Decreased accumulation of ultrasound contrast in the liver of nonalcoholic steatohepatitis rat model.

AIM: To investigate the diagnosis of nonalcoholic steatohepatitis (NASH) using contrast ultrasonography in the NASH rat model. METHODS: The liver in methionine choline-deficient diet (MCDD) rats, a NASH model constructed by feeding an MCDD, was examined by contrast ultrasonography at weeks 2, 4, 8, 12 and 16, with late phase images of contrast ultrasonography (Kupffer imaging) in which contrast enhancement was achieved by incorporation of a contrast agent by Kupffer cells (KCs), and images were compared to those in rats taking a regular chow. RESULTS: Decrease in contrast enhancement was observed first in MCDD rats at week 2. KCs were counted based on immunohistochemistry, but their numbers were not reduced and it was assumed that attenuation of contrast enhancement was attributable to reduced phagocytic activity of the KCs. CONCLUSION: It is suggested that clinical application of contrast ultrasonography may be valuable for non-invasive diagnosis of NASH.

[A case of splenic inflammatory pseudotumor].

A 74-year-old woman underwent abdominal echography at a local clinic and a splenic mass was found. She was hospitalized for detailed examinations and treatment. Splenectomy was performed to make a definite diagnosis and for treatment because a definitive diagnosis could not be made, despite various examinations. Histopathological examination revealed that the lesion was infiltrated by polyclonal lymphoid cells and contained proliferating spindle-shaped fibroblasts without any atypical cells, so the splenic mass was diagnosed as an inflammatory pseudotumor. Because some cases of inflammatory pseudotumor can be diagnosed from the clinical course and imaging findings, this possibility should also be considered in the differential diagnosis of a splenic mass.

Association of tannase-producing Staphylococcus lugdunensis with colon cancer and characterization of a novel tannase gene.

BACKGROUND: The relationship between Streptococcus (St.) bovis endocarditis and colon cancer is well known. In St. bovis, the biotype I strain (formerly, St. gallolyticus) produces tannase that degrades tannins. The aim of this study was to investigate the association of tannase-producing bacteria with colon cancer, and to identify the major tannase-producing bacteria and the gene involved. METHODS: Tannase-producing bacteria were isolated in tannic acid-treated selective agar medium from feces and rectal swabs of 357 patients who underwent colon endoscopy from 1999 to 2004. RESULTS: Tannase-producing bacteria were isolated more frequently from the colon cancer group (24.3%) than from the adenoma or normal groups (14.4%; P < 0.05). S. gallolyticus, Staphylococcus (S.) lugdunensis, Lactobacillus (L.) plantarum, and L. pentosus were all identified as tannase-producing bacteria. Of these, S. lugdunensis was significantly isolated from the advanced-stage cancer group (22.2%; P < 0.001) more than from the early-stage cancer (8.6%) or adenoma (4.9%) groups. The gene (tanA) for tannase in S. lugdunensis was cloned and sequenced. The tanA gene was associated with all S. lugdunensis but not with other bacteria by Southern blotting and polymerase chain reaction. CONCLUSIONS: Tannase-producing S. lugdunensis is associated with advanced-stage colon cancer, and the tanA gene is a useful marker for the detection of S. lugdunensis.

Phagocytosis of ultrasound contrast agent microbubbles by Kupffer cells.

Delayed parenchymal phase images of the liver more than 5 min after IV injection of ultrasound contrast agents are thought to be related to the phagocytosis of contrast agent microbubbles by macrophages. In this study, we examined whether liver-specific macrophages, Kupffer cells, phagocytosed the microbubbles and whether their elimination affected the delayed parenchymal images of the liver. Phase-contrast microscope observations showed that Kupffer cells phagocytosed various contrast agents in vitro. Among the contrast agents used, 99% of Sonazoid and Optison, and 47% of Levovist were phagocytosed, whereas only 7.3% of SonoVue and 0% of Imavist were phagocytosed. Elimination of Kupffer cells in vivo by gadolinium chloride (GdCl(3)) resulted in decreased intensity of the delayed parenchymal images with Sonazoid and Levovist, while SonoVue showed no changes compared with control. Our findings suggested that Kupffer cells phagocytosed contrast agents and they were responsible for the delayed images of contrast ultrasound in the liver.

Preoperative RFA simulation for liver cancer using a CT virtual ultrasound system.

We developed a computed tomography (CT) virtual ultrasound system (CVUS) as an imaging system to support treatment under percutaneous ultrasound (US) guidance. This prototype clinical system, produced in collaboration with Tokyo Medical University, uses display software developed by Toshiba Medical Systems. We examined the utility of this system by scheduling treatment plans preoperatively and simulating puncture and radiofrequency ablation (RFA) for liver cancer. The study enrolled 51 liver cancer patients with 66 nodules 0.8-8cm in diameter in which RFA was performed between June 2004 and December 2004. Virtual US and multiplanar reconstruction (MPR) images were constructed on the basis of DICOM CT data and puncture and ablation of liver cancer were simulated. The following were evaluated: (1) how to avoid complications and determine an appropriate puncture route by simulating puncture with C-mode MPR images; (2) determination of the three-dimensional location of the tumor for ablation, as well as the adjacent organs and vessels, by MPR rotation 360 degrees around the center of the tumor (center lock); and (3) how to determine the center and volume of ablation and avoid injuries to nearby organs and vessels by simulating ablation procedures. C -mode MPR images were effective for (1) determining and modifying the puncture route in 35 of 51 cases (69.6%) and (2) determining the spatial location of vessels and nearby organs in 50 of 51 cases (98.0%) by the center lock; and (3) simulating the ablation helped determine the center and volume of ablation by avoiding injuries to vessels and nearby organs in 45 or 51 cases (88.2%). Taken together, the CVUS allowed easy simulation of local treatment of liver cancer under US guidance using CT data alone and the preoperative simulation predicted an improvement in the safety of local therapy of liver cancer.

Anti-adipogenic regulation underlies hepatic stellate cell transdifferentiation.

Cirrhosis is the most important consequence of alcoholic liver disease for which liver transplantation is the only treatment option available. Transdifferentiation of hepatic stellate cells (HSC) to myofibroblastic cells (MF) is a central event in liver fibrogenesis, and understanding molecular mechanisms that underlie this cellular event provides pivotal insights into development of new therapeutic modalities for cirrhosis. To this end, the authors proposed several years ago that transdifferentiation of quiescent HSC to MF may be causally associated with transcriptional regulation known for adipocyte-preadipocytic fibroblast dedifferentiation. In support of this notion, the authors showed that adipogenic transcription factors and their downstream adipocyte specific genes are expressed abundantly in quiescent HSC and that this expression profile is lost in HM. Further, gain-of-function manipulations for adipogenic transcription factors such as peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and sterol regulatory element binding protein-1c have been shown to reverse culture-induced MF to quiescent HSC. The authors also demonstrated that tumor necrosis factor-alpha and Wnt, known mediators of anti-adipogenesis, also suppress the activity of PPAR-gamma and contribute to HSC-MF transdifferentiation. These results reinforce the concept of adipogenic regulation essential to the quiescent phenotype and the loss of such regulation underlying HSC-HM transdifferentiation. They also provide insights into the molecular basis for the use of PPAR-gamma agonists, which has been advocated for treatment of liver fibrosis.

Ultrasound contrast agent, Levovist microbubbles are phagocytosed by Kupffer cells-In vitro and in vivo studies.

Phase contrast microscopy showed that Levovist microbubbles were phagocytosed by cultured Kupffer cells and moved gradually toward the nucleus from the cell surface. They were diminished in size and disappeared within 30min by diffusion of the gas contained within the microbubbles. In an in vivo study, confocal laser scanning microscopy demonstrated that Levovist microbubbles were trapped and phagocytosed by Kupffer cells. No microbubbles were observed attached to the sinusoidal endothelium. Our findings suggest that liver-specific images obtained following intravenous injection of Levovist are composed of signals from microbubbles phagocytosed by Kupffer cells.

Diagnosis of NASH using delayed parenchymal imaging of contrast ultrasound.

BACKGROUND AND AIMS: Non alcoholic steatohepatitis (NASH) is one of the representative liver diseases in the developed countries. Diagnosis of NASH is dependent on histological findings from liver biopsy. Usefulness of contrast ultrasound with Levovist for diagnosis of NASH is described. METHODS AND MATERIALS: Clinical study: Ultrasound contrast agent, Levovist of 2.5g was injected intravenously. The liver was scanned at 5, 10, 15, 20, 30, 40, and 50min after Levovist injection in different planes using a contrast specific ultrasound mode. Changes in microbubble accumulation in the liver were evaluated. The signal intensity from regions of interest (ROI) on the contrast images was measured and accumulation and decrescence of microbubbles were estimated using the time intensity curves (TICs). The image data and TICs were evaluated by blind reviewers. Fifteen patients with NASH, 8 with alcoholic steatohepatitis (ASH), 45 with non alcoholic fatty liver (NAFL), 10 with chronic hepatitis C (CHC) and 10 healthy volunteers were studied. Animal study: Methionine-choline-deficient diet (MCDD) fed rats were used for NASH model. Correlation between microbubble accumulation and morphological and functional changes of sinusoidal endothelium and macrophage was evaluated. RESULTS: The maximum intensity of contrast ultrasound was decreased and time course decrescence was more rapid in NASH than the other groups. These changes were correlated to the degree of centrilobular and pericellular fibrosis but not to steatosis in histological study. Disturbance of microbubble accumulation was correlated with sinusoidal function rather than morphological changes such as fibrosis and parenchymatitis in the animal studies. CONCLUSIONS: The Levovist contrast study enables differential diagnosis between NASH and other diseases that provoke steatosis and fibrosis.

Differential diagnosis of hepatic nodules using delayed parenchymal phase imaging of levovist contrast ultrasound: comparative study with SPIO-MRI.

T2-weighted fast spin echo images and T2*-weighted gradient-echo images of superparamagnetic iron oxide magnetic resonance imaging (SPIO-MRI) have been reported to reflect the number and function of macrophages in reticuloendothelial organs and be useful to differentiate malignant tumors from benign nodules of liver. We tried to prove that contrast-enhanced ultrasound can diagnose hepatocellular carcinoma (HCC) by comparing the findings of SPIO-MRI and the findings of the liver parenchyma on the delayed parenchymal phase of ultrasound imaging using the intravenous contrast agent Levovist, not through the evaluation of vascular imaging. Forty-six patients (52 nodules) with histopathological diagnosis of hepatic tumors were studied. They consisted of 11 non-malignant nodules (six regenerative nodules and five dysplastic nodules) and 41 HCC. All the patients were examined by Levovist contrast-enhanced ultrasonography and SPIO-MRI. The delayed liver parenchymal images of contrast-enhanced ultrasound using the intravenous contrast agent Levovist were similar to those observed on SPIO-MRI. The similarity of both findings suggests that delayed phase imaging by Levovist is closely related to the number and function of Kupffer cells in liver tumors. The diagnostic accuracy of contrast-enhanced ultrasound for HCC was high (90.4%) demonstrating that it is as reliable as SPIO-MRI.

PPAR Gamma and Hepatic Stellate Cells.

Activation of Hepatic stellate cells (HSC) in fibrogenesis involves distinct morphological and biochemical changes. This activation requires the coordinated changes in activity of several transcription factors. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is one such factor whose activity is decreased in activated HSC. PPAR gamma ligands suppress several markers of HSC activation such as expression of collagen and alpha smooth muscle actin (alpha-SMA), cell proliferation and migration. Expression of PPAR gamma, per se, also inhibits HSC activation. These findings support the role of PPAR gamma in reversion of activated HSC toward their quiescent state.

IL-10 receptor and coreceptor expression in quiescent and activated hepatic stellate cells.

Interleukin (IL)-10 expression is induced in activated hepatic stellate cells (HSC) in vitro and in vivo. We analyzed expression of IL-10 receptor (IL-10R) and coreceptor cytokine receptor family (CRF2-4) in HSC. We aimed to clone and sequence partial cDNA for rat IL-10R and CRF2-4, determine their expression in activated rat HSC in vivo and in vitro, and examine the biological responsiveness of HSC to exogenous IL-10. PCR cloning and sequencing of partial rat IL-10R and CRF2-4 cDNAs revealed 86% homology with corresponding mouse sequences. In hepatic macrophages, Northern blot with cloned IL-10R cDNA detected an expected 3.5-kb transcript, and IL-10R and CRF2-4 mRNAs showed steady constitutive expression after in vitro lipopolysaccharide treatment or cholestatic liver injury. IL-10R mRNA expression, as confirmed by immunohistochemistry, was induced 20.1- and 8.6-fold in HSC from cholestatic livers and 7-day culture-activated HSC, respectively but CRF2-4 mRNA levels were unchanged. Under serum-free conditions, IL-10 had minimal effects on collagen production but reduced DNA synthesis, matrix metalloprotease-2 mRNA levels, and activity in HSC. With serum, IL-10 inhibited both collagen production and DNA synthesis but had no effect on procollagen-alpha(1)(I) mRNA levels. This shows concomitant induction of IL-10R but not CRF2-4 to that of IL-10 by activated HSC in vitro and in vivo and associated acquisition of the responsiveness to IL-10, entailing complex effects on HSC.