an ideal and promising source of hepatocytes for cell therapy and tissue engineering in treating liver disease. Copyright © 2012 Wiley Periodicals, Inc.

Results from a Survey of Current Practices for Sampling of Nervous System in Rodents and Non-rodents in General Toxicity Studies

EPA Science Inventory

A survey of current practices for sampling and examination of the nervous system in rodents and non-rodents for general and neurotoxicity (NT) studies was conducted by the Nervous System Sampling Subcommittee of the STP. For general toxicity studies most of those surveyed (>63%) ...

Hepatitis B virus evasion from cGAS sensing in human hepatocytes.

PubMed

Verrier, Eloi R; Yim, Seung-Ae; Heydmann, Laura; El Saghire, Houssein; Bach, Charlotte; Turon-Lagot, Vincent; Mailly, Laurent; Durand, Sarah C; Lucifora, Julie; Durantel, David; Pessaux, Patrick; Manel, Nicolas; Hirsch, Ivan; Zeisel, Mirjam B; Pochet, Nathalie; Schuster, Catherine; Baumert, Thomas F

2018-04-20

Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic GMP-AMP synthase (cGAS) was identified as a DNA sensor. In this study, we aimed to investigate the functional role of cGAS in sensing of HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss- and gain-of-function experiments combined with cGAS effector gene expression profiling in an infectious cell culture model, primary human hepatocytes and HBV-infected human liver chimeric mice. Here we show that cGAS is expressed in the human liver, primary human hepatocytes and human liver chimeric mice. While naked relaxed-circular HBV DNA is sensed in a cGAS-dependent manner in hepatoma cell lines and primary human hepatocytes, host cell recognition of viral nucleic acids is abolished during HBV infection, suggesting escape from sensing, likely during packaging of the genome into the viral capsid. While the hepatocyte cGAS pathway is functionally active, as shown by reduction of viral cccDNA levels in gain-of-function studies, HBV infection suppressed cGAS expression and function in cell culture models and humanized mice. HBV exploits multiple strategies to evade sensing and antiviral activity of cGAS and its effector pathways. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes

PubMed Central

Hiriart, Marcia; Sanchez-Soto, Carmen; Diaz-Garcia, Carlos Manlio; Castanares, Diana T.; Avitia, Morena; Velasco, Myrian; Mas-Oliva, Jaime; Macias-Silva, Marina; González-Villalpando, Clicerio; Delgado-Coello, Blanca; Sosa-Garrocho, Marcela; Vidaltamayo, Román; Fuentes-Silva, Deyanira

2014-01-01

It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l−1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance. PMID:24995000

Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes.

PubMed

Hiriart, Marcia; Sanchez-Soto, Carmen; Diaz-Garcia, Carlos Manlio; Castanares, Diana T; Avitia, Morena; Velasco, Myrian; Mas-Oliva, Jaime; Macias-Silva, Marina; González-Villalpando, Clicerio; Delgado-Coello, Blanca; Sosa-Garrocho, Marcela; Vidaltamayo, Román; Fuentes-Silva, Deyanira

2014-01-01

It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l(-1) insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

Extending hepatocyte functionality for drug-testing applications using high-viscosity alginate-encapsulated three-dimensional cultures in bioreactors.

PubMed

Miranda, Joana P; Rodrigues, Armanda; Tostões, Rui M; Leite, Sofia; Zimmerman, Heiko; Carrondo, Manuel J T; Alves, Paula M

2010-12-01

The maintenance of differentiated hepatocyte phenotype in vitro depends on several factors-in particular, on extracellular matrix interactions, for example, with three-dimensional (3D) matrices. Alginate hydrogel provides the cells with a good extracellular matrix due to the formation of a massive capsule with semi-permeable properties that allows for diffusion of the medium components into the cells as well as efficient waste product elimination. Simultaneously, alginate protects the cells from shear stress caused by the hydrodynamics when cultured in stirred systems such as bioreactors. We have previously developed a hepatocyte aggregate 3D culture system in a bioreactor where improved hepatocyte functionality could be maintained over longer periods (21 days). In this work, ultra-high-viscosity alginate was used for hepatocyte aggregates entrapment. Hepatocyte biotransformation (phase I and II enzymes), CYP450 inducibility, and secretory capacity (albumin and urea production) were monitored. The analyses were performed in both spinner vessels and bioreactors to test the effect of the pO(2) control, unavailable in the spinners. Performance of alginate-encapsulated hepatocyte aggregates in culture was compared with nonencapsulated aggregate cultures in both bioreactor (controlled environment) and spinner vessels. For both culture systems, hepatocytes' metabolic and biotransformation capacities were maintained for up to 1 month, and encapsulated cells in bioreactors showed the best performance. In particular, albumin production rate increased 2- and 1.5-fold in encapsulated aggregates compared with nonencapsulated aggregates in bioreactor and spinner vessels, respectively. Urea production rate increased twofold in encapsulated cultures compared with nonencapsulated cells, in both bioreactor and spinner vessels. Similarly, in both the bioreactor and the spinner system, cell encapsulation resulted in a 1.5- and 2.8-fold improvement of hepatocyte 7-ethoxycoumarin and

Impairment of Host Liver Repopulation by Transplanted Hepatocytes in Aged Rats and the Release by Short-Term Growth Hormone Treatment.

PubMed

Stock, Peggy; Bielohuby, Maximilian; Staege, Martin S; Hsu, Mei-Ju; Bidlingmaier, Martin; Christ, Bruno

2017-03-01

Hepatocyte transplantation is an alternative to whole liver transplantation. Yet, efficient liver repopulation by transplanted hepatocytes is low in livers of old animals. This restraint might be because of the poor proliferative capacity of aged donor hepatocytes or the regenerative impairment of the recipient livers. The age-dependent liver repopulation by transplanted wild-type hepatocytes was investigated in juvenile and senescent rats deficient in dipeptidyl-peptidase IV. Repopulation was quantified by flow cytometry and histochemical estimation of dipeptidyl-peptidase IV enzyme activity of donor cells in the negative host liver. As a potential pathway involved, expression of cell cycle proteins was assessed. Irrespective of the age of the donor hepatocytes, large cell clusters appeared in juvenile, but only small clusters in senescent host livers. Because juvenile and senescent donor hepatocytes were likewise functional, host-derived factor(s) impaired senescent host liver repopulation. Growth hormone levels were significantly higher in juvenile than in senescent rats, suggesting that growth hormone might promote host liver repopulation. Indeed, short-term treatment with growth hormone augmented senescent host liver repopulation involving the growth hormone-mediated release of the transcriptional blockade of genes associated with cell cycle progression. Short-term growth hormone substitution might improve liver repopulation by transplanted hepatocytes, thus augmenting the therapeutic benefit of clinical hepatocyte transplantation in older patients. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Evaluation of drug-metabolizing and functional competence of human hepatocytes incubated under hypothermia in different media for clinical infusion.

PubMed

Gómez-Lechón, María José; Lahoz, Agustín; Jiménez, Nuria; Bonora, Ana; Castell, José V; Donato, María Teresa

2008-01-01

Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. Key factors for clinical cell transplantation to progress is to prevent hepatocyte damage, loss of viability and cell functionality, factors that depend on the nature of the tissue used for isolation to a large extent. The main sources of tissue for hepatocyte isolation are marginal livers that are unsuitable for transplantation, and segments from reduced cadaveric grafts. Hepatocellular transplantation requires infusing human hepatocytes in suspension over a period of minutes to hours. The beneficial effect of hypothermic preservation of hepatocytes in infusion medium has been reported, but how critical issues towards the success of cell transplantation, such as the composition of infusion medium and duration of hepatocyte storage will affect hepatocyte quality for clinical cell infusion has not been systematically investigated. Infusion media composition is phosphate-buffered saline containing anticoagulants and human serum albumin. The supplementation of infusion media with glucose or N-acetyl-cystein, or with both components at the same time, has been investigated. After isolation, hepatocytes were suspended in each infusion medium and a sample at the 0 time point was harvested for cell viability and functional assessment. Thereafter, cells were incubated in different infusion media agitated on a rocker platform to simulate the clinical infusion technique. The time course of hepatocyte viability, funtionality (drug-metabolizing enzymes, ureogenic capability, ATP, glycogen, and GSH levels), apoptosis (caspase-3 activation), and attachment and monolayer formation were analyzed. The optimal preservation of cell viability, attaching capacity, and functionality, particularly GSH and glycogen levels, as well as drug-metabolizing cytochrome P450 enzymes, was found in infusion media supplemented with 2 mM N-acetyl-cystein and 15 mM glucose.

Novel developmental biology-based protocol of embryonic stem cell differentiation to morphologically sound and functional yet immature hepatocytes.

PubMed

Bukong, Terence N; Lo, Tracie; Szabo, Gyongyi; Dolganiuc, Angela

2012-05-01

Liver diseases are common in the United States and often require liver transplantation; however, donated organs are limited and thus alternative sources for liver cells are in high demand. Embryonic stem cells (ESC) can provide a continuous and readily available source of liver cells. ESC differentiation to liver cells is yet to be fully understood and comprehensive differentiation protocols are yet to be defined. Here, we aimed to achieve human (h)ESC differentiation into mature hepatocytes using defined recombinant differentiation factors and metabolites. Embryonic stem cell H1 line was sub-cultured on feeder layer. We induced hESCs into endodermal differentiation succeeded by early/late hepatic specification and finally into hepatocyte maturation using step combinations of Activin A and fibroblast growth factor (FGF)-2 for 7 days; followed by FGF-4 and bone morphogenic protein 2 (BMP2) for 7 days, succeeded by FGF-10 + hepatocyte growth factor 4 + epidermal growth factor for 14 days. Specific inhibitors/stimulators were added sequentially throughout differentiation. Cells were analysed by PCR, flow cytometry, microscopy or functional assays. Our hESC differentiation protocol resulted in viable cells with hepatocyte shape and morphology. We observed gradual changes in cell transcriptome, including up-regulation of differentiation-promoting GATA4, GATA6, POU5F1 and HNF4 transcription factors, steady levels of stemness-promoting SOX-2 and low levels of Nanog, as defined by PCR. The hESC-derived hepatocytes expressed alpha-antitrypsin, CD81, cytokeratin 8 and low density lipoprotein (LDL) receptor. The levels of alpha-fetoprotein and proliferation marker Ki-67 in hESC-derived hepatocytes remained elevated. Unlike stem cells, the hESC-derived hepatocytes performed LDL uptake, produced albumin and alanine aminotransferase and had functional alcohol dehydrogenase. We report a novel protocol for hESC differentiation into morphological and functional yet immature

Palm kernel cake extract exerts hepatoprotective activity in heat-induced oxidative stress in chicken hepatocytes.

PubMed

Oskoueian, Ehsan; Abdullah, Norhani; Idrus, Zulkifli; Ebrahimi, Mahdi; Goh, Yong Meng; Shakeri, Majid; Oskoueian, Armin

2014-10-02

Palm kernel cake (PKC), the most abundant by-product of oil palm industry is believed to contain bioactive compounds with hepatoprotective potential. These compounds may serve as hepatoprotective agents which could help the poultry industry to alleviate adverse effects of heat stress on liver function in chickens. This study was performed to evaluate the hepatoprotective potential of PKC extract in heat-induced oxidative stress in chicken hepatocytes. The nature of the active metabolites and elucidation of the possible mechanism involved were also investigated. The PKC extract possessed free radical scavenging activity with values significantly (p < 0.05) lower than silymarin as the reference antioxidant. Heat-induced oxidative stress in chicken hepatocyte impaired the total protein, lipid peroxidation and antioxidant enzymes activity significantly (p < 0.05). Treatment of heat-induced hepatocytes with PKC extract (125 μg/ml) and silymarin as positive control increased these values significantly (p < 0.05). The real time PCR and western blot analyses revealed the significant (p < 0.05) up-regulation of oxidative stress biomarkers including TNF-like, IFN-γ and IL-1β genes; NF-κB, COX-2, iNOS and Hsp70 proteins expression upon heat stress in chicken hepatocytes. The PKC extract and silymarin were able to alleviate the expression of all of these biomarkers in heat-induced chicken hepatocytes. The gas chromatography-mass spectrometry analysis of PKC extract showed the presence of fatty acids, phenolic compounds, sugar derivatives and other organic compounds such as furfural which could be responsible for the observed hepatoprotective activity. Palm kernel cake extract could be a potential agent to protect hepatocytes function under heat induced oxidative stress.

Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress.

PubMed

Mohammad, Mohammad K; Avila, Diana; Zhang, Jingwen; Barve, Shirish; Arteel, Gavin; McClain, Craig; Joshi-Barve, Swati

2012-11-15

Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. Also, it is produced endogenously via lipid peroxidation and cellular metabolism of certain amino acids and drugs. Acrolein is cytotoxic to many cell types including hepatocytes; however the mechanisms are not fully understood. We examined the molecular mechanisms underlying acrolein hepatotoxicity in primary human hepatocytes and hepatoma cells. Acrolein, at pathophysiological concentrations, caused a dose-dependent loss of viability of hepatocytes. The death was apoptotic at moderate and necrotic at high concentrations of acrolein. Acrolein exposure rapidly and dramatically decreased intracellular glutathione and overall antioxidant capacity, and activated the stress-signaling MAP-kinases JNK, p42/44 and p38. Our data demonstrate for the first time in human hepatocytes, that acrolein triggered endoplasmic reticulum (ER) stress and activated eIF2α, ATF-3 and -4, and Gadd153/CHOP, resulting in cell death. Notably, the protective/adaptive component of ER stress was not activated, and acrolein failed to up-regulate the protective ER-chaperones, GRP78 and GRP94. Additionally, exposure to acrolein disrupted mitochondrial integrity/function, and led to the release of pro-apoptotic proteins and ATP depletion. Acrolein-induced cell death was attenuated by N-acetyl cysteine, phenyl-butyric acid, and caspase and JNK inhibitors. Our data demonstrate that exposure to acrolein induces a variety of stress responses in hepatocytes, including GSH depletion, oxidative stress, mitochondrial dysfunction and ER stress (without ER-protective responses) which together contribute to acrolein toxicity. Our study defines basic mechanisms underlying liver injury caused by reactive aldehyde pollutants such as acrolein. Copyright © 2012 Elsevier Inc. All rights reserved.

Glycogen content in hepatocytes is related with their size in normal rat liver but not in cirrhotic one.

PubMed

Bezborodkina, Natalia N; Chestnova, Anna Yu; Vorobev, Mikhail L; Kudryavtsev, Boris N

2016-04-01

Hepatocytes differ from one another by the degree of the ploidy, size, position in the liver lobule, and level of the DNA-synthetic processes. It is believed, that the cell size exerts substantial influence on the metabolism of the hepatocytes and the glycogen content in them. The aim of the present study was to test this hypothesis. Dry weight of hepatocytes, their ploidy and glycogen content were determined in the normal and the cirrhotic rat liver. Liver cirrhosis in rats was produced by chronic inhalation of CCl4 vapours in the course of 6 months. A combined cytophotometric method was used. Dry weight of the cell, its glycogen and DNA content were successively measured on a mapped preparation. Hepatocytes of each ploidy class in the normal and the cirrhotic rat liver accumulated glycogen at the same rate. In the normal liver, there was a distinct correlation between the size of hepatocytes and glycogen content in them. This correlation was observed in each ploidy class, and was especially pronounced in the class of mononucleate tetraploid hepatocytes. In the cirrhotic liver, there was no correlation between the size of the cells and their glycogen content. The impairment of liver lobular structure probably explains the observed lack of correlation between hepatocyte size and their glycogen content in the cirrhotic liver. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Pluripotent stem cell derived hepatocyte like cells and their potential in toxicity screening.

PubMed

Greenhough, Sebastian; Medine, Claire N; Hay, David C

2010-12-30

Despite considerable progress in modelling human liver toxicity, the requirement still exists for efficient, predictive and cost effective in vitro models to reduce attrition during drug development. Thousands of compounds fail in this process, with hepatotoxicity being one of the significant causes of failure. The cost of clinical studies is substantial, therefore it is essential that toxicological screening is performed early on in the drug development process. Human hepatocytes represent the gold standard model for evaluating drug toxicity, but are a limited resource. Current alternative models are based on immortalised cell lines and animal tissue, but these are limited by poor function, exhibit species variability and show instability in culture. Pluripotent stem cells are an attractive alternative as they are capable of self-renewal and differentiation to all three germ layers, and thereby represent a potentially inexhaustible source of somatic cells. The differentiation of human embryonic stem cells and induced pluripotent stem cells to functional hepatocyte like cells has recently been reported. Further development of this technology could lead to the scalable production of hepatocyte like cells for liver toxicity screening and clinical therapies. Additionally, induced pluripotent stem cell derived hepatocyte like cells may permit in vitro modelling of gene polymorphisms and genetic diseases. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Branches of NF-κb signaling pathway regulate hepatocyte proliferation in rat liver regeneration.

PubMed

Chang, C F; Zhao, W M; Mei, J X; Zhou, Y; Pan, C Y; Xu, T T; Xu, C S

2015-07-13

Previous studies have demonstrated that the nuclear factor κB (NF-κB) pathway is involved in promoting cell proliferation. To further explore the regulatory branches and their sequence in the NF-κB pathway in the promotion of hepatocyte proliferation at the transcriptional level during rat liver regeneration, Rat Genome 230 2.0 array was used to detect the expression changes of the isolated hepatocytes. We found that many genes involved in the NF-κB pathway (including 73 known genes and 19 homologous genes) and cell proliferation (including 484 genes and 104 homologous genes) were associated with liver regeneration. Expression profile function (Ep) was used to analyze the biological processes. It was revealed that the NF-κB pathway promoted hepatocyte proliferation through three branches. Several methods of integrated statistics were applied to extract and screen key genes in liver regeneration, and it indicated that eight genes may play a vital role in rat liver regeneration. To confirm the above predicted results, Ccnd1, Jun and Myc were analyzed using qRT-PCR, and the results were generally consistent with that of microarray data. It is concluded that 3 branches and 8 key genes involved in the NF-κB pathway regulate hepatocyte proliferation during rat liver regeneration.

γγ coincidence spectrometer for instrumental neutron-activation analysis

NASA Astrophysics Data System (ADS)

Tomlin, B. E.; Zeisler, R.; Lindstrom, R. M.

2008-05-01

Neutron-activation analysis (NAA) is an important technique for the accurate and precise determination of trace and ultra-trace elemental compositions. The application of γγ coincidence counting to NAA in order to enhance specificity was first explored over 40 years ago but has not evolved into a regularly used technique. A γγ coincidence spectrometer has been constructed at the National Institute of Standards and Technology, using two HPGe γ-ray detectors and an all-digital data-acquisition system, for the purpose of exploring coincidence NAA and its value in characterizing reference materials. This paper describes the initial evaluation of the quantitative precision of coincidence counting versus singles spectrometry, based upon a sample of neutron-irradiated bovine liver material.

Neurobiology of rodent self-grooming and its value for translational neuroscience

PubMed Central

Kalueff, Allan V.; Stewart, Adam Michael; Song, Cai; Berridge, Kent C.; Graybiel, Ann M.; Fentress, John C.

2016-01-01

Self-grooming is a complex innate behaviour with an evolutionary conserved sequencing pattern and is one of the most frequently performed behavioural activities in rodents. In this Review, we discuss the neurobiology of rodent self-grooming, and we highlight studies of rodent models of neuropsychiatric disorders — including models of autism spectrum disorder and obsessive compulsive disorder — that have assessed self-grooming phenotypes. We suggest that rodent self-grooming may be a useful measure of repetitive behaviour in such models, and therefore of value to translational psychiatry. Assessment of rodent self-grooming may also be useful for understanding the neural circuits that are involved in complex sequential patterns of action. PMID:26675822

Hypothermic maintenance of hepatocyte spheroids.

PubMed

Lai, Pamela H; Meng, Qin; Sielaff, Timothy D; Hu, Wei-Shou

2005-01-01

Primary hepatocytes form spheroids under some culture conditions. These spheroids exhibit many tissue-like ultrastructures and retain many liver-specific functions over a long period of time. They are attractive for many applications employing liver cells. The ability to maintain their viability and functions at a reduced temperature to allow for transportation to the site of their application will facilitate their use. Furthermore, with their structural and functional similarity, they could possibly be used as a model system for studying various liver ischemias. The effect of hypothermic treatment was assessed by oxygen consumption rate, ATP, H2O2, and caspase 8 content, as well as albumin and urea synthesis, during and posttreatment. No single outcome variable gives a superlative quantification of hypothermic damage. Taken together, the hypothermic treatment can be seen as increasingly damaging as the temperature decreases from 21 degrees C to 15 degrees C and 4 degrees C. The addition of the chemical protectants glutathione, N-acetyl-L-cystein (NAC), and tauroursodeoxycholic acid (TUDCA) decreased the damaging effect of hypothermic treatment. This protection effect was even more profound when spheroids were preincubated with the protectant for 24 h, and was most prominent at 4 degrees C. The viability of the hypothermically treated hepatocyte spheroids was confirmed by laser scanning confocal microscopy. The method reported provides a means of maintaining spheroids' viability and may allow for their distribution to application sites at a distance.

Evaluation of the nutrient-upgraded rodent food bar for rodent spaceflight experiments.

PubMed

Sun, Gwo-Shing; Tou, Janet C; Liittschwager, Kurt; Herrera, Anna M; Hill, Esther L; Girten, Beverly; Reiss-Bubenheim, Debra; Vasques, Marilyn

2010-01-01

Selection of an appropriate diet for rodent spaceflight experiments is critical and may have significant effects on mission results. The National Aeronautics and Space Administration (NASA) rodent food bar (RFB) was reformulated and designated as the nutrient-upgraded RFB (NuRFB). The objectives of this study were to determine whether the NuRFB nutrient formulation meets the 1995 National Research Council (NRC) nutrient recommendations and whether the NuRFB can be used for short-term (45-d) and long-term (90-d) spaceflight experiments. Nutrient and moisture analyses of the NuRFB were performed. Young (age 13-14 wk) male Sprague-Dawley rats (n=16/group) were individually caged and fed a diet treatment consisting of 1) NuRFB, 2) RFB, or 3) modified AIN-93G containing 4% instead of the 7% fat for 45- or 90-d. At the end of the study, organs were weighted, and serum clinical chemistry indicators of organ function and hematologic measurements were determined. Chemical analysis of the diet ingredients showed that the NuRFB met the 1995 NRC nutrient recommendations for rats. Subsequent animal feeding studies showed that NuRFB was comparable to RFB and modified AIN-93G for supporting rat growth and body weight maintenance. In addition, the safety of the NuRFB for use as a spaceflight diet was indicated by the absence of changes in organ weight or function. Based on the study results, the NuRFB performed similarly to the RFB and met the criteria necessary for short-term and long-term rodent spaceflight experiments. Published by Elsevier Inc.

Accumulation of iron by primary rat hepatocytes in long-term culture: changes in nuclear shape mediated by non-transferrin-bound forms of iron.

PubMed Central

Cable, E. E.; Connor, J. R.; Isom, H. C.

1998-01-01

We have previously shown that hepatocytes in long-term dimethylsulfoxide (DMSO) culture, fed a chemically defined medium, are highly differentiated and an excellent in vitro model of adult liver. Hepatocytes in long-term DMSO culture can be iron loaded by exposure to non-transferrin-bound iron (NTBI) in the form of ferrous sulfate (FeSO4), ferric nitrilotriacetate, or trimethylhexanoyl (TMH)-ferrocene. Holotransferrin, at equivalent times and concentrations, was unable to load hepatocytes. Of the iron compounds tested, TMH-ferrocene most accurately simulated the morphological features of iron-loaded hepatocytes in vivo. When exposed to 25 micromol/L TMH-ferrocene, hepatocytes loaded increasing amounts of iron for 2 months before the cells died. When exposed to lower concentrations of TMH-ferrocene (as low as 2.5 micromol/L), hepatocytes continuously loaded iron and remained viable for more than 2 months. The cellular deposition of iron was different in hepatocytes exposed to TMH-ferrocene compared with those exposed to FeSO4; exposure to TMH-ferrocene resulted in the presence of more ferritin cores within lysosomes than were seen with FeSO4. When the concentration of TMH-ferrocene was increased, a greater number of ferritin cores were observed within the lysosome, and total cellular ferritin, as assessed by Western blot, increased. The formation of hemosiderin was also observed. Furthermore, nuclear shape was distorted in iron-loaded hepatocytes. The extent of deviation from circularity in the nucleus correlated with increasing concentrations of TMH-ferrocene and was greater in hepatocytes exposed to FeSO4 than an equivalent concentration of TMH-ferrocene. The deviation from circularity was smallest in hepatocytes that contained well formed ferritin cores and increased in hepatocytes that contained greater amounts of hemosiderin. Furthermore, in hepatocytes treated with FeSO4, a large amount of cell-associated iron was detected but without a significant increase in

Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells

PubMed Central

Peters, Derek T.; Henderson, Christopher A.; Warren, Curtis R.; Friesen, Max; Xia, Fang; Becker, Caroline E.; Musunuru, Kiran; Cowan, Chad A.

2016-01-01

ABSTRACT Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. PMID:27143754

The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas-permeable membranes.

PubMed

Xiao, Wenjin; Shinohara, Marie; Komori, Kikuo; Sakai, Yasuyuki; Matsui, Hitoshi; Osada, Tomoharu

2014-01-01

Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%-O2 (+)] or physiological oxygen concentrations [10%-O2 (+), 5%-O2 (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas-impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS-O2 (-)]. The results indicated that the hepatocytes under 10%-O2 (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS-O2 (-) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug-metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long-term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen-permeable membrane system to provide a simple method for in vitro studies. © 2014 American Institute of Chemical Engineers.

Regulation of ketogenesis during the suckling-weanling transition in the rat. Studies with isolated hepatocytes.

PubMed Central

Benito, M; Whitelaw, E; Williamson, D H

1979-01-01

The rates of ketogenesis from endogenous substrates, butyrate or oleate, have been measured in isolated hepatocytes from suckling and weanling rats. Ketogenesis from endogenous substrate and from oleate decreased on weaning, whereas the rate from butyrate remained unchanged. It is concluded that the major site of regulation of ketogenesis during this period of development involves the disposal of long-chain fatty acyl-CoA between the esterification and beta-oxidation pathways. Modulators of lipogenesis [dihydroxyacetone and 5-(tetradecyloxy)-2-furoic acid] did not alter the rate of ketogenesis in hepatocytes from suckling rats, and it is suggested that this is due to the low rate of lipogenesis in these cells. Hepatocytes from fed weanling rats have a high rate of lipogenesis and evidence is presented for a reciprocal relationship between ketogenesis and lipogenesis, and ketogenesis, and esterification in these cells. Dibutyryl cyclic AMP stimulated ketogenesis from oleate in hepatocytes from fed weanling rats, even in the presence of an inhibitor of lipogenesis [5-(tetradecyloxy)-2-furoic acid], but not in cells from suckling rats. It is suggested that cyclic AMP may act via inhibition of esterification and that in hepatocytes from suckling rats ketogenesis is already maximally stimulated by the high basal concentrations of cyclic AMP [Beaudry, Chiasson & Exton (1977) Am. J. Physiol. 233, E175--E180]. PMID:226064

Myristoylated PreS1-domain of the hepatitis B virus L-protein mediates specific binding to differentiated hepatocytes.

PubMed

Meier, Anja; Mehrle, Stefan; Weiss, Thomas S; Mier, Walter; Urban, Stephan

2013-07-01

Chronic infection with the human hepatitis B virus (HBV) is a global health problem and a main cause of progressive liver diseases. HBV exhibits a narrow host range, replicating primarily in hepatocytes. Both host and hepatocyte specificity presumably involve specific receptor interactions on the target cell; however, direct evidence for this hypothesis is missing. Following the observation that HBV entry is specifically blocked by L-protein-derived preS1-lipopeptides, we visualized specific HBV receptor/ligand complexes on hepatic cells and quantified the turnover kinetics. Using fluorescein isothiocyanate-labeled, myristoylated HBV preS1-peptides we demonstrate (1) the presence of a highly specific HBV receptor on the plasma membrane of HBV-susceptible primary human and tupaia hepatocytes and HepaRG cells but also on hepatocytes from the nonsusceptible species mouse, rat, rabbit and dog; (2) the requirement of a differentiated state of the hepatocyte for specific preS1-binding; (3) the lack of detectable amounts of the receptor on HepG2 and HuH7 cells; (4) a slow receptor turnover at the hepatocyte membrane; and (5) an association of the receptor with actin microfilaments. The presence of the preS1-receptor in primary hepatocytes from some non-HBV-susceptible species indicates that the lack of susceptibility of these cells is owed to a postbinding step. These findings suggest that HBV hepatotropism is mediated by the highly selective expression of a yet unknown receptor* on differentiated hepatocytes, while species specificity of the HBV infection requires selective downstream events, e.g., the presence of host dependency or the absence of host restriction factors. The criteria defined here will allow narrowing down reasonable receptor candidates and provide a binding assay for HBV-receptor expression screens in hepatic cells. Copyright © 2012 American Association for the Study of Liver Diseases.

Demonstration of nuclear compartmentalization of glutathione in hepatocytes.

PubMed Central

Bellomo, G; Vairetti, M; Stivala, L; Mirabelli, F; Richelmi, P; Orrenius, S

1992-01-01

The intracellular distribution of glutathione (GSH) in cultured hepatocytes has been investigated by using the compound monochlorobimane (BmCl), which interacts specifically with GSH to form a highly fluorescent adduct. Image analysis of BmCl-labeled hepatocytes predominantly localized the fluorescence in the nucleus; the nuclear/cytoplasmic concentration gradient was approximately three. This concentration gradient was collapsed by treatment of the cells with ATP-depleting agents. The uneven distribution of BmCl fluorescence was not attributable to (i) nonspecific interaction of BmCl with protein sulfhydryl groups, (ii) any selective nuclear localization of the GSH transferase(s) catalyzing formation of the GSH-BmCl conjugate, or (iii) any apparent alterations in cell morphology from culture conditions, suggesting that this distribution did, indeed, reflect a nuclear compartmentalization of GSH. That the nuclear pool of GSH was found more resistant to depletion by several agents than the cytoplasmic pool supports the assumption that GSH is essential in protecting DNA and other nuclear structures from chemical injury. Images PMID:1584774

Mitochondrial Impairment as a Key Factor for the Lack of Attachment after Cold Storage of Hepatocyte Suspensions

PubMed Central

Pless-Petig, Gesine; Walter, Björn; Bienholz, Anja

2018-01-01

Isolated primary hepatocytes, which are widely used for pharmacological and clinical purposes, usually undergo certain periods of cold storage in suspension during processing. While adherent hepatocytes were shown previously to suffer iron-dependent cell death during cold (4 °C) storage and early rewarming, we previously found little iron-dependent hepatocyte death in suspension but severely decreased attachment ability unless iron chelators were added. Here, we focus on the role of mitochondrial impairment in this nonattachment of hepatocyte suspensions. Rat hepatocyte suspensions were stored in a chloride-poor, glycine-containing cold storage solution with and without iron chelators at 4 °C. After 1 wk of cold storage in the basic cold storage solution, cell viability in suspension was unchanged, while cell attachment was decreased by >80%. In the stored cells, a loss of mitochondrial membrane potential (MMP), a decrease in adenosine triphosphate (ATP) content (2 ± 2 nmol/106 cells after cold storage, 5 ± 3 nmol/106 cells after rewarming vs. control 29 ± 6 nmol/106 cells), and a decrease in oxygen consumption (101 ± 59 pmol sec−1 per 106 cells after rewarming vs. control 232 ± 83 pmol sec−1 per 106 cells) were observed. Addition of iron chelators to the cold storage solution increased cell attachment to 53% ± 20% and protected against loss of MMP, and cells were able to partially regenerate ATP during rewarming (15 ± 10 nmol/106 cells). Increased attachment could also be achieved by addition of the inhibitor combination of mitochondrial permeability transition, trifluoperazine + fructose. Attached hepatocytes displayed normal MMP and mitochondrial morphology. Additional experiments with freshly isolated hepatocytes confirmed that impaired energy production—as elicited by an inhibitor of the respiratory chain, antimycin A—can decrease cell attachment without decreasing viability. Taken together, these results suggest that mitochondrial impairment

Mitochondrial Impairment as a Key Factor for the Lack of Attachment after Cold Storage of Hepatocyte Suspensions.

PubMed

Pless-Petig, Gesine; Walter, Björn; Bienholz, Anja; Rauen, Ursula

2017-12-01

Isolated primary hepatocytes, which are widely used for pharmacological and clinical purposes, usually undergo certain periods of cold storage in suspension during processing. While adherent hepatocytes were shown previously to suffer iron-dependent cell death during cold (4 °C) storage and early rewarming, we previously found little iron-dependent hepatocyte death in suspension but severely decreased attachment ability unless iron chelators were added. Here, we focus on the role of mitochondrial impairment in this nonattachment of hepatocyte suspensions. Rat hepatocyte suspensions were stored in a chloride-poor, glycine-containing cold storage solution with and without iron chelators at 4 °C. After 1 wk of cold storage in the basic cold storage solution, cell viability in suspension was unchanged, while cell attachment was decreased by >80%. In the stored cells, a loss of mitochondrial membrane potential (MMP), a decrease in adenosine triphosphate (ATP) content (2 ± 2 nmol/10 6 cells after cold storage, 5 ± 3 nmol/10 6 cells after rewarming vs. control 29 ± 6 nmol/10 6 cells), and a decrease in oxygen consumption (101 ± 59 pmol sec -1 per 10 6 cells after rewarming vs. control 232 ± 83 pmol sec -1 per 10 6 cells) were observed. Addition of iron chelators to the cold storage solution increased cell attachment to 53% ± 20% and protected against loss of MMP, and cells were able to partially regenerate ATP during rewarming (15 ± 10 nmol/10 6 cells). Increased attachment could also be achieved by addition of the inhibitor combination of mitochondrial permeability transition, trifluoperazine + fructose. Attached hepatocytes displayed normal MMP and mitochondrial morphology. Additional experiments with freshly isolated hepatocytes confirmed that impaired energy production-as elicited by an inhibitor of the respiratory chain, antimycin A-can decrease cell attachment without decreasing viability. Taken together, these results suggest that mitochondrial impairment

Quantitative structure toxicity relationships for phenols in isolated rat hepatocytes.

PubMed

Moridani, Majid Y; Siraki, Arno; O'Brien, Peter J

2003-05-06

Quantitative structure toxicity relationship (QSTR) equations were obtained to predict and describe the cytotoxicity of 31 phenols using logLD(50) as a concentration to induce 50% cytotoxicity of isolated rat hepatocytes in 2 h and logP as octanol/water partitioning: logLD(50) (microM)=-0.588(+/-0.059)logP+4.652(+/-0.153) (n=27, r(2)=0.801, s=0.261, P<1 x 10(-9)). Hydroquinone, catechol, 4-nitrophenol, and 2,4-dinitrophenol were outliers for this equation. When the ionization constant pK(a) was considered as a contributing factor a two-parameter QSTR equation was derived: logLD(50) (microM)=-0.595(+/-0.051)logP+0.197(+/-0.029)pK(a)+2.665(+/-0.281) (n=28, r(2)=0.859, s=0.218, P<1 x 10(-6)). Using sigma+, the Brown variation of the Hammet electronic constant, as a contributing parameter, the cytotoxicity of phenols towards hepatocytes were defined by logLD(50) (microM)=-0.594(+/-0.052)logP-0.552(+/-0.085)sigma+ +4.540(+/-0.132) (n=28, r(2)=0.853, s=0.223, P<1 x 10(-6)). Replacing sigma+ with the homolytic bond dissociation energy (BDE) for (X-PhOH+PhO.-->X-PhO.+PhOH) led to logLD(50) (microM)=-0.601(+/-0.066)logP-0.040(+/-0.018)BDE+4.611(+/-0.166) (n=23, r(2)=0.827, s=0.223, P<0.05). Hydroquinone, catechol and 2-nitrophenol were outliers for the above equations. Using redox potential and logP led to a new correlation: logLD(50) (microM)=-0.529(+/-0.135)logP+2.077(+/-0.892)E(p/2)+2.806(+/-0.592) (n=15, r(2)=0.561, s=0.383, P<0.05) with 4-nitrophenol as an outlier. Our findings indicate that phenols with higher lipophilicity, BDE, or sigma+ values or with lower pK(a) and redox potential were more toxic towards hepatocytes. We also showed that a collapse of hepatocyte mitochondrial membrane potential preceded the cytotoxicity of most phenols. Our study indicates that one or a combination of mechanisms; i.e. mitochondrial uncoupling, phenoxy radicals, or phenol metabolism to quinone methides and quinones, contribute to phenol cytotoxicity towards hepatocytes depending on

[Effects of dietary fibers on hepatocyte apoptosis in rats with alimentary polyhypovitaminosis].

PubMed

Trushina, É N; Mustafina, O K; Beketova, N A; Vrzhesinskaia, O A; Kodentsova, V M

2014-01-01

The effect of dietary fibers (DF) of wheat bran on hepatocyte apoptosis in rats adequately provided with vitamins or insufficiently supplied with vitamins has been investigated. 48 male Wistar rats (initial body mass--58.1 +/- 0.5 g) were randomly divided into 6 groups and fed with semi-synthetic diet, containing 100% or 20% of vitamin mixture (Vit) with or without addition of DF in the dose corresponding to the upper allowable level of its consumption (5% of diet mass) for 4 weeks. The animals of the 1 group received 100% of vitamin mixture (100% Vit); 2 group--100% Vit + DF; 3 group--20% of vitamin mixture with full exclusion of vitamins E, B1 and B2 (20% Vit); 4 group--20% of vitamin mixture and DF (20% Vit + DF). The next 5 days rats from vitamin-deficient groups were fed with diets supplemented with 80% of vitamins from their content in control group: (5 group--20% Vit + 80% Vit; 6 group--20% Vit + DF + 80% Vit). The suspension of hepatocytes was received by Becton Dickinson Medimachine System (USA). Hepatocyte apoptosis was assessed by the method of flow cytometry using Beckman Coulter FC 500 (USA) cytometer by stained cells with Annexin V-FITC/ 7-Amino-Actinomycin D Kit (Beckman Coulter, USA). In rats fed complete semi-synthetic diet supplemented with DF (100% Vit + DF) the hepatocyte apoptosis was higher by 22% (p < 0.10) than that in rats of control group (4.99 +/- 1.82%). In rats fed diets with low vitamin content (groups: 20% Vit and 20% Vit + DF) the hepatocyte apoptosis was significantly higher (p < 0.05) than that in the control group and reached 7.03 +/- 1.74 and 7.26 +/- 1.13% accordingly. Normalization of vitamin content in the diets of rats from deficient groups during 5 days had no effect on the severity of apoptosis regardless from presence (8.02 +/- 2.18%) or absence of the DF (8.04 +/- 1.66%). Adding DF in dose corresponding to the upper allowable level of consumption, on the background of adequate vitamin content in the diet is accompanied by

Differences in the half-lives of some mitochondrial rat liver enzymes may derive partially from hepatocyte heterogeneity.

PubMed

Vargas, J L; Roche, E; Knecht, E; Grisolía, S

1987-11-16

The different turnover rates of rat liver mitochondrial enzymes make autophagy unlikely to be the main mechanism for degradation of mitochondria. Although alternatives have been presented, hepatocyte heterogeneity has not been considered. Lighter hepatocytes isolated in a discontinuous Percoll gradient contain more glutamate dehydrogenase (GDH) (half-life 1 day) and a more active autophagic system than heavier hepatocytes. The latter contain more carbamoyl phosphate synthase (CPS) and ornithine carbamoyl transferase (OTC) (half-lives 8 days) but less lysosomal activity. As expected, isolated autophagic vacuoles contain, relative to the mitochondrial content, 3-times less OTC and CPS than GDH, probably reflecting a faster lysosomal engulfment of mitochondria in the light hepatocytes (which contain more GDH). These data may explain some of the half-life differences of the enzymes studied.

Chemical carcinogenesis: Too many rodent carcinogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ames, B.N.; Gold, L.S.

1990-10-01

The administration of chemicals at the maximum tolerated dose (MTD) in standard animal cancer tests is postulated to increase cell division (mitogenesis), which in turn increases rates of mutagenesis and thus carcinogenesis. The animal data are consistent with this mechanism, because a high proportion{endash}about half{endash}of all chemicals tested (whether natural or synthetic) are indeed rodent carcinogens. The authors conclude that at the low doses of most human exposures, where cell killing does not occur, the hazards to humans of rodent carcinogens may be much lower than is commonly assumed.

Cytochrome P450 peroxidase/peroxygenase mediated xenobiotic metabolic activation and cytotoxicity in isolated hepatocytes.

PubMed

Anari, M R; Khan, S; Liu, Z C; O'Brien, P J

1995-12-01

Cytochrome P450 (P450) can utilize organic hydroperoxides and peracids to support hydroxylation and dealkylation of various P450 substrates. However, the biological significance of this P450 peroxygenase/peroxidase activity in the bioactivation of xenobiotics in intact cells has not been demonstrated. We have shown that tert-butyl hydroperoxide (tBHP) markedly enhances 3-20-fold the cytotoxicity of various aromatic hydrocarbons and their phenolic metabolites. The tBHP-enhanced hepatocyte cytotoxicity of 4-nitroanisole (4-NA) and 4-hydroxyanisole (4-HA) was also accompanied by an increase in the hepatocyte O-demethylation of 4-NA and 4-HA up to 7.5- and 21-fold, respectively. Hepatocyte GSH conjugation by 4-HA was also markedly increased by tBHP. An LC/MS analysis of the GSH conjugates identified hydroquinone-GSH and 4-methoxy-catechol:GSH conjugates as the predominant adducts. Pretreatment of hepatocytes with P450 inhibitors, e.g., phenylimidazole, prevented tBHP-enhanced 4-HA metabolism, GSH depletion, and cytotoxicity. In conclusion, hydroperoxides can therefore be used by intact cells to support the bioactivation of xenobiotics through the P450 peroxidase/peroxygenase system.

Tularemia and plague survey in rodents in an earthquake zone in southeastern Iran

PubMed Central

Gyuranecz, Miklós

2015-01-01

OBJECTIVES: Earthquakes are one the most common natural disasters that lead to increased mortality and morbidity from transmissible diseases, partially because the rodents displaced by an earthquake can lead to an increased rate of disease transmission. The aim of this study was to evaluate the prevalence of plague and tularemia in rodents in the earthquake zones in southeastern Iran. METHODS: In April 2013, a research team was dispatched to explore the possible presence of diseases in rodents displaced by a recent earthquake magnitude 7.7 around the cities of Khash and Saravan in Sistan and Baluchestan Province. Rodents were trapped near and in the earthquake zone, in a location where an outbreak of tularemia was reported in 2007. Rodent serums were tested for a serological survey using an enzyme-linked immunosorbent assay. RESULTS: In the 13 areas that were studied, nine rodents were caught over a total of 200 trap-days. Forty-eight fleas and 10 ticks were obtained from the rodents. The ticks were from the Hyalomma genus and the fleas were from the Xenopsylla genus. All the trapped rodents were Tatera indica. Serological results were negative for plague, but the serum agglutination test was positive for tularemia in one of the rodents. Tatera indica has never been previously documented to be involved in the transmission of tularemia. CONCLUSIONS: No evidence of the plague cycle was found in the rodents of the area, but evidence was found of tularemia infection in rodents, as demonstrated by a positive serological test for tularemia in one rodent. PMID:26602769

Hepatocyte-mediated cytotoxicity and host defense mechanisms in the alcohol-injured liver.

PubMed

McVicker, Benita L; Thiele, Geoffrey M; Tuma, Dean J; Casey, Carol A

2014-09-01

The consumption of alcohol is associated with many health issues including alcoholic liver disease (ALD). The natural history of ALD involves the development of steatosis, inflammation (steatohepatitis), fibrosis and cirrhosis. During the stage of steatohepatitis, the combination of inflammation and cellular damage can progress to a severe condition termed alcoholic hepatitis (AH). Unfortunately, the pathogenesis of AH remains uncharacterized. Some modulations have been identified in host defense and liver immunity mechanisms during AH that highlight the role of intrahepatic lymphocyte accumulation and associated inflammatory cytokine responses. Also, it is hypothesized that alcohol-induced injury to liver cells may significantly contribute to the aberrant lymphocytic distribution that is seen in AH. In particular, the regulation of lymphocytes by hepatocytes may be disrupted in the alcoholic liver resulting in altered immunologic homeostasis and perpetuation of disease. In recent studies, it was demonstrated that the direct killing of activated T lymphocytes by hepatocytes is facilitated by the asialoglycoprotein receptor (ASGPR). The ASGPR is a well-characterized glycoprotein receptor that is exclusively expressed by hepatocytes. This hepatic receptor is known for its role in the clearance of desialylated glycoproteins or cells, yet neither its physiological function nor its role in disease states has been determined. Interestingly, alcohol markedly impairs ASGPR function; however, the effect alcohol has on ASGPR-mediated cytotoxicity of lymphocytes remains to be elucidated. This review discusses the contribution of hepatocytes in immunological regulation and, importantly, how pathological effects of ethanol disrupt hepatocellular-mediated defense mechanisms.

[Permeability of isolated rat hepatocyte plasma membranes for molecules of dimethyl sulfoxide].

PubMed

Kuleshova, L G; Gordienko, E A; Kovalenko, I F

2014-01-01

We have studied permeability of isolated rat hepatocyte membranes for molecules of dimethyl sulfoxide (DMSO) at different hypertonicity of a cryoprotective medium. The permeability coefficient of hepatocyte membranes κ1 for DMSO molecules was shown to be the differential function of osmotic pressure between a cell and an extracellular medium. Ten-fold augmentation of DMSO concentration in the cryoprotective medium causes the decrease of permeability coefficients κ1 probably associated with the increased viscosity in membrane-adjacent liquid layers as well as partial limitations appeared as a result of change in cell membrane shape after hepatocyte dehydration. We have found out that in aqueous solutions of NaCl (2246 mOsm/l) and DMSO (2250 mOsm/l) the filtration coefficient L(p) in the presence of a penetrating cryoprotectant (L(pDMSO) = (4.45 ± 0.04) x 10(-14) m3/Ns) is 3 orders lower compared to the case with electrolyte (L(pNaCl) = (2.25 ± 0.25) x 10(-11) m3/Ns). This phenomenon is stipulated by the cross impact of flows of a cryoprotectant and water at the stage of cell dehydration. Pronounced lipophilicity of DMSO, geometric parameters of its molecule as well as the presence of large aqueous pores in rat hepatocyte membranes allow of suggesting the availability of two ways of penetrating this cryoprotectant into the cells by non-specific diffusion through membrane lipid areas and hydrophilic channels.

Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

PubMed

Orsini, Malina; Sperber, Saskia; Noor, Fozia; Hoffmann, Esther; Weber, Susanne N; Hall, Rabea A; Lammert, Frank; Heinzle, Elmar

2018-01-01

Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile.

PubMed

Heslop, James A; Rowe, Cliff; Walsh, Joanne; Sison-Young, Rowena; Jenkins, Roz; Kamalian, Laleh; Kia, Richard; Hay, David; Jones, Robert P; Malik, Hassan Z; Fenwick, Stephen; Chadwick, Amy E; Mills, John; Kitteringham, Neil R; Goldring, Chris E P; Kevin Park, B

2017-01-01

The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation. This phenomenon reduces many unique hepatocyte functions, limiting their use in drug disposition and toxicity assessment. The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells. We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation. Cells were cultured for 0, 24, 72 and 168 h as a monolayer in order to permit unrestricted hepatocyte dedifferentiation, so as to reveal the causative signalling pathways and factors in this process, by pathway analysis. A total of 3430 proteins were identified with a false detection rate of <1 %, of which 1117 were quantified at every time point. Increasing numbers of significantly differentially expressed proteins compared with the freshly isolated cells were observed at 24 h (40 proteins), 72 h (118 proteins) and 168 h (272 proteins) (p < 0.05). In particular, cytochromes P450 and mitochondrial proteins underwent major changes, confirmed by functional studies and investigated by pathway analysis. We report the key factors and pathways which underlie the loss of hepatic phenotype in vitro, particularly those driving the large-scale and selective remodelling of the mitochondrial and metabolic proteomes. In summary, these findings expand the current understanding of dedifferentiation should facilitate further development of simple and complex hepatic culture systems.

Effect of copper chloride exposure on the membrane potential and cytosolic free calcium in primary cultured chicken hepatocytes.

PubMed

Jia, Xuexia; Chen, Long; Li, Jingtao; Su, Rongsheng; Shi, Dayou; Tang, Zhaoxin

2012-09-01

This study was conducted to examine the effects of copper on membrane potential and cytosolic free calcium in isolated primary chicken hepatocytes which were exposed to different concentration of Cu(2+) (0, 10, 50, 100 μM) or a mixture of Cu(2+) and vitamin C (50 and 50 μM, respectively). Viability, membrane potential, and cytosolic free Ca(2+) of monolayer cultured hepatocytes were investigated at the indicated time point. Results showed that, among the different concentrations of Cu(2+) exposure, the viability of hepatocytes treated with 100 μM Cu(2+) was the worst at the 12th and 24th hours. The effects of Cu(2+) on viability and proliferation were time and dose dependent. Further investigation indicated that Cu(2+) exposure significantly enhanced cytosolic free Ca(2+) in hepatocytes, compared to that in control group, at the 24th hour. Meanwhile, membrane potential was noticeably reduced in hepatocytes increasing concentration of Cu(2+). Taking these results together, we have shown that Cu(2+) can cause toxicity to primary chicken hepatocytes in excessive dose and the effect of Cu(2+) exposure on membrane potential is not site specific, which is probably mediated by the changes of cytosolic free Ca(2+).

Functional hepatocyte clusters on bioactive blend silk matrices towards generating bioartificial liver constructs.

PubMed

Janani, G; Nandi, Samit K; Mandal, Biman B

2018-02-01

The creation of in vitro functional hepatic tissue simulating micro-environmental niche of native liver is a keen area of research due to its demand in bioartificial liver (BAL) and cell-based tissue engineering. Here, we investigated the potential of novel blend (BA) silk scaffold fabricated by blending mulberry (Bombyx mori, BM) silk fibroin with cell adhesion motif (RGD) rich non-mulberry (Antheraea assamensis, AA) silk fibroin, in generating a functional liver construct. Three-dimensional (3D) porous silk scaffolds (BM, AA and BA) were physico-chemically characterized and functionally evaluated using human hepatocarcinoma cells (HepG2) and primary neonatal rat hepatocytes. The growth and distribution of hepatocytes within the scaffolds were tracked by FESEM, alamar blue proliferation assay and live/dead staining. Hemocompatible BA scaffolds supported the formation of high density hepatocyte clusters, facilitating cell-matrix and cell-cell interactions. Blend scaffolds evinced enhanced liver-specific functions of cultured hepatocytes in terms of albumin synthesis, urea synthesis and cytochrome P450 enzyme activity over 21 days. Subcutaneous implantation of scaffolds demonstrated minimal macrophage infiltration in blend scaffolds. These findings substantiate that the integral property of blend (BA) scaffold offers a befitting environment by influencing spheroidal growth of hepatocytes with enhanced biological activity. Collectively, the present study provides a new 3D bio-matrix niche for growing functional liver cells that would have future prospects in BAL as well as regenerative medicine. An end stage liver disease called cirrhosis perturbs the self-healing ability and physiological functions of liver. Due to the scarcity of healthy donors, a functional in vitro hepatic construct retaining the liver-specific functions is in great demand for its prospects in bioartificial liver (BAL) and cell-based tissue engineering. Physicochemical attributes of a matrix