Haruhide Shinzawa

Empty virus‐like particle‐based enzyme‐linked immunosorbent assay for antibodies to hepatitis E virus

Hepatitis E, an enterically transmitted non‐A, non‐B hepatitis, is a serious viral infection that occasionally causes large epidemics in developing countries. In developed countries, the disease only appears sporadically due to the transmission routes, and it is considered to be less important. The hepatitis E virus (HEV) cannot grow in cultured cells and no reliable assay system has ever been developed. In addition, the present diagnostic are not perfect, and actual rates of HEV infection may be underestimated. Highly purified empty virus‐like particles (VLPs) of HEV have been produced by the use of a recombinant baculovirus vector in insect cells. Using these VLPs as an antigen, an enzyme‐linked immunosorbent assay (ELISA) for antibodies to HEV was developed. A panel of 164 sera that were randomized and coded, and sera collected periodically from three patients with hepatitis E were used for the evaluation. The sensitivity of the assay was shown to be equal to or better than that obtained in previous research that used the same serum panel. The ELISA demonstrated that the serum IgM level of the patients was highest at the onset of the clinical illness and then rapidly decreased. In contrast, a high level of circulating IgG antibody titers lasted for more than 4 years. In Japan, a non‐endemic country, the prevalence of the IgG class antibody to HEV in healthy individuals was found to range from 1.9% to 14.1%, depending on the geographical area. Only one out of 900 (0.1%) serum samples was IgM‐positive. The IgM class antibody to HEV was detected in 10.8% of non‐A, non‐B, and non‐C acute hepatitis patients in northeast China, whereas none of the patients in Korea had the IgM antibody. The ELISA utilizing the VLPs is sensitive and specific in its detection of the IgM and IgG antibodies to HEV. The ELISA is therefore useful for diagnosing HEV infection and for seroepidemiological study of hepatitis E. J. Med. Virol. 62:327–333, 2000.

Simultaneous measurements of serum α-fetoprotein and protein induced by vitamin K absence for detecting hepatocellular carcinoma

Simultaneous measurements of serum α-fetoprotein and protein induced by vitamin K absence for detecting hepatocellular carcinoma

Simultaneous measurements of serum alpha-fetoprotein and protein induced by vitamin K absence for detecting hepatocellular carcinoma. South Tohoku District Study Group.

OBJECTIVE:We evaluated the measurements of serum α-fetoprotein (AFP) and the protein induced by vitamin K absence (PIVKA-II) in 734 patients with chronic hepatitis (CH) and liver cirrhosis (LC) who had been followed-up for the development of hepatocellular carcinoma (HCC).METHODS:Serum AFP and PIVKA-II were measured every month and abdominal ultrasonography was performed every 3 months. Youdens index (sensitivity + specificity −1) was calculated.RESULTS:On an average follow-up period of 374.5 days, HCC was detected in three HBsAg-positive LC patients (10.0%/yr), four anti-HCV-positive CH patients (1.35%/yr), 21 anti-HCV-positive LC patients (7.8%/yr), and one patient with both HBsAg- and anti-HCV-positive LC (22.7%/yr). At the time of HCC detection, the size of HCC was 4.7 ± 0.6 (mean ± SD) cm in HBsAg-positive patients and 2.4 ± 1.3 cm in anti-HCV-positive patents. Cut-off values of 20 ng/ml for AFP (Youdens index = 0.422) and 60 mAU/ml for PIVKA-II (Youdens index = 0.316) gave the highest index for each marker. When these two markers were combined, cut-off values of 40 ng/ml for AFP and 80 mAU/ml for PIVKA-II gave the highest index (Youdens index = 0.500, sensitivity = 65.5%, specificity = 85.5%, positive predictable value = 14.8%, negative predictable value = 98.3%). The levels of AFP or PIVKA-II increased within three months before the detection of HCC.CONCLUSIONS:Simultaneous measurements of serum AFP and PIVKA-II levels that are performed every 3 months are useful for detecting a developing HCC. The optimal cut-off values for AFP and PIVKA-II may be 40 ng/ml and 80 mAU/ml, respectively.

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.

Inhibition of Corynebacterium parvum-primed and lipopolysaccharide-induced hepatic necrosis in rats by selective depletion of neutrophils using a monoclonal antibody.

To examine whether neutrophils are involved in the pathogenesis of experimental liver dysfunction, we observed the effect of selective in vivo neutrophil depletion by a monoclonal antibody (RP‐3) on the pathogenesis of acute experimental hepatic necrosis in rats induced by a preparative injection of heat‐killed Corynebacterium parvum and a challenging dose of lipopolysaccharide (LPS) (positive control group). The serum transaminase titer 8 h after LPS injection was reduced by selective depletion of peripheral blood neutrophils as a result of RP‐3 treatment (RP‐3 group). A kinetic study showed that the serum transaminase titer of the RP‐3 group was significantly lower than that of the positive control group from 4 to 24 h after LPS injection. The transaminase level was significantly lower in the group with less than 400/mm3 peripheral blood neutrophils than in the group with a greater number. The effect of RP‐3 on the transaminase level was due neither to the injection of cancer ascites nor to RP‐3 injection with the accompanying decrease in complement titer. These results suggest that neutrophils play an important role in the induction of liver dysfunction in this system.

Activities of free oxygen radical scavenger enzymes in human liver

Activities of superoxide dismutase, catalase and glutathione peroxidase were measured in liver biopsy specimens from patients with various liver diseases, including six with chronic persistent hepatitis, nine with chronic active hepatitis, nine with non-alcoholic cirrhosis, eight with alcoholic cirrhosis and eight with acute hepatitis. Measurements from ten patients without liver disease were used as controls. Levels of total superoxide dismutase activity in the chronic active hepatitis and non-alcoholic cirrhosis groups were significantly lower than those in the controls (p less than 0.01 and p less than 0.01, respectively). The level of total superoxide dismutase activity in the acute hepatitis group was significantly higher than that in the control group (p less than 0.01). The levels of Cu,Zn-superoxide dismutase activity in all the experimental groups, except for the chronic persistent hepatitis group, were significantly lower than those in the controls (p less than 0.01 in all groups). The levels of Mn-superoxide dismutase activity in the alcoholic cirrhosis and acute hepatitis groups were significantly higher than those in the controls (p less than 0.01 and p less than 0.01, respectively), although no difference in the level of this enzyme was seen among the controls, chronic persistent hepatitis, chronic active hepatitis and non-alcoholic cirrhosis groups. The levels of catalase activity in the groups with chronic active hepatitis, non-alcoholic and alcoholic cirrhosis and acute hepatitis were significantly lower than those in the controls (p less than 0.01 in all groups). Glutathione peroxidase activity showed no difference among the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations of the X region of hepatitis B virus and their clinical implications

Nucleotide (nt) sequences of the X region of more than 130 hepatitis B virus (HBV) Isolates were determined and derived from patients with a variety of clinical features. Correlation of not substitutions with clinicopathological characteristies was attempted. The X region (465nt) Is crucial for the replication and expression of HBV because the X protein trans‐activates the HBV genes and this region contains the core promoter, enhancer II, and two direct repeats. There are several mutational hotspots, some of which seem to relate to immunological epitopes of the X protein. Two kinds of mutations which have important clinical significances were found. One is an 8‐nt deletion between nt 1770 and 1777, which truncates 20 amino acids from the carboxyl terminus of the X protein. This deletion leads to the suppression of replication and expression of HBV DNA, resulting In immunoserological marker (HBsAg) negativity. This silent HBV infection Is responsible for the majority of non A to non‐E hepatitis. The other mutation substituting T for C (nt 1655), T for A (nt 1764) and A for G (nt 1766) seems to relate to fulminant hepatitis. Further sequencing studies and In vitro mutagenesis experiments will clarify the significance of other mutations of the X region.