Hitoshi Togashi

Association of visceral fat accumulation and plasma adiponectin with colorectal adenoma: evidence for participation of insulin resistance.

Purpose: Colorectal carcinogenesis is thought to be related to abdominal obesity and insulin resistance. To investigate whether visceral fat accumulation contributes to colorectal carcinogenesis, we examined its accumulation and the levels of the adipose tissue–derived hormone adiponectin in Japanese patients with colorectal adenoma. Experimental Design: Fifty-one consecutive Japanese patients ages ≥40 years and with colorectal adenoma were subjected to measurement of visceral fat area by computed tomography scanning and plasma adiponectin concentration. The patients also underwent the 75-g oral glucose tolerance test. Insulin resistance was calculated by the homeostasis metabolic assessment (HOMA-IR) method. The controls were 52 Japanese subjects ages ≥40 years and without colorectal polyp. Cigarette smokers and subjects who consumed alcohol (≥30 g ethanol/d) were excluded. Results: The patients with colorectal adenoma showed significantly more visceral fat area and significantly less plasma adiponectin concentration in comparison with the controls [odds ratio (OR), 2.19; 95% confidence interval (95% CI), 1.47-3.28; P < 0.001 and OR, 0.24; 95% CI, 0.14-0.41; P < 0.001, respectively] by logistic regression analysis. HOMA-IR index was also associated with colorectal adenoma (OR 2.60; 95% CI, 1.20-5.64; P = 0.040). Visceral fat area and adiponectin were associated with adenoma number (1, 2, ≥ 3), the size of the largest adenoma (<10 and ≥10 mm), and adenoma histology (tubular and tubulovillous/villous). Conclusions: These results suggest an association of visceral fat accumulation and decreased plasma adiponectin concentration with colorectal adenoma in Japanese patients. This study may offer a new insight to understanding the relationship of colorectal carcinogenesis with abdominal obesity and insulin resistance.

NAD(P)H oxidase plays a crucial role in PDGF-induced proliferation of hepatic stellate cells.

The proliferation of hepatic stellate cells (HSCs) is a critical step in hepatic fibrogenesis. Platelet‐derived growth factor (PDGF) is the most potent mitogen for HSCs. We investigated the role of nonphagocytic NAD(P)H oxidase–derived reactive oxygen species (ROS) in PDGF‐induced HSC proliferation. The human HSC line, LI‐90 cells, murine primary‐cultured HSCs, and PDGF‐BB were used in this study. We examined the mechanism of PDGF‐BB‐induced HSC proliferation in relation to the role of a ROS scavenger and diphenylene iodonium, an inhibitor of NAD(P)H oxidase. We also measured ROS production with the aid of chemiluminescence. We showed that PDGF‐BB induced proliferation of HSCs through the intracellular production of ROS. We also demonstrated that HSCs expressed key components of nonphagocytic NAD(P)H oxidase (p22phox, gp91phox, p47phox, and p67phox) at both the messenger RNA and protein levels. Diphenylene iodonium suppressed PDGF‐BB–induced ROS production and HSC proliferation. Coincubation of H2O2 and PDGF‐BB restored the proliferation of HSCs that was inhibited by diphenylene iodonium pretreatment. Phosphorylation of the mitogen‐activated protein kinase (MAPK) family constitutes a signal transduction pathway of cell proliferation. Our data demonstrate that NAD(P)H oxidase–derived ROS induce HSC proliferation mainly through the phosphorylation of p38 MAPK. Moreover, an in vivo hepatic fibrosis model also supported the critical role of NAD(P)H oxidase in the activation and proliferation of HSCs. In conclusion, NAD(P)H oxidase is expressed in HSCs and produces ROS via activation of NAD(P)H oxidase in response to PDGF‐BB. ROS further induce HSC proliferation through the phosphorylation of p38 MAPK. (HEPATOLOGY 2005;41:1272–1281.)

Differentiation of bone marrow cells into cells that express liver-specific genes in vitro: implication of the Notch signals in differentiation

Bone marrow (BM) stem cells have been shown to differentiate into liver cells. It remains difficult to sort and culture BM stem cells, and the gene expression of liver-specific proteins in these cells has not been fully investigated. We used a negative selective magnetic cell separation system to obtain stem cell-enriched BM cells. The cells obtained were cultured with hepatocytes or with hepatocyte growth factor (HGF), and the differentiation of BM cells into cells expressing liver-specific genes, hepatocyte nuclear factor (HNF) 1alpha, cytokeratin (CK) 8, alpha-fetoprotein (AFP), and albumin was investigated by the reverse transcription-polymerase chain reaction. We also investigated the gene expressions of Notch receptor-1 (Notch-1) and its ligand Jagged-1 in BM cell differentiation. Sorted BM cells showed positive for Sca-1 (Ataxin-1) by immunofluorescence staining. Fluorescence activated cell sorter analysis showed that 32.6% of sorted BM cells had a high level of expression of the hematopoietic stem cell marker CD90 (Thy-1). When cultured with hepatocytes, these cells expressed the liver-specific genes HNF1alpha and CK8 on culture day 3, AFP and albumin on culture day 7. When cultured with HGF (20ng/ml), the cells expressed HNF1alpha on day 3 and CK8 on day 7. Gene expressions of Notch-1 and Jagged-1 were detected in cultured BM cells on day 3. These results suggest that the negative selective magnetic cell separation system is useful for the rapid preparation of stem cell-enriched BM cells, and that the Notch signaling pathway plays a role in BM cell differentiation into a hepatocyte lineage in vitro.

Possible contribution of circulating transforming growth factor-beta1 to immunity and prognosis in unresectable hepatocellular carcinoma.

Transforming growth factor‐β1 (TGF‐β1) has been implicated in tumor progression. The relationship of this cytokine as measured in plasma to anti‐tumor immunity and prognosis was investigated.

Augmentation effect of postprandial hyperinsulinaemia on growth of human hepatocellular carcinoma

Background: Cirrhotic patients with hepatocellular carcinoma (HCC) frequently have impaired glucose metabolism. Aims: To investigate whether impaired glucose metabolism affects the growth rate of the tumour. Patients and methods: Tumour doubling time (DT), assessed by ultrasound imaging analysis, was measured in 60 patients with single small HCC (diameter <30 mm). DT was compared with plasma insulin and glucose concentrations following the oral glucose tolerance test (OGTT). The effect of continuous infusion of octreotide (a somatostatin analogue 200 μg/day) for three months on DT in five cases was assessed. Results: The 60 patients were divided into two groups because the median DT was 140 days: rapid growth group (DT ≤140 days, n=30) and slow growth group (DT >140 days, n=30). Fasting plasma insulin concentration and area under the plasma insulin curve (AUCins) of the OGTT (10.4 (6.2) μU/ml and 262 (152) μU/ml/h, respectively; mean (SD)) in the rapid growth group were significantly higher than those in the slow growth group (7.6 (4.3) and 146 (140), respectively) (p=0.041 and p=0.0006, respectively). In contrast, fasting plasma glucose concentration and area under the plasma glucose curve (AUCgluc) in the rapid growth group were significantly lower than those in the slow growth group (p=0.0003 and p=0.0012, respectively). Univariate and multivariate analyses of logistic regression models demonstrated that AUCins was a significant factor contributing to the growth rate of HCC (p=0.001 and p=0.016, respectively). AUCins significantly decreased after octreotide treatment (p<0.02) but AUCgluc did not significantly change. DT after treatment increased in three of the five patients and could not be calculated in the remaining two patients because of no change in the diameter of the tumour. Conclusions: These data suggest that postprandial hyperinsulinaemia is associated with accelerated HCC growth.

Potential therapeutic application of intravenous autologous bone marrow infusion in patients with alcoholic liver cirrhosis.

The present study was conducted to evaluate the application and efficacy of autologous bone marrow infusion (ABMi) for improvement of liver function in patients with alcoholic liver cirrhosis (ALC). Five subjects and 5 control patients with ALC who had abstained from alcohol intake for 24 weeks before the study were enrolled. Autologous bone marrow cells were washed and injected intravenously, and the changes in serum liver function parameters, and the level of the type IV collagen 7S domain as a marker of fibrosis, were monitored for 24 weeks. The distribution of activated bone marrow was assessed by indium-111-chloride bone marrow scintigraphy. The number of cells infused was 8.0±7.3×10(9) (mean±standard error). The serum levels of albumin and total protein and the prothrombin time were significantly higher during the follow-up period after ABMi than during the observation period in treated patients, whereas no such changes were observed in the controls. In the patients who received ABMi, the Child-Pugh score decreased in all 3 who were classified as class B; the serum levels of type IV collagen 7S domain improved in 4 of the 5 patients; and bone marrow scintigraphy demonstrated an increase of indium-111-chloride uptake in 3 of the 4 patients tested. ABMi for patients with ALC helps improve liver function parameters in comparison with observation during abstinence and ameliorates the degree of fibrosis in terms of serum markers and bone marrow activation in most cases.

Analysis of hepatic oxidative stress status by electron spin resonance spectroscopy and imaging

Real-time detection of free radicals generated within the body may contribute to clarify the pathophysiological role of free radicals in disease processes. Of the techniques available for studying the generation of free radicals in biological systems, electron spin resonance (ESR) has emerged as a powerful tool for detection and identification. This article begins with a review of spin trapping detection of oxygen-centered radicals using X-band ESR spectroscopy and then describes the detection of superoxide and hydroxyl radicals by the spin trap 5,5-dimethyl-1-pyrroline-N-oxide and ESR spectroscopy in the perfusate from isolated perfused rat livers subjected to ischemia/reperfusion. This article also reviews the current status of ESR for the in vivo detection of free radicals and in vivo imaging of exogenously administered free radicals. Moreover, we show that in vivo ESR-computed tomography with 3-carbamoyl-2,2,5, 5-tetramethylpyrrolidine-1-oxyl may be useful for noninvasive anatomical imaging and also for imaging of hepatic oxidative stress in vivo.

Spontaneous regression of hepatocellular carcinoma and review of literature

A 68‐year‐old man presented with multiple hepatocellular carcinoma, which was considered to be unresectable at the first admission in January 1994. Pathological diagnosis was made by biopsy of the one lesion among them. From January 1994 to December 1997, 10 transarterial chemoembolizations and six percutaneous ethanol injection therapies were performed on the tumours in the cirrhotic liver. In February 1998 the tumour situated in the right lobe began to increase in size. The maximum tumour diameter was 6.3 cm measured by computed tomography (CT). In the beginning of May 1998 moderate ascites was present and mild hepatic encephalopathy was noticed. The patient was in the terminal stage of hepatocellular carcinoma and no further treatment was possible at that time. However, serum α‐fetoprotein and protein induced by vitamin K absence or antagonist II dramatically decreased in June 1998. The CT scan also showed that the tumour had completely regressed without specific treatment. In February 1999 a new biopsy‐proven hepatocellular carcinoma, 2 cm in diameter, developed in the lateral segment of the liver. It was well treated by percutaneous ethanol injection therapy. The patient was alive in good condition without any symptoms or tumour recurrence in June 1999. It was concluded that a rare case of spontaneous regression of hepatocellular carcinoma had occurred.

Subcutaneous seeding of small hepatocellular carcinoma after fine needle aspiration biopsy

Ultrasonically guided fine needle (21 gauge) aspiration biopsy (FNAB) was performed on a patient with a hepatocellular carcinoma (HCC) measuring 1.5 × 1.5 cm in segment VI of the liver. The tumour was located just beneath the liver surface. Subsegmentectomy of segment VI was performed. Twelve months after the biopsy and 10 months after the operation, levels of alpha‐fetoprotein (AFP) and protein induced by Vitamin K absence or antagonist‐II (PIVKA‐II) increased gradually without any evidence of recurrence of HCC in the liver. Thirteen months after the biopsy, the patient palpated a hard subcutaneous nodule 1.5 cm in diameter in the right lower anterior chest wall at the insertion site of the biopsy needle. A subcutaneous tumour was excised and histological examination revealed moderately differentiated HCC. The levels of AFP and PIVKA‐II normalized thereafter. These tumour markers were therefore useful for diagnosing the subcutaneous nodule as a metastatic HCC. The patient is currently doing well without further recurrence of HCC or needle‐tract seeding 23 months after subsegmentectomy and 11 months after excision of the subcutaneous tumour.

Quantitative DNA analysis of low-level hepatitis B viremia in two patients with serologically negative chronic hepatitis B.

Low‐level viremia due to hepatitis B virus (HBV) was demonstrated in the sera of two patients diagnosed previously as having non‐B, non‐C chronic hepatitis. Both patients had a “silent” HBV infection, because they were negative for both hepatitis B surface antigen (HBsAg) and anti‐hepatitis B core antibody. The TaqMan chemistry polymerase chain reaction (PCR) amplified the HBV DNA, enabling quantitation of the virus in their sera. Their serum HBV DNA concentrations were low: the amount of each HBV S or X gene amplified showed there were approximately 103 copies/ml and HBV DNA was detected occasionally during clinical follow‐up. Positive HBsAg staining in liver tissues was demonstrated by an immunoperoxidase technique. Vertical transmission of silent HBV from one patient to her daughter was confirmed. Direct nucleotide sequencing of the amplified HBV X region revealed several mutations, suggesting reduced viral replication. One patient had a T‐to‐C mutation at the extreme 5′‐terminus of the direct repeat 2 region and the other exhibited a coexisting X region with a 155‐nucleotide deletion. These findings suggest that HBV replication is suppressed considerably in patients with silent hepatitis B. J. Med. Virol. 58:325–331, 1999.