Osteodystrophy in Cholestatic Liver Diseases Is Attenuated by Anti-γ-Glutamyl Transpeptidase Antibody

Background

Cholestatic liver diseases exhibit higher levels of serum γ-glutamyl transpeptidase (GGT) and incidence of secondary osteoporosis. GGT has been identified as a novel bone-resorbing factor that stimulates osteoclast formation. The aim of this study was to elucidate the interaction of elevated GGT levels and cholestatic liver disease-induced bone loss.

Methods

Wistar rats were divided into three groups: sham-operated control (SO) rats, bile duct ligation (BDL) rats, and anti-GGT antibody-treated BDL rats (AGT). Serum GGT level was measured. Bone mineral density (BMD) was analyzed by dual-energy X-ray absorptiometry. Bone morphometric parameters and microarchitectural properties were determined by micro-computed tomography and histomorphometry of the distal metaphysis of femurs. Alterations of bone metabolism-related factors were evaluated by cytokine array. Effects of GGT on osteoblasts or stromal cells were evaluated by RT-PCR, enzyme activity, and mineralization ability.

Results

Serum levels of GGT were significantly elevated in the BDL-group. In the BDL group, BMD, bone mass percentage, and osteoblast number were significantly decreased, whereas osteoclast number was significantly increased. These alterations were markedly attenuated in the AGT group. The mRNA levels of vascular endothelial growth factor-A, LPS-induced CXC chemokine, monocyte chemoattractant protein-1, tumor necrosis factor-α interleukin-1β and receptor activator of nuclear factor-kappa B ligand were upregulated, and those of interferon-γ and osteoprotegerin were downregulated in the GGT-treated stromal cells. Furthermore, GGT inhibited mineral nodule formation and expression of alkaline phosphatase and bone sialo-protein in osteoblastic cells.

Conclusion

Our results indicate that elevated GGT level is involved in hepatic osteodystrophy through secretion of bone resorbing factor from GGT-stimulated osteoblasts/bone marrow stromal cells. In addition, GGT also possesses suppressive effects on bone formation. Managing elevated GGT levels by anti-GGT antibody may become a novel therapeutic agent for hepatic osteodystrophy in chronic liver diseases.     

Unregulated expression of the imprinted genes H19 and Igf2r in mouse uniparental fetuses.

The present study shows that the H19 and Igf2r genes, which are imprinted and expressed solely from maternal alleles, are expressed in an unregulatable manner in mouse uniparental, androgenetic, and parthenogenetic fetuses at day 9.5 of gestation. In the androgenetic fetuses, the H19 and Igf2r genes were respectively expressed at 12 and 40% of the levels in biparental fetuses. In addition, the expression of both genes was excessive (1259 and 482%, respectively) in the parthenotes. These expressions of the imprinted genes were not regulated by methylation in the regulatory regions. Moreover, the expression of the antisense Igf2r RNA (Air) was also excessive and was not correlated with Igf2r gene expression in the uniparental fetuses. Taken together, these results indicate that the parental specific expression of imprinted genes is not maintained in particular genes in uniparental embryos, which in turn suggests that both parental genomes are required to establish maternal specific expression of the H19 and Igf2r genes by trans-acting mechanisms.     

Identification of cancer stem cell markers in human malignant mesothelioma cells

Malignant mesothelioma (MM) is an aggressive and therapy-resistant neoplasm arising from the pleural mesothelial cells and usually associated with long-term asbestos exposure. Recent studies suggest that tumors contain cancer stem cells (CSCs) and their stem cell characteristics are thought to confer therapy-resistance. However, whether MM cell has any stem cell characteristics is not known. To understand the molecular basis of MM, we first performed serial transplantation of surgical samples into NOD/SCID mice and established new cell lines. Next, we performed marker analysis of the MM cell lines and found that many of them contain SP cells and expressed several putative CSC markers such as CD9, CD24, and CD26. Interestingly, expression of CD26 closely correlated with that of CD24 in some cases. Sorting and culture assay revealed that SP and CD24⁺ cells proliferated by asymmetric cell division-like manner. In addition, CD9⁺ and CD24⁺ cells have higher potential to generate spheroid colony than negative cells in the stem cell medium. Moreover, these marker-positive cells have clear tendency to generate larger tumors in mouse transplantation assay. Taken together, our data suggest that SP, CD9, CD24, and CD26 are CSC markers of MM and could be used as novel therapeutic targets.     

New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication

Background

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed.

Methodology/Principal Findings

Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.

Conclusions/Significance

We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin.     

Quantitation and sensory properties of three newly identified pyroglutamyl oligopeptides in sake

Three new peptides: (pGlu)L-ethyl, (pGlu)LFGP-ethyl and (pGlu)LFNP-ethyl, were identified in the search for pyroglutamyl oligopeptide ethyl esters in sake. The ethyl esterified peptides in sake were quantitated using stable isotope dilution analysis and additional quantitation of (pGlu)L was performed using an external standard method. The concentrations of (pGlu)L-ethyl and (pGlu)L in 33 commercial sake samples ranged from 0.16 to 1.57 mg/L and 1.49 to 7.55 mg/L, respectively. The sensory properties of the pyroglutamyl oligopeptide ethyl esters and corresponding non-esterified peptides were examined: the estimated difference threshold of (pGlu)L (2.0 mg/L) and (pGlu)L-ethyl (0.267 mg/L) was exceeded in 32 and 26 samples, respectively. Estimated thresholds of (pGlu)LFGP-ethyl and (pGlu)LFNP-ethyl were often lower than the levels in quantitated sake samples. The sensory effects of these pyroglutamyl dipeptides on a model sake quality may be negative because of their unpleasant taste, however, (pGlu)LFNP-ethyl may be positive because of its mild taste.     

Gray and Red Fragments of the Egg of the Ascidian Ciona savignyi: Preferential Development of Muscle Cells from Gray Fragments

The egg of the ascidian Ciona savignyi is pinkish red with brownish myoplasm that contains the putative determinants responsible for differentiation of muscle cells. When dechorionated unfertilized eggs were centrifuged at moderate speed, eggs were divided into centripetal, small gray fragments and centrifugal, large red fragments. The former contained the female pronucleus and clear cytoplasm, while most of the latter was filled with yolk granules. An antibody raised against the myoplasm of C. intestinalis eggs extensively stained the cortical region of gray fragments, while the antibody stained only small regions of the red fragments. After insemination, both fragments cleaved and gave rise to partial embryos. When development of muscle and epidermal cells in the partial embryos was examined with specific antibodies, muscle development was conspicuous in gray partial embryos, while epidermal differentiation was extensive in red partial embryos. Furthermore, when expression of markers of differentiation was examined in cleavage-arrested gray and red fragments, the number of arrested gray fragments exhibiting the muscle marker was about three-fold greater than in controls. These results suggest that putative muscle determinants are concentrated into gray fragments.     

A Proton Beam Therapy System Dedicated to Spot-Scanning Increases Accuracy with Moving Tumors by Real-Time Imaging and Gating and Reduces Equipment Size

Purpose

A proton beam therapy (PBT) system has been designed which dedicates to spot-scanning and has a gating function employing the fluoroscopy-based real-time-imaging of internal fiducial markers near tumors. The dose distribution and treatment time of the newly designed real-time-image gated, spot-scanning proton beam therapy (RGPT) were compared with free-breathing spot-scanning proton beam therapy (FBPT) in a simulation.

Materials and Methods

In-house simulation tools and treatment planning system VQA (Hitachi, Ltd., Japan) were used for estimating the dose distribution and treatment time. Simulations were performed for 48 motion parameters (including 8 respiratory patterns and 6 initial breathing timings) on CT data from two patients, A and B, with hepatocellular carcinoma and with clinical target volumes 14.6 cc and 63.1 cc. The respiratory patterns were derived from the actual trajectory of internal fiducial markers taken in X-ray real-time tumor-tracking radiotherapy (RTRT).

Results

With FBPT, 9/48 motion parameters achieved the criteria of successful delivery for patient A and 0/48 for B. With RGPT 48/48 and 42/48 achieved the criteria. Compared with FBPT, the mean liver dose was smaller with RGPT with statistical significance (p<0.001); it decreased from 27% to 13% and 28% to 23% of the prescribed doses for patients A and B, respectively. The relative lengthening of treatment time to administer 3 Gy (RBE) was estimated to be 1.22 (RGPT/FBPT: 138 s/113 s) and 1.72 (207 s/120 s) for patients A and B, respectively.

Conclusions

This simulation study demonstrated that the RGPT was able to improve the dose distribution markedly for moving tumors without very large treatment time extension. The proton beam therapy system dedicated to spot-scanning with a gating function for real-time imaging increases accuracy with moving tumors and reduces the physical size, and subsequently the cost of the equipment as well as of the building housing the equipment.     

Vinculin Is Indispensable for Repopulation by Hematopoietic Stem Cells,Independent of Integrin Function

Vinculin is a highly conserved actin-binding protein that is localized in integrin-mediated focal adhesion complexes. Although critical roles have been proposed for integrins in hematopoietic stem cell (HSC) function, little is known about the involvement of intracellular focal adhesion proteins in HSC functions. This study showed that the ability of c-Kit⁺Sca1⁺Lin⁻ HSCs to support reconstitution of hematopoiesis after competitive transplantation was severely impaired by lentiviral transduction with short hairpin RNA sequences for vinculin. The potential of these HSCs to differentiate into granulocytic and monocytic lineages, to migrate toward stromal cell-derived factor 1α, and to home to the bone marrow in vivo were not inhibited by the loss of vinculin. However, the capacities to form long term culture-initiating cells and cobblestone-like areas were abolished in vinculin-silenced c-Kit⁺Sca1⁺Lin⁻ HSCs. In contrast, adhesion to the extracellular matrix was inhibited by silencing of talin-1, but not of vinculin. Whole body in vivo luminescence analyses to detect transduced HSCs confirmed the role of vinculin in long term HSC reconstitution. Our results suggest that vinculin is an indispensable factor determining HSC repopulation capacity, independent of integrin functions.     

HNK-1 Epitope-carrying Tenascin-C Spliced Variant Regulates the Proliferation of Mouse Embryonic Neural Stem Cells

Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into neuronal and glial cells. NSCs are the source for neurogenesis during central nervous system development from fetal and adult stages. Although the human natural killer-1 (HNK-1) carbohydrate epitope is expressed predominantly in the nervous system and involved in intercellular adhesion, cell migration, and synaptic plasticity, the expression patterns and functional roles of HNK-1-containing glycoconjugates in NSCs have not been fully recognized. We found that HNK-1 was expressed in embryonic mouse NSCs and that this expression was lost during the process of differentiation. Based on proteomics analysis, it was revealed that the HNK-1 epitopes were almost exclusively displayed on an extracellular matrix protein, tenascin-C (TNC), in the mouse embryonic NSCs. Furthermore, the HNK-1 epitope was found to be present only on the largest isoform of the TNC molecules. In addition, the expression of HNK-1 was dependent on expression of the largest TNC variant but not by enzymes involved in the biosynthesis of HNK-1. By knocking down HNK-1 sulfotransferase or TNC by small interfering RNA, we further demonstrated that HNK-1 on TNC was involved in the proliferation of NSCs via modulation of the expression level of the epidermal growth factor receptor. Our finding provides insights into the function of HNK-1 carbohydrate epitopes in NSCs to maintain stemness during neural development.