[Development of a Lead-covered Case for a Wireless X-ray Output Analyzer to Perform CT Half-value Layer Measurements].

Measurement of the half-value layer (HVL) is a difficult task in computed tomography (CT) , because a nonrotating X-ray tube must be used. The purpose of this study is to develop a lead-covered case, which enables HVL measurements with a rotating CT X-ray tube. The lead-covered case was manufactured from acrylic and lead plates, which are 3 mm thick and have a slit. The slit-detector distance can be selected between 14 mm and 122 mm. HVL measurements were performed using a wireless X-ray output analyzer "Piranha." We used the following exposure conditions: tube voltages of 80, 100, and 120 kV; a tube current of 550 mA; and an exposure time of 1.0 s. The HVLs were measured by using the following two methods: (a) Nonrotating method-a conventional method that uses the nonrotating exposure mode. (b) Rotating method-a new method that uses the lead-covered case and the rotating exposure mode. As a result, when the slit-detector distance was 58 mm, the HVL values obtained by the nonrotating and rotating methods were 4.38 and 4.24 mmAl at 80 kV, 5.51 and 5.37 mmAl at 100 kV, 6.61 and 6.48 mmAl at 120 kV, respectively. A lead-covered case, which enables the measurement of the HVL in a rotating X-ray tube, was developed. The case is useful in measuring the HVLs at facilities that cannot fix the X-ray tube.

Involvement of bone marrow-derived stromal cells in gastrointestinal cancer development and metastasis.

BACKGROUND: The involvement of bone marrow (BM) in tumor-stroma reactions or tumor development has not been examined in a cancer allograft, which has otherwise been appropriate for assessing therapeutic modalities. We investigated the fate of BM-derived cells in colon cancer allografts and liver metastases in mice. METHODS: C57BL/6 mice were irradiated and rescued by BM transplantation from green fluorescent protein (GFP)-transgenic mice. MC38 colon cancer cells were stably transfected with the pDsRed gene in order to identify tumor cells by fluorescence. These were inoculated into the mice to generate subcutaneous allografted tumors or liver metastases. The tumors were observed under confocal microscopy and fluorescent immunohistochemistry to determine the fate of tumor versus BM-derived cells. RESULTS: GFP-positive (GFP(+)) cells were consistently identified as vimentin(+), alpha-smooth muscle actin (alphaSMA)(+), spindle-shaped stromal cells in both the subcutaneous tumors and the liver metastases. GFP(+) cells of leukocyte lineage also infiltrated the tumors. Neither GFP(+) CD31(+) endothelial cells nor GFP(+) DsRed(+) cells were detected in the tumor. CONCLUSIONS: BM-derived cells frequently and consistently infiltrated the tumor allografts and metastases as interstitial cells and leukocytes. Cells derived from the fusion of BM cells and tumor cells were not observed. This model may be appropriate for the clarification of the effects of anticancer therapies and the study of BM-derived cells in tumor-host interactions.

Suppressive effect on hepatocyte differentiation of hepatitis C virus core protein.

The influence of hepatitis C virus (HCV) protein(s) on cellular differentiation remains to be clarified. Using murine normal liver epithelial cells, we investigated whether HCV core protein affects differentiation into hepatocytes. Mock and HCV core-expressing cells were stimulated with oncostatin M (OSM) and dexamethasone, and the degree of differentiation was evaluated by measuring the expression of albumin and tyrosine aminotransferase (TAT). Lower amounts after stimulation were found in HCV core-expressing cells than in mock cells. Phosphorylation of the signal transducer and activator transcription factor 3 (STAT3) was prevented by the HCV core under OSM stimulation. Reporter gene assay revealed that the HCV core/Janus kinase (JAK) interaction directly suppressed the OSM-dependent JAK-STAT signal transduction. Furthermore, expression of OSM receptor beta (OSMRbeta) after stimulation was prevented by the HCV core. In conclusion, the HCV core may suppress differentiation into hepatocytes via inhibition of the JAK-STAT pathway and OSMRbeta expression.

Viral covalently closed circular DNA in a non-transgenic mouse model for chronic hepatitis B virus replication.

BACKGROUND/AIMS: The lack of small animal models supporting chronic hepatitis B virus (HBV) infection impedes the assessment of anti-viral drugs in the whole animal. Although transgenic mice have been used for this purpose, these models are clearly different from natural infection, because HBV is produced from the integrated HBV sequence harbored in all hepatocytes. METHODS: Balb/cA nude mice were hydrodynamically injected with a plasmid having 1.5-fold over-length of HBV DNA and analyzed for HBV replication. RESULTS: Hydrodynamically injected mice showed substantial levels of antigenemia and viremia for more than 1 year. Covalently closed circular DNA (cccDNA), the template of viral replication in natural infection, was produced in the livers and was critically involved in the long-term HBV production, because disruption of the pol gene of the inoculated DNA resulted in transient expression of HBV genes for less than 2 months. Administration of the IFNalpha gene transiently suppressed HBV DNA replication, but was not capable of eliminating HBV in this model. CONCLUSIONS: In vivo gene transfer of a plasmid encoding HBV DNA can establish chronic viral replication in mice, which involves, at least in part, new synthesis of the HBV cccDNA episome, thus recapitulating a part of human HBV infection.

Negative regulation of NK cell activities by inhibitory receptor CD94/NKG2A leads to altered NK cell-induced modulation of dendritic cell functions in chronic hepatitis C virus infection.

NK cells are potent activators of dendritic cells (DCs), but it remains obscure how third-party cells affect the ability of NK cells to modulate DC functions. We show here that NK cells derived from healthy donors (N-NK), when cocultured with human liver epithelial cells, induced maturation as well as activation of DCs, such as increased migratory capacity as well as T cell stimulatory activity. In contrast, NK cells from chronic hepatitis C virus-infected donors (HCV-NK) were not capable of activating DCs under the same conditions. In comparison to N-NK, HCV-NK showed higher expression of CD94/NKG2A and produced IL-10 and TGFbeta when cultured with hepatic cells, most of which express HLA-E, a ligand for CD94/NKG2A. Blockade of NKG2A restored the ability of HCV-NK to activate DCs, which appeared to result from the reduced NK cell production of IL-10 and TGFbeta. The blockade also endowed HCV-NK with an ability to drive DCs to generate Th1-polarized CD4+ T cells. These findings show that NK cell modulation of DCs is regulated by third-party cells through NK receptor and its ligand interaction. Aberrant expression of NK receptors may have an impact on the magnitude and direction of DC activation of T cells under pathological conditions, such as chronic viral infection.

Concanavalin a injection activates intrahepatic innate immune cells to provoke an antitumor effect in murine liver.

Concanavalin A (ConA), directly injected into mice, induces T cell-mediated liver injury. However, it remains unclear whether ConA injection can activate innate immune cells, including natural killer (NK) cells and natural killer T (NKT) cells, both of which exist abundantly in the liver. Here we report that ConA injection stimulated interferon (IFN)-gamma production from liver NKT cells as early as 2 hours after injection and augmented YAC-1 cytotoxicity of liver NK cells. ConA-induced NK cell activation required other types of immune cells and critically depended on IFN-gamma. Because a nonhepatotoxic low dose of ConA was capable of fully activating both NKT cells and NK cells, we next addressed the possibility of ConA injection displaying an antitumor effect in the liver without liver injury. A nonhepatotoxic low-dose ConA injection augmented the cytotoxicity of liver NK cells against Colon-26 colon cancer cells and suppressed hepatic metastasis of Colon-26 cells in a NK cell- and IFN-gamma-dependent manner. In conclusion, a nonhepatotoxic low dose of ConA might serve as an immunomodulator that can preferentially activate the innate immune cells to induce an antitumor effect against metastatic liver tumor.

Hepatocyte-specific disruption of Bcl-xL leads to continuous hepatocyte apoptosis and liver fibrotic responses.

BACKGROUND & AIMS: Recent research has suggested that apoptosis could be involved in the development of fibrosis, although it is generally considered to be a mechanism of cell removal without consequences to the tissue. Bcl-xL , an antiapoptotic member of the Bcl-2 family, is expressed in hepatocytes and up-modulated during various pathologic conditions. The aim of this study was to explore the function of Bcl-xL in hepatocytes using the Cre-loxP system and to analyze the consequences of long-term apoptosis in hepatocytes. METHODS: Hepatocytes isolated from mice homozygous for a floxed bcl-x allele (bcl-x fl/fl) were infected with recombinant adenovirus expressing the Cre recombinase gene (AdexCre). Bcl-x fl/fl mice were crossed with Alb-Cre transgenic mice, which express Cre under regulation of the albumin gene promoter to generate hepatocyte-specific Bcl-xL-deficient mice. RESULTS: On AdexCre infection, primary cultured bcl-x fl/fl hepatocytes reduced their expression of Bcl-xL and rapidly underwent apoptosis associated with mitochondrial damage. In vivo hepatocyte-specific disruption of Bcl-xL resulted in spontaneous apoptosis of hepatocytes for more than 6 months. The Bcl-xL -deficient mice showed liver fibrosis with advanced age that was preceded by an increase in hepatic transforming growth factor beta production. In vitro, macrophages and hepatocytes produced transforming growth factor beta on exposure to apoptotic hepatocytes. CONCLUSIONS: The present study identified Bcl-xL as a critical apoptosis antagonist in hepatocytes. Furthermore, it offers proof that persistent apoptosis of parenchymal cells is sufficient to induce fibrotic responses and suggests a mechanistic link between apoptosis and fibrosis.

Little evidence of bone marrow-derived hepatocytes in the replacement of injured liver.

We have tested the ability of bone marrow (BM) cells (BMCs) to form hepatocytes in liver injury models. We used three models: (i) carbon tetrachloride (CCl4) treatment, (ii) albumin-urokinase transgenic mouse [TgN(Alb1Plau)], and (iii) hepatitis B transgenic mouse [TgN(Alb1HBV)]. As a nonselective liver injury model, irradiated C57BL/6 (B6) mice were transplanted with BMCs from GFP transgenic mouse [TgN(ActbEGFP)] or beta-galactosidase transgenic mouse [TgN(MtnLacZ)] followed by the administration of CCl4. Irradiated TgN(Alb1HBV) and TgN(Alb1Plau) were also transplanted with BMCs from TgN(ActbEGFP) or TgN(MtnLacZ). Approximately 1.5 x 106 hepatocytes per liver were analyzed for GFP-positive cells, and the whole livers were inspected for beta-galactosidase expression. No GFP-positive hepatocytes and no gross blue staining of the livers with 5-bromo-4-chloro-3-indolyl beta-d-galactoside in any of the 18 recipient mice analyzed were detected. The livers from female animals with gender-mismatched BM transplantation were also tested with Y chromosome fluorescent in situ hybridization analysis to detect donor-derived cells. A total of five isolated hepatocytes were positive for Y chromosome in 4.1 x 105 hepatocytes analyzed. Our results demonstrate that there is little or no contribution of BMCs to the replacement of injured livers in these models. We conclude that BM-derived cells cannot generally lead to a cure of liver damage.

Gene therapy of Fanconi anemia: preclinical efficacy using lentiviral vectors.

Fanconi anemia (FA) is an inherited cancer susceptibility syndrome caused by mutations in a DNA repair pathway including at least 6 genes (FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG). The clinical course of the disease is dominated by progressive, life-threatening bone marrow failure and high incidence of acute myelogenous leukemia and solid tumors. Allogeneic bone marrow transplantation (BMT) is a therapeutic option but requires HLA-matched donors. Gene therapy holds great promise for FA, but previous attempts to use retroviral vectors in humans have proven ineffective given the impaired proliferation potential of human FA hematopoietic progenitors (HPCs). In this work, we show that using lentiviral vectors efficient genetic correction can be achieved in quiescent hematopoietic progenitors from Fanca(-/-) and Fancc(-/-) mice. Long-term repopulating HPCs were transduced by a single exposure of unfractionated bone marrow mononuclear cells to lentivectors carrying the normal gene. Notably, no cell purification or cytokine prestimulation was necessary. Resistance to DNA- damaging agents was fully restored by lentiviral transduction, allowing for in vivo selection of the corrected cells with nonablative doses of cyclophosphamide. This study strongly supports the use of lentiviral vectors for FA gene therapy in humans.