Gene expression profiling of hepatocarcinogenesis in a mouse model of chronic hepatitis B

<div><p>Background</p><p>Hepatocellular carcinoma (HCC) is a common complication of chronic viral hepatitis. In support of this notion, we have reported that hepatitis B surface antigen (HBsAg)-specific CD8⁺ T lymphocytes critically contribute to inducing chronic liver cell injury that exerts high carcinogenic potential in a hepatitis B virus (HBV) transgenic mouse model. The dynamics of the molecular signatures responsible for hepatocellular carcinogenesis are not fully understood. The current study was designed to determine the serial changes in gene expression profiles in a model of chronic immune-mediated hepatitis.</p><p>Methods</p><p>Three-month-old HBV transgenic mice were immunologically reconstituted with bone marrow cells and splenocytes from syngeneic nontransgenic donors. Liver tissues were obtained every three months until 18 months at which time all mice developed multiple liver tumors. Nitrative DNA lesions and hepatocyte turnover were assessed immunohistochemically. Gene expression profiles were generated by extracting total RNA from the tissues and analyzing by microarray.</p><p>Results</p><p>The nitrative DNA lesions and the regenerative proliferation of hepatocytes were increased during the progression of chronic liver disease. In a gene expression profile analysis of liver samples, the chemokine- and T cell receptor (TCR)-mediated pathways were enhanced during chronic hepatitis, and the EGF- and VEGF-mediated pathways were induced in HCC. Among these molecules, the protein levels of STAT3 were greatly enhanced in all hepatocyte nuclei and further elevated in the cytoplasm in HCC tissue samples at 18 months, and the levels of phosphorylated TP53 (p-p53-Ser 6 and -Ser 15) were increased in liver tissues.</p><p>Conclusions</p><p>HBV-specific immune responses caused unique molecular signatures in the liver tissues of chronic hepatitis and triggered subsequent carcinogenic gene expression profiles in a mouse model. The results suggest a plausible molecular basis responsible for HBV-induced immune pathogenesis of HCC.</p></div

Image_2_Magnetic resonance imaging arterial spin labeling hypoperfusion with diffusion-weighted image hyperintensity is useful for diagnostic imaging of Creutzfeldt–Jakob disease.TIF

Background and objectivesMagnetic resonance imaging with arterial spin labeling (ASL) perfusion imaging is a noninvasive method for quantifying cerebral blood flow (CBF). We aimed to evaluate the clinical utility of ASL perfusion imaging to aid in the diagnosis of Creutzfeldt–Jakob disease (CJD).MethodsThis retrospective study enrolled 10 clinically diagnosed with probable sporadic CJD (sCJD) based on the National CJD Research & Surveillance Unit and EuroCJD criteria and 18 healthy controls (HCs). Diffusion-weighted images (DWIs), CBF images obtained from ASL, N-isopropyl-(123I)-p-iodoamphetamine (123IMP)-single-photon emission computed tomography (SPECT) images, and 18F-fluorodeoxyglucose (18FDG)-positron emission tomography (PET) images were analyzed. First, the cortical values obtained using volume-of-interest (VOI) analysis were normalized using the global mean in each modality. The cortical regions were classified into DWI-High (≥ +1 SD) and DWI-Normal (ResultsThe mean values of ASL-CBF (N = 10), 123IMP-SPECT (N = 8), and 18FDG-PET (N = 3) in DWI-High regions were significantly lower than those in the DWI-Normal regions (p DiscussionPatients with CJD showed ASL hypoperfusion in lesions with DWI hyperintensity, suggesting that ASL-CBF could be beneficial for the diagnostic aid of CJD.</p

STAT3 expression in the model of chronic hepatitis B.

<p>(A) STAT3 levels in the liver tissues at the indicated time points (two representatives of each), the tumor tissues (18M-Tum) and HCC cell lines (HepG2 and PLC/PRF/5) were evaluated by Western blotting, and normalized to β-actin. (B) The liver tissues were stained with hematoxylin and eosin, and analyzed immunohistochemically. #282 and #283 mice (0M: unmanipuladed) were 24-month-old. Original magnifications, x 200 (bar represents 50 μm).</p

Table_1_Magnetic resonance imaging arterial spin labeling hypoperfusion with diffusion-weighted image hyperintensity is useful for diagnostic imaging of Creutzfeldt–Jakob disease.pdf

Background and objectivesMagnetic resonance imaging with arterial spin labeling (ASL) perfusion imaging is a noninvasive method for quantifying cerebral blood flow (CBF). We aimed to evaluate the clinical utility of ASL perfusion imaging to aid in the diagnosis of Creutzfeldt–Jakob disease (CJD).MethodsThis retrospective study enrolled 10 clinically diagnosed with probable sporadic CJD (sCJD) based on the National CJD Research & Surveillance Unit and EuroCJD criteria and 18 healthy controls (HCs). Diffusion-weighted images (DWIs), CBF images obtained from ASL, N-isopropyl-(123I)-p-iodoamphetamine (123IMP)-single-photon emission computed tomography (SPECT) images, and 18F-fluorodeoxyglucose (18FDG)-positron emission tomography (PET) images were analyzed. First, the cortical values obtained using volume-of-interest (VOI) analysis were normalized using the global mean in each modality. The cortical regions were classified into DWI-High (≥ +1 SD) and DWI-Normal (ResultsThe mean values of ASL-CBF (N = 10), 123IMP-SPECT (N = 8), and 18FDG-PET (N = 3) in DWI-High regions were significantly lower than those in the DWI-Normal regions (p DiscussionPatients with CJD showed ASL hypoperfusion in lesions with DWI hyperintensity, suggesting that ASL-CBF could be beneficial for the diagnostic aid of CJD.</p

Image_1_Magnetic resonance imaging arterial spin labeling hypoperfusion with diffusion-weighted image hyperintensity is useful for diagnostic imaging of Creutzfeldt–Jakob disease.TIF

Background and objectivesMagnetic resonance imaging with arterial spin labeling (ASL) perfusion imaging is a noninvasive method for quantifying cerebral blood flow (CBF). We aimed to evaluate the clinical utility of ASL perfusion imaging to aid in the diagnosis of Creutzfeldt–Jakob disease (CJD).MethodsThis retrospective study enrolled 10 clinically diagnosed with probable sporadic CJD (sCJD) based on the National CJD Research & Surveillance Unit and EuroCJD criteria and 18 healthy controls (HCs). Diffusion-weighted images (DWIs), CBF images obtained from ASL, N-isopropyl-(123I)-p-iodoamphetamine (123IMP)-single-photon emission computed tomography (SPECT) images, and 18F-fluorodeoxyglucose (18FDG)-positron emission tomography (PET) images were analyzed. First, the cortical values obtained using volume-of-interest (VOI) analysis were normalized using the global mean in each modality. The cortical regions were classified into DWI-High (≥ +1 SD) and DWI-Normal (ResultsThe mean values of ASL-CBF (N = 10), 123IMP-SPECT (N = 8), and 18FDG-PET (N = 3) in DWI-High regions were significantly lower than those in the DWI-Normal regions (p DiscussionPatients with CJD showed ASL hypoperfusion in lesions with DWI hyperintensity, suggesting that ASL-CBF could be beneficial for the diagnostic aid of CJD.</p

Serial changes of signaling pathways in a model of chronic Hepatitis B.

<p>Serial changes of signaling pathways in a model of chronic Hepatitis B.</p

Tumor-specific changes of signaling pathways.

<p>Tumor-specific changes of signaling pathways.</p

Table_2_Magnetic resonance imaging arterial spin labeling hypoperfusion with diffusion-weighted image hyperintensity is useful for diagnostic imaging of Creutzfeldt–Jakob disease.pdf

Background and objectivesMagnetic resonance imaging with arterial spin labeling (ASL) perfusion imaging is a noninvasive method for quantifying cerebral blood flow (CBF). We aimed to evaluate the clinical utility of ASL perfusion imaging to aid in the diagnosis of Creutzfeldt–Jakob disease (CJD).MethodsThis retrospective study enrolled 10 clinically diagnosed with probable sporadic CJD (sCJD) based on the National CJD Research & Surveillance Unit and EuroCJD criteria and 18 healthy controls (HCs). Diffusion-weighted images (DWIs), CBF images obtained from ASL, N-isopropyl-(123I)-p-iodoamphetamine (123IMP)-single-photon emission computed tomography (SPECT) images, and 18F-fluorodeoxyglucose (18FDG)-positron emission tomography (PET) images were analyzed. First, the cortical values obtained using volume-of-interest (VOI) analysis were normalized using the global mean in each modality. The cortical regions were classified into DWI-High (≥ +1 SD) and DWI-Normal (ResultsThe mean values of ASL-CBF (N = 10), 123IMP-SPECT (N = 8), and 18FDG-PET (N = 3) in DWI-High regions were significantly lower than those in the DWI-Normal regions (p DiscussionPatients with CJD showed ASL hypoperfusion in lesions with DWI hyperintensity, suggesting that ASL-CBF could be beneficial for the diagnostic aid of CJD.</p

TP53 expression in the model of chronic hepatitis B.

<p>Total TP53 levels and phosphorylated TP53 at serines 6 and 15 (p-p53-Ser 6 and -Ser 15) in the liver tissues at the indicated time points (two representatives of each) and the tumor tissues (18M-Tum) were evaluated by Western blotting, and normalized to β -actin.</p

Course of liver disease in a transgenic mouse model of chronic hepatitis B.

<p>(A) Kinetics of serum alanine aminotransferase (ALT) in HBV transgenic mice (open circles; n = 40) were monitored after transferring spleen cells from syngeneic nontransgenic donors that had been previously immunized with recombinant vaccinia virus HBs-vac. All results were compared with unmanipulated age- and sex-matched transgenic mice (filled triangles; n = 20). Adoptive transfer of splenocytes was performed on day 0. Serum ALT activity was monitored to evaluate liver injury. Results were expressed as mean units per liter ± S.D. of ALT activity. (B) Inflammation-induced, reactive nitrogen species and regenerative proliferation of hepatocytes were estimated immunohistochemically using a double immunofluorescence technique for 8-nitroguanine (8-NG) (nuclear, red) and inducible nitric oxide synthase (iNOS) (cytoplasmic, green) expression and anti-proliferating cell nuclear antigen (PCNA) solution at 0 (untreated), 9 and 18 months. PCNA(+) hepatocyte nuclei are indicated with arrows. Original magnifications: x 100 (8-NG / iNOS) and x 400 (PCNA). (C) Quantitative morphometric analysis of PCNA⁺ hepatocytes. Fifty high power (x 400) fields representing 2 mm² of liver tissue were examined. Results are expressed means ± S.E. per 1,000 hepatocytes. Each group represents three animals.</p