[Evaluation of Sorafenib for Hepatocellular Carcinoma with Low α-Fetoprotein by Arrival Time Parametric Imaging Using Contrast-Enhanced Ultrasonography with Sonazoid].

We aimed to determine the usefulness of arrival time parametric imaging (AtPI) using contrast-enhanced ultrasonography (CEUS)with Sonazoid in the evaluation of early response to sorafenib for hepatocellular carcinoma (HCC). Thirteen ad- vanced HCC patients with low a / -fetoprotein (AFP) level (≤35 ng/mL) who received sorafenib for at least 4 weeks were enrolled in this study. CEUS was performed before and after treatment (2 weeks), and the images of the target lesion in the arterial phase were analyzed by AtPI. In the color mapping images obtained by AtPI, the mean arrival time of the contrast agent in the target lesion from the starting point (mean time: MT) was calculated. In each patient, differences between MT before and MT 2 weeks after treatment were compared. MT (+) and MT(-) groups were designated as such if the difference was 0 or greater(blood flow velocity of the lesion was reduced)and less than 0 sec(blood flow velocity of the lesion was increased), respectively. The overall survival was evaluated between the 2 groups. In the MT (+) group (7 patients) and MT (-) group (6 patients), the median survival times were 307 and 208 days, respectively, which was statistically significant. We suggest AtPI is useful for evaluating early response to sorafenib in advanced HCC patients with low AFP level.

Evaluation of hemodynamics in focal steatosis and focal spared lesion of the liver using contrast-enhanced ultrasonography with sonazoid.

We aim to investigate the hemodynamics in focal steatosis and focal spared lesion of the liver using contrast-enhanced ultrasonography (CEUS) with Sonazoid. The subjects were 47 patients with focal steatosis and focal spared lesion. We evaluated enhancement patterns (hyperenhancement, isoenhancement, and hypoenhancement) in the vascular phase and the presence or absence of a hypoechoic area in the postvascular phase for these lesions using CEUS. Of the 24 patients with focal steatosis, the enhancement pattern was isoenhancement in 19 and hypoenhancement in 5. Hypoechoic areas were noted in the postvascular phase in 3 patients. Of the 23 patients with focal spared lesions, the enhancement pattern was isoenhancement in 18 and hyperenhancement in 5. No hypoechoic areas were noted in the postvascular phase in any patient. The hemodynamics in focal steatosis and focal spared lesions in nondiffuse fatty liver can be observed using low-invasive procedures in real-time by CEUS. It was suggested that differences in the dynamics of enhancement in the vascular phase of CEUS were influenced by the fat deposits in the target lesion, the surrounding liver parenchyma, and the third inflow.

Human immunodeficiency virus protease inhibitors modulate Ca2+ homeostasis and potentiate alcoholic stress and injury in mice and primary mouse and human hepatocytes.

UNLABELLED: A portion of human immunodeficiency virus (HIV)-infected patients undergoing protease inhibitor (PI) therapy concomitantly consume or abuse alcohol leading to hepatic injury. The underling mechanisms are not known. We hypothesize that HIV PIs aggravate alcohol-induced liver injury through an endoplasmic reticulum (ER) stress mechanism. To address this, we treated mice, primary mouse hepatocytes (PMHs), and primary human hepatocytes (PHHs) with alcohol and the HIV PIs ritonavir (RIT) and lopinavir (LOP). In mice, RIT and LOP induced mild ER stress and inhibition of sarco/ER calcium-ATPase (SERCA) without significant increase in serum alanine aminotransferase (ALT) levels. However, a single dose of alcohol plus the two HIV PIs caused a more than five-fold increase in serum ALT, a synergistic increase in alcohol-induced liver lipid accumulation and ER stress response, and a decrease of SERCA. Mice treated with chronic HIV PIs and alcohol developed moderate liver fibrosis. In PMHs, the HIV drugs plus alcohol also inhibited SERCA expression and increased expression of glucose-regulated protein 78, C/EBP homologous protein, sterol regulatory element-binding protein 1c, and phosphorylated c-Jun N-terminal kinase 2, which were accompanied by a synergistic increase in cell death compared with alcohol or the HIV drugs alone. In PHHs, treatment with RIT and LOP or alcohol alone increased messenger RNA of spliced X box-binding protein 1 and decreased SERCA, which were accompanied by reduced levels of intracellular calcium. Alcohol combined with the HIV drugs significantly reduced intracellular calcium levels and potentiated cell death, which was comparable to the cell death caused by the SERCA inhibitor thapsigargin. CONCLUSION: Our findings suggest the possibility that HIV PIs potentiate alcohol-induced ER stress and injury through modulation of SERCA and maintaining calcium homeostasis could be a therapeutic aim for better care of HIV patients.

Differences in betaine-homocysteine methyltransferase expression, endoplasmic reticulum stress response, and liver injury between alcohol-fed mice and rats.

UNLABELLED: Chronic ethanol infusion resulted in greater serum alanine aminotransferase elevation, lipid accumulation, necroinflammation, and focal hepatic cell death in mice than rats. Mice exhibited a remarkable hyperhomocysteinemia but no increase was seen in rats. Similarly, a high-methionine low-folate diet (HMLF) induced less steatosis, serum alanine aminotransferase increase, and hyperhomocysteinemia in rats than in mice. Western blot analysis of betaine homocysteine methyltransferase (BHMT) expression showed that rats fed either ethanol or HMLF had significantly increased BHMT expression, which did not occur in mice. Nuclear factor-kappaB p65 was increased in mouse in response to alcohol feeding. The human BHMT promoter was repressed by homocysteine in mouse hepatocytes but not rat hepatocytes. BHMT induction was faster and greater in primary rat hepatocytes than mouse hepatocytes in response to exogenous homocysteine exposure. Mice fed ethanol intragastrically exhibited an increase in glucose-regulated protein 78 and inositol-requiring enzyme 1, which was not seen in the rat, and sterol regulatory element binding protein 1 was increased to a greater extent in mice than rats. Thus, rats are more resistant to ethanol-induced steatosis, endoplasmic reticulum stress, and hyperhomocysteinemia, and this correlates with induction of BHMT in rats. CONCLUSION: These findings support the hypothesis that a critical factor in the pathogenesis of alcoholic liver injury is the enhanced ability of rat or impaired ability of mouse to up-regulate BHMT which prevents hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury.

[Pulmonary hypertension in a patient on chronic hemodialysis].

A 52-year-old woman with end-stage renal disease and long-term hemodialysis complained of worsening exertional dyspnea. A chest X-ray showed cardiomegaly. She was admitted to our hospital because an echocardiogram suggested pulmonary hypertension. Right heart catheterization revealed pulmonary hypertension, but pulmonary perfusion scintigraphy with Tc-99m-MAA showed no evidence of pulmonary thromboembolism. We gave her a diagnosis of pulmonary arterial hypertension associated with Sjögren syndrome on the basis of a positive serological test (SS-A. SS-B) and the findings of lip biopsy. After four months of therapy with bosentan, her 6-minute walk distance, estimated pulmonary arterial pressure and brain natriuretic peptide (BNP) improved. Bosentan is mainly cleared by hepatic elimination and its dialysis clearance is low. Bosentan for the treatment of pulmonary hypertension was safe as well as effective in this patient with end-stage renal disease and hemodialysis. In consideration of the relationship between pulmonary hypertension and end-stage renal disease, and hemodialysis, bosentan was considered to be a reasonable and effective treatment.

Effect of transgenic extrahepatic expression of betaine-homocysteine methyltransferase on alcohol or homocysteine-induced fatty liver.

BACKGROUND: Chronic alcohol feeding induces hyperhomocysteinemia (HHcy). Previously, we reported a protective role of betaine-homocysteine methyltransferase (BHMT) in homocysteine-induced injury in cultured hepatocytes. In this study, we investigated the direct role of BHMT in alcohol or homocysteine-induced liver injury. METHODS: Betaine-homocysteine methyltransferase transgenic (Tg) mice were generated. Comparisons were made between the Tg and wild type (WT) mice in their response to intragastric alcohol infusion or to oral feeding of a high methionine low folate diet (HMLF). RESULTS: Expression of the Tg BHMT was increased in organs peripheral to the liver. The alcohol infusion for 4 weeks increased: plasma ALT by 5-fold in WT mice and 2.7-fold in Tg mice; plasma homocysteine by 7-fold in WT mice and 2-fold in Tg mice; liver triglycerides by 4-fold in WT mice and 2.5-fold in Tg mice. The alcohol-induced fatty liver was more severe in WT than in Tg mice based on H&E staining. The HMLF feeding for 4 weeks increased plasma ALT by 2-fold in WT mice and 1-fold in Tg mice; plasma homocysteine by 21-fold in WT mice and 3.3-fold in Tg mice; liver triglycerides by 2.5-fold in WT mice and 1.5-fold in Tg mice. HMLF induced accumulation of macro fat droplets in WT but not Tg mice. Betaine supplementation decreased partially the alcohol or HMLF-induced increase of ALT, homocysteine and liver lipids in WT mice. However, Tg mice were normal when fed both HMLF and betaine. In WT mice, both alcohol and HMLF induced moderate increase of sterol regulatory element binding protein 1 (SREBP1) protein which was partially reduced by betaine supplementation. In Tg mice, alcohol but not HMLF increased SREBP1. Carbohydrate responsive element-binding protein was increased by alcohol in either WT or Tg mice which was not affected by betaine supplementation. Ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) was reduced by 50% in WT and by 20% in Tg mice fed alcohol. Ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) was reduced in WT but not Tg mice fed alcohol. Changes in PE methyltransferase activities were not detected in response to alcohol or HMLF feeding but were increased by betaine. CONCLUSIONS: The BHMT Tg mice are resistant to alcohol or HMLF-induced HHcy and liver steatosis indicating that peripheral metabolism of homocysteine protected the liver without a direct effect of BHMT in the liver. Multiple mechanisms are involved in protection by betaine including increased SAM/SAH and PC/PE ratios.

Endoplasmic reticulum stress and liver injury.

Endoplasmic reticulum stress, initiated by protein overload or malfolding, activates a complex network of interacting and parallel responses that dampen the stress. However, when the protective response is insufficient, a set of responses leads to apoptosis. Coupled with the latter are promotion of lipid synthesis and proinflammatory responses. Evidence has been mounting for an important role of the endoplasmic reticulum (ER) stress response in the pathogenesis of chronic viral hepatitis, insulin resistance and nonalcoholic fatty liver disease, ischemia-reperfusion injury, genetic disorders of protein malfolding, and alcoholic liver disease. In the latter, a key candidate for inducing ER stress is hyperhomocysteinemia. Betaine treatment promotes removal of homocysteine and prevents ER stress, fatty liver, and apoptosis in a mouse model of alcohol-induced liver disease. With increasing interest in the potential role of ER stress in liver disease, greater understanding of pathophysiology, prevention, and treatment of liver disease is anticipated.

Mechanisms of protection by the betaine-homocysteine methyltransferase/betaine system in HepG2 cells and primary mouse hepatocytes.

UNLABELLED: Betaine-homocysteine methyltransferase (BHMT) regulates homocysteine levels in the liver. We previously reported that the alteration of BHMT is associated with alcoholic liver steatosis and injury. In this study, we tested whether BHMT protects hepatocytes from homocysteine-induced injury and lipid accumulation. Both BHMT transfectants of HepG2 cells and primary mouse hepatocytes with suppressed BHMT were generated. Comparisons were made between the cell models with respect to their response to homocysteine treatments. Homocysteine metabolism was impaired in HepG2 cells, and the expression of BHMT in HepG2 cells ameliorated the impairment and stabilized the levels of intracellular homocysteine after the addition of exogenous homocysteine. BHMT expression inhibited homocysteine-induced glucose-regulated protein 78 (GRP78) and C/EBP-homologous protein (CHOP) and homocysteine-induced cell death. A betaine treatment protected primary mouse hepatocytes from a homocysteine-induced increase in GRP78 and cell death but not a tunicamycin-induced increase. Homocysteine induced greater CHOP expression (2.7-fold) in BHMT small interfering RNA (siRNA)-transfected cells than in a control (1.9-fold). Homocysteine-induced cell death was increased by 40% in the siRNA-treated cells in comparison with the control. Apolipoprotein B (apoB) expression was higher and triglycerides and cholesterol were lower in HepG2 expressing BHMT. In primary mouse hepatocytes, homocysteine induced the accumulation of triglycerides and cholesterol, which was reduced in the presence of betaine. Betaine partially reduced homocysteine-induced sterol regulatory element binding protein 1 expression in HepG2 cells and increased S-adenosylmethionine in primary mouse hepatocytes. CONCLUSION: The BHMT/betaine system directly protects hepatocytes from homocysteine-induced injury but not tunicamycin-induced injury, including an endoplasmic reticulum stress response, lipid accumulation, and cell death. This system also exhibits a more generalized effect on liver lipids by inducing ApoB expression and increasing S-adenosylmethionine/S-adenosylhomocysteine.

Early immune-mediated response to ribavirin combined with IFN in patients with chronic hepatitis C.

AIM: This study was to investigate the immunomodulatory effects of ribavirin combined with interferon (IFN)-alpha 2b (R+IFN) compared with consensus IFN monotherapy (IFN-Con) in chronic hepatitis C (CHC) patients. PATIENTS AND METHODS: Thirty-four adult patients with biopsy-proven CHC, who were infected with HCV genotype 2a or 2b, were studied. A 24-week regimen of IFN-alpha 2b (6MU daily for 2 weeks followed by 6MU tiw for 22 weeks) and ribavirin (600-800mg/day for 24 weeks) was given to 17 patients. The other 17 patients were treated with a 24-week regimen of IFN-Con (18MU daily for 2 weeks followed by 18MU tiw for 22 weeks). Flow cytometric determination of cytoplasmic IFN-gamma and IL-4 expression in peripheral blood CD4+ T cells was performed, and the percentage of IFN-gamma+ and IL-4- (Th1), IFN-gamma- and IL-4+ (Th2) cells were calculated before and 3, 7, 14, and 28 days after the start of therapy. RESULTS: In the R+IFN group, the percentage of Th1 cell peaked on day 3, and then decreased to near baseline by day 14, while the percentage of Th2 cell did not change. In the IFN-Con group, the percentage of Th1 cell peaked on day 14 and the percentage of Th2 cell peaked on day 3, and then decreased to near baseline by day 14. CONCLUSIONS: Our results suggest that ribavirin induces an early immune response by peripheral blood CD4+ T cells in CHC patients.

Disappearance of HCV after cessation of immunosuppression in a patient with ulcerative colitis and renal transplantation.

We report a patient, a 45-year-old Japanese woman, who underwent living-related donor renal transplantation in 1986 and 1988, with the second procedure being successful. Ulcerative colitis (UC) was diagnosed in 1987 while she was receiving immunosuppressive therapy after the renal transplantation. She became positive for serum anti-hepatitis C virus (HCV) in November 1990, although her serum aminotransferase levels were normal. In June 2001, she had frequent episodes of melena with abdominal pain, as control of her UC deteriorated. In July 2001, she was admitted to the Department of Surgery at our hospital, and her daily dose of prednisolone was increased from 40 mg to 80 mg. After 2 weeks of high-dose prednisolone therapy, there was a significant increase of serum aminotransferases, and serum HCV-RNA rose above 850 KIU/ml (by reverse transcription-polymerase chain reaction [RT-PCR]). Control of UC was still poor, so cyclosporine A (CyA) was added at a dose that maintained a high serum concentration. The daily dose of prednisolone was tapered and leukapheresis was performed three times weekly. As result, serum aminotransferases decreased to the normal range. However, total colectomy and colostomy were required because the UC could not be controlled by these therapies. Serum aminotransferase levels increased transiently 2 months after the cessation of immunosuppressive therapy (prednisolone, azathioprine [AZP], and CyA). Subsequently, serum aminotransferases rapidly declined below normal, and the serum level of HCV-RNA (by RT-PCR) fell from 480 KIU/ml to less than 0.5 KIU/ml. She was discharged on April 25, 2002. During follow-up as an outpatient, serum HCV-RNA became negative and remained negative for 7 months. To confirm clearance of HCV, liver biopsy was performed, and no HCV-RNA was detected in the liver tissue by RT-PCR. These findings suggested that HCV was cleared by the cessation of immunosuppressive therapy, as a rebound effect.

Mycobacterium peregrinum infection in a patient with AIDS.

The patient, a 30-year-old housewife, visited a nearby doctor in mid August 2002 because of weight loss and neck swelling. HIV tests done at the hospital were positive. She was referred to and admitted to our hospital on October 2 for detailed examination and treatment of the neck tumor. A coat of epithelial debris extended from the oral cavity to the pharynx and an abscess and a fistula were found in the left tonsil. After hospitalization, an abscess culture revealed the presence of acid-fast bacteria, which was identified as Mycobacterium peregrinum. Treatment with imipenem and clarithromycin resulted in the normalization of CRP (0.1 mg/dl), on day 5 of treatment. The patient was discharged from the hospital after treatment for 2 weeks with imipenem and clarithromycin. Thereafter, the patient received continuous treatment with faropenem and clarithromycin for 4 more weeks, and has shown no signs of recurrence for 11 months to date. Only a few cases of infection with this bacterial strain have been reported. This infection is difficult to treat because most antituberculosis agents are not effective against it and there is limited availability of effective antibiotics. Medical treatment of infection caused by Mycobacterium peregrinum may be useful in such cases.

Investigation of liver parenchymal flow using contrast-enhanced ultrasound in patients with alcoholic liver disease.

BACKGROUND: Peripheral hepatic blood flow has not been investigated using contrast-enhanced ultrasound. The aim of this study was to assess the characteristics of peripheral blood flow within the liver parenchyma using Levovist in patients with alcoholic liver disease (ALD). METHODS: The study population comprised 160 patients: 7 normal control subjects (NC), 89 with chronic viral hepatitis (CVH), 8 with alcoholic liver injury (ALI), 18 with viral liver cirrhosis without portosystemic shunting (VLCwoPSS) plus 11 with PSS (VLCwPSS), and 12 with alcoholic liver cirrhosis without PSS (ALCwoPSS) plus 15 with PSS (ALCwPSS). First, continuous spectral Doppler ultrasound was performed to assess peripheral blood flow within the liver parenchyma. We defined the arrival time (AT) as the time when signals began to increase in the region of interest after the injection of Levovist, whereas the peak time (PT) was the time between the AT and the maximum signal. Second, stimulated acoustic emission imaging was compared between the liver parenchyma and the right kidney at 20 sec, 90 sec, and 5 min after injection of Levovist. RESULTS: The average AT was as follows: NC, 18.4 sec; CVH, 18.7 sec; ALI, 15.5 sec; VLCwoPSS, 18.2 sec; VLCwPSS, 12.5 sec; ALCwoPSS, 13.5 sec; and ALCwPSS, 12.7 sec. In patients with ALI and cirrhosis (excluding VLCwoPSS), the AT was earlier than in NC and CVH. The average PT was as follows: NC, 18.3 sec; CVH, 19.4 sec; ALI, 20.3 sec; VLCwoPSS, 19.3 sec; VLCwPSS, 9.0 sec; ALCwoPSS, 10.7 sec; and ALCwPSS, 4.9 sec. In patients with cirrhosis (excluding VLCwoPSS), the PT was earlier than in the other groups. As PSS developed, the PT of ALC became much earlier. In NC, the stimulated acoustic emission study showed no enhancement of the liver parenchyma at 20 sec. However, most of the patients with ALD showed marked enhancement of the liver at 20 sec. It is interesting that only slight enhancement of the liver was seen in ALCwPSS, although NC showed marked liver enhancement at 5 min. CONCLUSIONS: Our results suggest that ALD is accompanied by regional hepatic and systemic hemodynamic changes, such as hyperdynamic circulation and arterialization of the liver, before progression to cirrhosis.

Determination of peripheral color signal density using contrast-enhanced color Doppler ultrasonography in diffuse liver disease.

PURPOSE: The study aimed to quantify hepatic vascular changes that accompany the development of chronic liver disease using contrast-enhanced color Doppler ultrasonography and histopathological examination. METHODS: A series of 62 patients with biopsy-proven chronic liver disease (31 chronic hepatitis, 31 liver cirrhosis) and 8 healthy controls were studied. Altogether, 22 livers (9 chronic hepatitis, 13 liver cirrhosis) obtained at surgery or autopsy were subjected to histopathological examination. Patients with cardiopulmonary disease or intrahepatic tumors were excluded. Intrahepatic color Doppler signals were scanned and counted at the liver surface in a 10 × 30 mm rectangle from liver segment V using color Doppler sonography (SSA 380 A) before and after contrast enhancement with SHU 508A (Levovist). Small arteries 30-1000 µm in diameter were counted on the histopathlogical specimen by microscopy. RESULTS: The number of color Doppler signals increased significantly after contrast enhancement in both patients and controls. The number of color Doppler signals was elevated before and after contrast enhancement when chronic liver disease was present, especially in cases of Child-Pugh grade C liver cirrhosis. Histologically, more arteries 30-125 µm in diameter were present in patients with chronic hepatitis than in those with liver cirrhosis, whereas more arteries ≦ 125 µm in diameter were present in patients with liver cirrhosis than in those with chronic hepatitis. CONCLUSION: Intrahepatic color Doppler signals are probably derived from peripheral arteries larger than 125 µm in diameter, and the signal density in these arteries increases with progression of the chronic liver disease.