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The natural course of hepatitis C virus (HCV) infection has not been fully elucidated. To investigate whether HCV is spontaneously eliminated in chronic carriers, a long‐term population‐based cohort study was conducted on 435 chronic HCV carriers. Individual characteristics, serum HCV RNA, and liver function tests were analyzed, and ultra sonography (US) was performed in all subjects. Subjects were followed up for 7.2 ± 2.4 years (mean ± SD). Serum HCV RNA was spontaneously eliminated in 16/435 (3.7%) individuals during this period; thus, the incidence of spontaneous elimination of serum HCV RNA was 0.5%/year/person. Multivariate analysis revealed that both a low value of ZTT and no US finding of chronic liver disease were associated with spontaneous viral elimination in HCV carriers. Three of these 16 individuals had chronic hepatitis, and 13 of them had normal ALT levels. When the neutralization of binding (NOB) assay that evaluates inhibition of the HCV envelope‐2 protein binding to human cells was examined using sera from these 16 individuals, the NOB antibody was detected in only 3 cases with chronic hepatitis. These results suggest that serum HCV RNA is spontaneously eliminated in chronic HCV carriers in a population, and that the development of NOB antibody is associated with a natural resolution of chronic hepatitis in the minority of them. J. Med. Virol. 71:56–61, 2003. © 2003 Wiley‐Liss, Inc.

We developed a highly sensitive enzyme immunoassay (EIA), the p‐AP/HHTIO method, that detects serum hepatitis B surface antigen (HBsAg) by measuring stabilized nitroxide radicals using a novel electron spin resonance technique [Matsuo et al. (1998) Free Radic Biol Med 25:929–935]. To demonstrate the clinical significance of this method and to reveal occult hepatitis B virus (HBV) infection in patients, we used the method to analyze serum samples of 30 patients with acute or fulminant hepatitis who were negative for HBsAg by standard EIA, and those of seven chronic HBV carriers who became negative for HBsAg during a follow‐up period by standard EIA. We also examined serum HBV DNA by amplification of the HBV S gene, using the polymerase chain reaction (PCR) technique. The p‐AP/HHTIO method showed that 9 of 20 (45%) patients with acute hepatitis and 2 of 10 (20%) with fulminant hepatitis were positive for HBsAg; PCR detected HBV DNA in these HBsAg‐positive patients. Antibody against hepatitis B core antigen was detected in one patient with fulminant hepatitis. The p‐AP/HHTIO method demonstrated prolonged seropositivity of HBsAg even after standard EIA showed a loss of HBsAg in all seven HBV carriers. Our p‐AP/HHTIO method is useful for screening and diagnosing HBV infection in patients with liver diseases who are negative for conventional HBV‐related serological markers. J. Med. Virol. 66:166–170, 2002. © 2002 Wiley‐Liss, Inc.

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. © 2000 Wiley‐Liss, Inc.

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. © 2000 Wiley-Liss, Inc.

The extent of population diversity among GB virus C (GBV-C)/hepatitis G virus (HGV) within a persistently infected individual (Iw) was investigated by sequence analysis of multiple clones generated from polymerase chain reaction (PCR)-amplified products of cDNA analogous to fragments of 5′ non-coding region (5′NC), envelope region 1/2 (E1/E2) and non-structural region 3 (NS3) of viral genome. Although nucleotide substitutions were more common in coding regions than in the 5′NC region, there was no region corresponding to the hypervariable region of hepatitis C virus in the E1/E2 region. Transition substitution exceeded transversion by 7 to 12-fold, and 79.4% of substitutions were synonymous. This bias against substitutions producing amino acid replacements and the use of Pfu DNA polymerase with an error rate 10 times lower than the observed frequency of substitution, suggests that most substitutions were not artefactual. This data suggests that individual genomes of HGV within an infected individual may differ from each other at 0.23–0.84% nucleotide position and at 0.42–0.61% amino acid position.

A controlled trial was conducted to compare the efficacy of interferon (IFN) between two groups of patients with type C liver. Thirty-five patients were randomly assigned to group A (17 patients) or group B (18 patients). The former received 3 megaunits (MU) of human lymphoblastoid IFN six days per week for two weeks, followed by three days per week for 50 weeks; the latter group received 6 MU six days per week for two weeks followed by three days per week for 24 weeks. The percentages of biological sustained responders (B-SR) and virological sustained responders (V-SR) were 29.4 and 23.5%, respectively, in group B, and 17.6% for both in group A. The therapeutic effects were not different between two groups. HCV genotype 2 accounted for significantly higher percentage of B-SR and V-SR (both 57.1%, respectively). These findings indicate that IFN is effective in type C cirrhosis with genotype 2.

Carbohydrate-deficient transferrin (CDT) has been established as a valuable biological marker for detecting chronic alcohol abuse. To improve the diagnostic efficiency, we studied new CDT determination procedures involving the use of lectin affinity chromatography with Allomyrina dichotomaagglutinin (allo A) and Trichosanthes japonicaagglutinin I (TJA-I) to isolate the CDT isoforms CDT-allo A and CDT-TJA, respectively. These procedures, based on detection of the CDT-allo A and CDT-TJA isoforms in sera, showed high sensitivity (100% and 98%, respectively) and high specificity (93% and 85%, respectively). These results demonstrate that the new procedures involving the use of lectin affinity chromatography are more useful for isolating markers in the CDT test than the conventional charge-based separation method.

Carbohydrate‐deficient transferrin (CDT) has been established as a valuable biological marker for detecting chronic alcohol abuse. To improve the diagnostic efficiency, we studied new CDT determination procedures involving the use of lectin affinity chromatography with Allomyrina dichotomaagglutinin (allo A) and Trichosanthes japonicaagglutinin I (TJA‐I) to isolate the CDT isoforms CDT‐allo A and CDT‐TJA, respectively. These procedures, based on detection of the CDT‐allo A and CDT‐TJA isoforms in sera, showed high sensitivity (100% and 98%, respectively) and high specificity (93% and 85%, respectively). These results demonstrate that the new procedures involving the use of lectin affinity chromatography are more useful for isolating markers in the CDT test than the conventional charge‐based separation method.

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 103copies/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. © 1999 Wiley‐Liss, Inc.

A novel DNA virus, TT virus (TTV), was identified in a Japanese patient with posttransfusion hepatitis. The epidemiology and etiological role of this virus have not been elucidated. We investigated the epidemiology of TTV infection in hepatitis C virus (HCV) high endemic and low endemic areas, R town and M town, respectively. The seroprevalence, potential risk factors, and laboratory features of TTV in relation to those of HCV were analyzed. TTV DNA was detected using a seminested polymerase chain reaction and the TTV genotypes were determined by a direct sequencing method. TTV DNA was detected in 16.1% of the subjects in R town and 17.5% of those in M town. The TTV DNA positivity rates of the 2 areas did not differ significantly. A history of blood transfusion was not a specific risk factor for TTV infection. The mean serum alanine aminotransferase (ALT) level of the anti‐HCV‐positive subjects was significantly higher than that of the TTV DNA‐positive subjects, most of whom had normal ALT levels. The TTV genotype distributions of these 2 distinct areas differed. These results suggest that TTV infection is widespread with a geographical genotypic distribution independent of HCV infection and that the ALT abnormalities are not attributable to TTV but to HCV infection in the general population. J. Med. Virol. 58:235–238, 1999. © 1999 Wiley‐Liss, Inc.

The nucleotide sequence of hepatitis G virus (HGV) genome was determined by analysis of cDNA clones obtained by long reverse transcription-polymerase chain reaction (long RT PCR) and 5′- and 3′-rapid amplification of cDNA ends (RACE) from a Japanese patient (Iw) with non A-E hepatitis. The HGV-Iw genome, consisting of 9375 nucleotides, contains a long open reading frame encoding 2873 amino acid residues. Comparison of HGV-Iw with two American isolates of HGV and one African isolate of GB virus C (GBV-C) indicates that although the nucleotide sequences of these isolates were considerably divergent (86.2% to 93.3%), the deduced amino acid sequences shared an extremely high degree of identity (96.1% to 100%). It was also found that HGV-Iw was more closely related to the HGV isolates from USA than to the GBV-C isolate from Africa.

Nine patients with 16 symptomatic nonneoplastic congenital hepatic cysts were treated prospectively by ultrasonically guided percutaneous minocycline hydrochloride injection, and the usefulness of this treatment was evaluated. Seven of the patients had multiple hepatic cysts, and two solitary cysts. All the patients were women, ranging in age from 36 to 81 years. After cystic fluid had been aspirated with a 21-gauge PTC needle, minocycline hydrochloride was injected into all the cysts. The minocycline hydrochloride was dissolved in saline at a concentration of 200 mg in 9 ml, and mixed with 1 ml of 2% mepivacaine hydrochloride. The total quantity of minocycline hydrochloride injected varied from 100 mg to 1200 mg per hepatic cyst, depending on its size. Total or subtotal regression of the cysts was observed in all patients during follow-up periods ranging from 15 to 35 months. Seven patients became symptom-free, one showed symptom reduction, and one showed no change in symptoms. Minor side effects, eg, transient abdominal pain, slight right shoulder pain, and temperature elevation, were noted in three patients respectively. On the basis of these results, we conclude that ultrasonically guided percutaneous minocycline hydrochloride injection is useful for the treatment of symptomatic hepatic cysts.

Cholecystokinin (CCK)-like activities in the duodenal mucosa were measured by bioassay in patients with duodenal ulcer, cholelithiasis and some other gastrointestinal diseases as well as in healthy normal subjects. The mean CCK-like activity of the duodenal mucosa in patients with duodenal ulcer and cholelithiasis was 0.405 Ivy dog units per milligram and 0.376 Ivy dog units per milligram of dry weight of the duodenal mucosa (IDU/mg d.w.), respectively. Both these figures are significantly higher than 0.180 IDU/mg d.w. in normal subjects (p<0.005, p<0.005, respectively). It can be speculated that the CCK-level of the duodenal mucosa in patients with cholelithiasis may be regulated by a feedback mechanism and CCK in patients with duodenal ulcer may act physiologically for the cure of duodenal ulcer.

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 parvumand 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/mm3peripheral 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.

A 65‐year‐old female received recombinant interferon (IFN) α‐2b daily for the treatment of chronic hepatitis C. Fever (39°C or higher) developed 14 days after the start of administration. Abdominal computed tomography suggested multiple liver abscesses, which had not been detected before IFN administration. An autopsy revealed an amoebic liver abscess. A subclinical infection of Entamoeba histolyticain this case developed into amoebic liver abscess during IFN administration.

In rats with experimental liver injuries and humans with various liver diseases, fluorescence from the liver surface after intravenous injection of 10% fluorescein-sodium was investigated. Our improved fluorescence laparoscopy allowed clear fluorescent images. In normal rats and in cases of nonspecific reactive hepatitis, fluorescence was first found in the portal area of the liver surface; it was then observed spreading into the lobules towards the central veins. In damaged livers of both rats and humans, bright fluorescence was observed in arterioles, necrotic foci, and fibrotic areas on the liver surface. The patterns of fluorescence which appeared in the early phase after intravenous injection of fluorescein-sodium seemed to reflect the shapes and distribution patterns of the portal areas. These results suggest that observations with improved fluorescence laparoscopy are useful for diagnoses of liver diseases.

To clarify the characteristics of cell surface Pre-S2 Ag, HBcAg and HBeAg immunohistochemically and to explore their relationship with a cellular immune target antigen, 31 liver biopsy specimens from chronic HBV carriers were examined by immunoperoxidase staining. By immune light microscopy, Pre-S2 Ag was detected on the liver cell membrane in 18 (58%) of the 31 cases, HBcAg in 4 cases (13%) and HBeAg in 4 cases (13%). Pre-S2 Ag frequently showed a honeycomb-like membrane expression pattern which was present regardless of liver inflammation, whereas HBcAg and HBeAg exhibited a scattered membrane expression pattern detected in areas of marked inflammation. Of the 18 cases showing a honeycomb-like Pre-S2 Ag expression, 3 concomitantly showed a scattered membrane expression pattern. Immunoelectron microscopy revealed these two distinct membrane expression patterns. In areas showing a honeycomb-like membrane expression pattern, Pre-S2 Ag was demonstrated in the intercellular space and on the basolateral membranes of hepatocytes, but was not detected on the cell membranes in areas of the intercellular space lacking an immunoreaction. Cytoplasmic expression of Pre-S2 Ag was less extensive in these hepatocytes. These findings suggest that the honeycomb-like membrane expression of Pre-S2 Ag results from attachment of extracellular antigen to the liver cell membrane. In contrast, in areas showing a scattered membrane expression pattern, Pre-S2 Ag, HBcAg and HBeAg were each detected as single-layered linear deposits along the cell membrane, but were absent in the intercellular space. Each antigen was also expressed abundantly in the cytoplasm, and the immunoproducts appeared to fuse with the cell membrane. These findings suggest that the scattered membrane expression of these antigens results from intrahepatic transfer of antigen synthesized in the liver cell to the cell membrane, possibly serving as a target for the host immune-mediated response in connection with inflammation.

Computerized morphometrical measurements were made of liver cells and their nuclei taken from livers of healthy persons and from patients with acute hepatitis (AH), chronic hepatitis (CH), and liver cirrhosis (LC). Liver biopsies were performed on 127 people (24 convalescing from acute hepatitis, 20 with chronic inactive hepatitis (CIH), 24 with chronic active hepatitis (CAH), 38 with liver cirrhosis (LC) and 21 who were healthy controls (HC)). The specimens were stained with hematoxylin-eosin, then light microscopic pictures were obtained.

Thirty three resected gastric lesions were examined histologically to analyze the relationship between histologie and cytologie diagnoses of the so-called atypical epithelial growth based on biopsy specimens. Of the 33 lesions twenty were histologically verified atypical epithelial growth and the remaining thirteen were, in the same manner, diagnosed with some equivocation as papillary adenocarcinoma. The results of both histological and cytological diagnoses of the biopsy materials from the above mentioned 33 lesions were as follows; for the twenty atypical epithelial growths, 18 were diagnosed as benign, one was as suspicious of malignancy and the other one was as cancer by biopsy histology, whereas these lesions were diagnosed as benign in 16, suspicious in two and cancer in two by cytology; for the thirteen papillary adenocarcinomas, 8 were diagnosed as benign, 4 were as suspicious of malignancy and one was as cancer by biopsy histology. On the other hand their diagnoses were malignant in six, suspicious of cancer in four and benign in three by biopsy cytology. The data indicate that biopsy cytology favors malignancy for atypical epithelial growth and papillary adenocarcinoma when compared with biopsy histology.

A 54‐year‐old woman who was being treated with 10 million units (mu) of natural interferon (IFN)‐α per day for chronic active hepatitis C at a local clinic, developed coma on the fourth day of treatment. On admission to Yamagata University Hospital, she was still in a state of semicoma with severe hyponatraemia (122 mEq/L) and hypochloraemia (89 mEq/L). After the administration of electrolytes, her condition improved remarkably. Endocrinological loading tests showed a hypofunction of the anterior pituitary gland. In consideration of these results, and her past experiences of haemorrhage during childbirth and subsequent amenorrhoea, we diagnosed her illness as a coma as a result of Sheehan's syndrome which had become overt during IFN therapy. She recovered completely after treatment with hydrocortisone and 1‐thyroxine.