development-hepatitis-b-virus-reporter-system-to-monitor-early-stages

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Japan Tobacco Inc.

The demand for clinical use of hepatocytes is escalating because cell transplantation will be an alternative to orthotopic liver transplantation and the shortage of liver donors is a serious problem throughout the world. However, the supply of fresh differentiated hepatocytes is limited and methods for cryopreservation of hepatocytes that can proliferate with hepatic functions are not satisfactorily established.

Proliferation of rat small hepatocytes after long-term cryopreservation.

Small hepatocytes (SHs), which are known to be hepatic progenitor cells, were isolated from an adult rat liver. SHs in a colony sometimes change their shape from small to large and from flat to rising/piled-up. The aim of the present study is to clarify whether the alteration of cell shape is correlated with the maturation of SHs and whether extracellular matrix (ECM) can induce the morphological changes of SHs. We used liver-enriched transcription factors (LETFs) such as hepatocyte nuclear factor (HNF) 4 alpha, HNF6, CCAAT/enhancer binding proteins (C/EBP) alpha, and C/EBP beta, tryptophan 2,3-dioxygenase (TO), and serine dehydratase (SDH) as markers of hepatic maturation. To enrich the number of SH colonies, the colonies were isolated from dishes and replated. Replated colonies proliferated and the average number of cells per colony was about five times larger at day 9 than at day 1. When the cells were treated with laminin, type IV collagen, a mixture of laminin and type IV collagen, Matrigel or collagen gel (CG), only the cells treated with Matrigel dramatically changed their shape within several days and had reduced growth activity, whereas the cells treated with other ECM did not. HNF4 alpha, HNF6, C/EBP alpha, C/EBP beta, and TO were well expressed in the cells treated with Matrigel. Furthermore, addition of both glucagon and dexamethasone dramatically induced the expression of SDH mRNA and protein in the cells treated with Matrigel. In conclusion, morphological changes of SHs may be correlated with hepatic maturation and basement membrane (BM)-like structure may induce the morphological changes of SHs.

Morphological changes induced by extracellular matrix are correlated with maturation of rat small hepatocytes.

Surgeons sometimes encounter hyperbilirubinemia without mechanical obstruction of the biliary tree postoperatively. Many of these patients have bacterial infections and endotoxemia. Kupffer's cells stimulated by endotoxin secrete inflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. We hypothesized that TNF-alpha and IL-6 might be involved in the pathogenesis of hyperbilirubinemia.

Tumor necrosis factor-alpha and interleukin-6 reduce bile canalicular contractions of rat hepatocytes.

Hybrid bioartificial liver devices supporting a large mass of metabolically active hepatocytes are thought to be necessary for the successful treatment of patients with severe acute liver failure. However, it is very difficult to obtain cells with both growth activity and differentiated functions. Rat small hepatocytes (SHs), which are hepatic progenitor cells, can differentiate into mature hepatocytes and reconstruct a hepatic organoid by interacting with hepatic nonparenchymal cells (NPCs).

Rapid formation of hepatic organoid in collagen sponge by rat small hepatocytes and hepatic nonparenchymal cells.

The morphogenesis and movement of bile canaliculi (BC) are not well understood. This is because culture of hepatocytes that maintain polarity of cell membranes and possess highly differentiated functions has never been successful. We found that small hepatocytes (SHs), which are known to be hepatic progenitor cells, could proliferate and differentiate into mature hepatocytes and that BC-like structures developed between rising/piled-up cells. We investigated how BC-like structures developed with maturation of SHs and whether the structures were functionally active as BC. Hepatic cells, including SHs, were isolated from an adult rat liver and cultured. Immunocytochemistry and immunoblotting for BC proteins, such as ectoATPase, 5'-nucleotidase, dipeptidylpeptidase IV, and multidrug-resistance associated protein 2, were examined and time-lapse microscopy was used for the observation of BC contractions. Secretion of bilirubin into the reconstructed BC was also observed. The results of immunocytochemistry, immunoblots, and immunoelectron micrographs revealed that BC proteins were localized in the intercellular space that coincided with BC-like structures reconstructed between rising/piled-up cells. Tight junction-associated protein ZO-1 was also expressed along the BC-like structures. Bilirubin added to the medium were secreted into BC-like structure and accumulated without leakage. Time-lapse microscopy showed continuous contractions of reconstructed BC. In conclusion, BC-like structures reconstructed by SHs may be functional with membrane polarity, secretory ability, and motility. These results show that this culture system may suitable for investigating the mechanism of the formation of BC and their functions.

Bile canalicular formation in hepatic organoid reconstructed by rat small hepatocytes and nonparenchymal cells.

We examined whether small hepatocytes (SHs), which are hepatic progenitor cells, could be isolated from a normal human liver and whether human hepatic cells could form hepatic organoids in a collagen sponge. Normal liver tissues were obtained from resected specimens from nine patients who underwent hepatic resection. Isolated hepatic cells were plated on dishes and a collagen sponge. More than 1 month later, SH-like cells appeared and proliferated on the dishes, whereas cell aggregates were formed in the sponge and showed characteristic tissue architecture: columnar and/or cuboidal epithelial cells lined the surface of the sponge. Clusters of epithelial cells with a large cytoplasm and ductular structures were observed under the lining cells. The lining and ductular cells were positive for cytokeratins 7 and 19, which indicated they were biliary epithelial cells (BECs), and the epithelial cells forming clusters were positive for the anti-human hepatocyte antibody, identifying them as hepatocytes. Some lining cells were positive for both the hepatic marker and the BEC markers. The cells in the collagen sponge actively proliferated and the hepatocytes excreted albumin into the medium. Thus, hepatic organoids could be reconstructed in a collagen sponge by normal human liver cells.

Hepatic organoid formation in collagen sponge of cells isolated from human liver tissues.

Small hepatocytes (SH), which are hepatic progenitor cells, were isolated from an adult rat liver. SH in a colony sometimes change their shape from small to large and from flat to rising/piled-up. The morphological changes of SH may be correlated with hepatic maturation. Cytochrome P450s (CYP) are drug-metabolizing enzymes and the expression is one of hepatic differentiated functions. However, it is well known that the re-expression and maintenance of CYP activity are very difficult in cultured hepatocytes. We investigated the expression of CYP and the enzymatic activities in long-term cultured SH.

Expression of cytochrome P450 enzymes in hepatic organoid reconstructed by rat small hepatocytes.

Hyperbaric oxygen therapy (HBO) has been used for many clinical treatments, including primary liver non-function. However, the cellular mechanism by which HBO treatment ameliorates liver function is not understood. Therefore, the purpose of this study was to elucidate this cellular mechanism using primary cultured rat hepatocytes in in vitro studies. Hepatocytes were treated with HBO at 1 day after plating, and the morphological and functional characteristics of bile canaliculi formed in cultured hepatocytes were observed by time-lapse microscopy. Multidrug resistance protein-2 localization was observed by confocal laser microscopy. In cultured hepatocytes, the labeling index in the HBO group at 2 days after treatment was significantly higher than that in the control group. In addition, the proliferating cellular nuclear antigen level in the HBO group was significantly higher than that in the control group. The contraction of the bile canaliculi in the HBO group was slower than in the control group and the dilatation of bile canaliculi in the HBO group was much larger than in the control group. Multidrug resistance protein-2 in the HBO group was localized at the apical membrane. These results show that HBO stimulates hepatocytes to proliferate and HBO normalizes multidrug resistance protein-2 localization to the apical membrane, which could dilate bile canaliculi.

Hyperbaric oxygen stimulates cell proliferation and normalizes multidrug resistance protein-2 protein localization in primary rat hepatocytes.

Bacteria isolated from infections in abdominal surgery during the period from April 2008 to March 2009 were investigated in a multicenter study in Japan, and the following results were obtained. In this series, 712 strains including 18 strains of Candida spp. were isolated from 173 (80.5%) of 215 patients with surgical infections. Three hundred and sixty-six strains were isolated from primary infections, and 346 strains were isolated from postoperative infections. From primary infections, anaerobic Gram-negative bacteria were predominant, followed by aerobic Gram-negative bacteria, while from postoperative infections aerobic Gram-positive bacteria were predominant, followed by anaerobic Gram-negative bacteria. Among aerobic Gram-positive bacteria, the isolation rate of Enterococcus spp. was highest, followed by Streptococcus spp., and Staphylococcus spp. in this order, from primary infections, while Enterococcus spp. was highest, followed by Staphylococcus spp. from postoperative infections. Among aerobic Gram-negative bacteria, Escherichia coli was the most predominantly isolated from primary infections, followed by Klebsiella pneumoniae and Pseudomonas aeruginosa, in this order, and from postoperative infections, P aeruginosa was most predominantly isolated, followed by E. coli, Enterobacter cloacae, and K. pneumoniae. Among anaerobic Gram-positive bacteria, the isolation rate of Eggerthella lenta was the highest from primary infections, followed by Parvimonas micra, Streptococcus constellatus and Gemella morbillorum, and from postoperative infections, E. lenta was most predominantly isolated. Among anaerobic Gram-negative bacteria, the isolation rate of Bacteroides fragilis was the highest from primary infections, followed by Bacteroides thetaiotaomicron, Bacteroides ovatus and Bilophila wadsworthia, and from postoperative infections, B. fragilis was most predominantly isolated, followed by B. thetaiotaomicron, B. wadsworthia and B. ovatus, in this order. In this series, we noticed no vancomycin-resistant methicillin-resistant S. aureus, and Enterococcus spp., nor multidrug-resistant P aeruginosa. We should carefully follow up B. wadsworthia which was resistant to various antibiotics, and also Bacteroides spp. which was resistant to many beta-lactam antibiotics.

[Bacteria isolated from surgical infections and their susceptibilities to antimicrobial agents --special references to bacteria isolated between April 2008 and March 2009].

The combination of docetaxel, cisplatin, and S-1 (DCS) chemotherapy is expected to be a promising regimen for advanced gastric cancer. This study was performed to evaluate the efficacy and safety of neoadjuvant DCS chemotherapy for locally advanced resectable gastric cancer.

A phase II study of neoadjuvant combination chemotherapy with docetaxel, cisplatin, and S-1 for locally advanced resectable gastric cancer: nucleotide excision repair (NER) as potential chemoresistance marker.

Nef is one of the accessory proteins of human immunodeficiency viruses. Here, we noted that the relative expression level of Nef(NL4-3) is much lower than that of NefJR-CSF in HEK293 cells. By evaluating the expression level using a Nef mutant, it was indicated that amino acids 129-206 of Nef(NL4-3), that is, the C-terminal region named NLAA129-206, could contain the region responsible for the induction of the low protein expression level. In addition, the expression levels of the enhanced green fluorescent protein and Renilla luciferase became extremely low with the fusion of NLAA129-206. Interestingly, the NLAA129-206-corresponding sequences of other Nef variants with relatively high expression levels also induced the extremely low protein expression level by fusion. These results suggest that the C-terminal region of Nef can generally induce an extremely low protein expression level. Here, we propose that the C-terminal region of Nef could become an excellent tool for the induction of an extremely low expression level of arbitrary proteins by attachment as fusion proteins.

Induction of extremely low protein expression level by fusion of C-terminal region of Nef.

Nef is one of the accessory proteins of the human immunodeficiency virus type 1 (HIV-1). Nef is translated from multiple-spliced mRNAs transcribed from the viral genome, whose mRNAs have a relatively long 5' untranslated region (5'UTR). Here, we identified a cis element in the 5'UTR of Nef mRNA essential for efficient Nef translation, which was named the Nef-translation essential region (NER). Mutants with a deleted NER in the 5'UTR of the HIV-1 NL4-3 strain showed an almost undetectable Nef expression owing to a low Nef translation efficiency. The NER of the NL4-3 strain was predicted to form putative stem loops. Although the 5'UTR showed significant but relatively low internal ribosome entry site (IRES) activity, the mechanism of 5'cap-dependent translation mainly contributed to the Nef translation from its Nef mRNA. Altogether, it was clarified that not only the 5' cap but also the NER in the 5'UTR is an essential cis element for efficient Nef translation, which is not a typical 5'-cap-dependent mechanism, and that there must be an as yet unknown mechanism using the NER for efficient Nef translation.

Identification of essential cis element in 5'UTR of Nef mRNA for Nef translation.

One of the major functions of Nef is in the enhancement of the infectivity of the human and simian immunodeficiency viruses (HIV and SIV, respectively). However, the detailed mechanism of the enhancement of viral infectivity by Nef remains unclear. Additionally, studies of mechanisms by which Nef enhances the infectivity of SIV are not as intensive as those of HIV-1.

Clearly different mechanisms of enhancement of short-lived Nef-mediated viral infectivity between SIV and HIV-1.

A recombinant hepatitis B virus (HBV) expressing NanoLuc (NL) (HBV/NL) was produced by cotransfecting a plasmid containing a 1.2-fold HBV genome carrying the NL gene with a plasmid bearing a packaging-defective 1.2-fold HBV genome into a human hepatoma cell line, HepG2. We found that NL activity in HBV/NL-infected primary hepatocytes or sodium taurocholate cotransporting polypeptide-transduced human hepatocyte-derived cell lines increased linearly for several days after infection and was concordant with HBV RNA levels in the cells. Treatment of the virus-infected cells with HBV inhibitors reduced NL activity in a dose-dependent manner. Detection of HBV/NL infection, monitored by NL activity, was highly sensitive and less expensive than detection using the conventional method to evaluate HBV infection. In addition, because we also studied host factors, this system is applicable not only for studying the HBV life cycle, but also for exploring agent(s) that regulate HBV proliferation.

Novel reporter system to monitor early stages of the hepatitis B virus life cycle.

Tension gastrothorax is a kind of obstructive shock with prolapse and distention of the stomach into the thoracic cavity. Progressive gastric distension leads to mediastinal shift, reduced venous return, decreased cardiac output, and ultimately cardiac arrest. Therefore, it is crucial to decompress the stomach distension for the initial resuscitation of tension gastrothorax.

Article	Year
Proliferation of rat small hepatocytes after long-term cryopreservation. Journal of hepatology\| PubMed ID: 12076856	2002
Morphological changes induced by extracellular matrix are correlated with maturation of rat small hepatocytes. Journal of cellular biochemistry\| PubMed ID: 12210718	2002
Tumor necrosis factor-alpha and interleukin-6 reduce bile canalicular contractions of rat hepatocytes. Surgery\| PubMed ID: 12563244	2003
Rapid formation of hepatic organoid in collagen sponge by rat small hepatocytes and hepatic nonparenchymal cells. Journal of hepatology\| PubMed ID: 14568252	2003
Bile canalicular formation in hepatic organoid reconstructed by rat small hepatocytes and nonparenchymal cells. Journal of cellular physiology\| PubMed ID: 15040008	2004
Hepatic organoid formation in collagen sponge of cells isolated from human liver tissues. Tissue engineering\| PubMed ID: 15869439	2005
Expression of cytochrome P450 enzymes in hepatic organoid reconstructed by rat small hepatocytes. Journal of gastroenterology and hepatology\| PubMed ID: 15946133	2005
Hyperbaric oxygen stimulates cell proliferation and normalizes multidrug resistance protein-2 protein localization in primary rat hepatocytes. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society\| PubMed ID: 16283870	2005
[Bacteria isolated from surgical infections and their susceptibilities to antimicrobial agents --special references to bacteria isolated between April 2008 and March 2009]. The Japanese journal of antibiotics\| PubMed ID: 20919496	2010
A phase II study of neoadjuvant combination chemotherapy with docetaxel, cisplatin, and S-1 for locally advanced resectable gastric cancer: nucleotide excision repair (NER) as potential chem... Cancer chemotherapy and pharmacology\| PubMed ID: 23338051	2013
Induction of extremely low protein expression level by fusion of C-terminal region of Nef. Biotechnology and applied biochemistry\| PubMed ID: 23586835	2012
Identification of essential cis element in 5'UTR of Nef mRNA for Nef translation. Current HIV research\| PubMed ID: 24981044	2014
Clearly different mechanisms of enhancement of short-lived Nef-mediated viral infectivity between SIV and HIV-1. Virology journal\| PubMed ID: 25519983	2014
Novel reporter system to monitor early stages of the hepatitis B virus life cycle. Cancer science\| PubMed ID: 26310603	2015
How to manage tension gastrothorax: a case report of tension gastrothorax with multiple trauma due to traumatic diaphragmatic rupture. International journal of emergency medicine\| PubMed ID: 28127711	2017

Keisuke Harada

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