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A case of acute hepatitis A associated with fibrin-ring granulomas in the liver is presented. Because a relationship between acute hepatitis A infection and granuloma formation had not previously been established, liver specimens were examined from both the hepatitic and recovery phases. Numerous fibrin-ring granulomas were observed in the parenchyma during the hepatitic phase. The cellular components of the granulomas were largely macrophages and CD4-positive T-cells. Granulomas had disappeared completely by the recovery phase. These results suggest that fibrin-ring granulomas were caused by hepatitis A virus infection. This virus may activate macrophages and CD4-positive T-cells through an as-yet undetermined mechanism.

In ileal epithelial cells, apical sodium-dependent bile acid transporter (ASBT) is responsible for the uptake of bile acids from the lumen. Furthermore, ASBT is expressed in the apical plasma membrane of intrahepatic bile duct cells (BECs). Using cultured immortalized mouse intrahepatic BECs that form monolayers or cysts, vectorial transport of bile acids was studied. [3H]-taurocholic acid ([3H]-TCA) was transported through monolayers transcellularly almost exclusively from the apical to the basolateral side in a Na(+)- and a temperature-dependent manner. Transport of [3H]-TCA was inhibited by 59.3+/-18.6% in the presence of taurochenodeoxycholic acid. Uptake of lysyl fluorescein-conjugated bile acid, Cholyl-[Nepsilon-NBD]-lysine, was seen in a Na(+)- and a temperature-dependent manner from the apical side of BECs that form monolayer or cysts. Reverse transcription-polymerase chain reaction for mRNAs in the cells showed presence of mRNAs for ASBT and farnesoid X receptor (FXR), a nuclear bile acid receptor. In conclusion, intrahepatic BECs transport bile acids mainly from the apical to the basolateral side in concert with ASBT and maybe FXR in the cells.

BACKGROUND/AIMS: In primary biliary cirrhosis (PBC), cytokines from CD4+ T lymphocytes were suggested to contribute to the intralobular bile duct damage together with cellular immunity by CD8+ T lymphocytes. Recently, we reported that immunolocalization of 7H6--a tight junction (TJ)-associated protein--was significantly diminished in cholangiocytes in the PBC liver. In this study, we examined the direct effects of several cytokines--tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-2 and 4 (IL-2 and 4)--on TJ in immortalized mouse cholangiocytes. Moreover, we examined the inhibitory effect of ursodeoxycholic acid (UDCA) on cytokine-induced changes in paracellular permeability. METHODS: Barrier function of TJ was evaluated by measuring transepithelial electrical resistance (TER) and 3H-inulin flux. We also performed immunostaining and immunoblotting for TJ-associated proteins--claudin-1 and -3, occludin, zonula occluden-1 (ZO-1) and 7H6. RESULTS: TNF-alpha and IFN-gamma, but neither IL-2 nor IL-4, significantly decreased TER (P < 0.005). 3H-inulin flux studies confirmed IFN-alpha-induced increases in paracellular permeability of cholangiocytes (P < 0.001). In immunostaining and immunoblotting studies, TJ-associated proteins were well preserved in TNF-alpha- or IFN-gamma-treated cells. Ursodeoxycholic acid has been found to have no inhibitory effect on increased paracellular permeability induced by TNF-alpha or IFN-gamma. CONCLUSION: These findings show that TNF-alpha and IFN-gamma disrupt barrier function of TJ in cholangiocytes without major structural changes to TJ and suggest that disruption of TJ function and subsequent leakage of the bile constituents may influence the aggravation of cholestasis in PBC.

Gene expression of interleukin 12-receptor beta2 chain mRNA in peripheral blood mononuclear cells (PBMCs) was examined in patients with chronic hepatitis C (n=7) and in healthy control subjects (n=6) by semi-quantitative RT-PCR. The level of interleukin 12-receptor beta2 chain mRNA was higher in patients with chronic hepatitis C than in healthy subjects (P=0.032). The level of interleukin 12-receptor beta2 chain mRNA had a weak correlation with the ratio of Th1 to Th2 populations (r=0.714, P=0.020). There was a tendency for the level of interleukin 12-receptor beta2 mRNA to increase both in chronic hepatitis C (P=0.109) and in healthy volunteers (P=0.144) after the incubation of PBMCs with interferon-alpha in vitro. During interferon-alpha administration to the patients with chronic hepatitis C, the level of interleukin 12-receptor beta2 chain mRNA in PBMCs was increased in all four cases. Although this is a preliminary study with a small sample size, our results suggest that the level of interleukin 12-receptor beta2 chain mRNA is higher than normal in patients with chronic hepatitis C and can be further enhanced by interferon therapy.

It has been reported in Germany that seroconversion to anti-HBe or anti-HBs is frequently associated with genotype changes of hepatitis B virus (HBV) from genotype A to genotype D. We previously reported that the HBeAg-negative state in Japan was significantly more common in patients infected with genotype B HBV than those infected with genotype C HBV. To determine whether the high prevalence of genotype B in the HBeAg-negative state is due to a change from genotype C to genotype B, 72 pairs of serum samples before and after HBe seroconversion were examined for nucleotide sequences in the S gene (amino acids 42-164) among Japanese HBV carriers. No one was identified to have undergone genotype change during HBe seroconversion. A total of 71 codon mutations were observed. Sixty-two of these 71 codon mutations (87.3%) were non-synonymous. Genotype B HBV had no mutational hot spots. In genotype C, there was a mutational hot spot at amino acid 126 of the S protein, and it was suggested that Thr126 before HBe seroconversion was more susceptible to becoming an asymptomatic carrier after HBe seroconversion than Ile126. In conclusion, genotype changes during HBe seroconversion were not found to be common in Japan.

Background: The cyclin dependent kinase p21(waf1) plays a crucial role in the regulation of cell cycle. The family of p53 proteins has the ability to induce p21(waf1), whereas p16(INK4a) modulates post-transcriptionally the expression of p21(waf1). Methods: Total 36 hepatocellular carcinomas (HCCs) and 24 paired adjacent liver tissues were evaluated for the following: (1) expression of p21(waf1) and p16(INK4a); (2) that of p21(waf1), p73 and p63 mRNAs; (3) genomic mutations and the loss of heterozygosity of p73 and p53; and (4) frequency of methylation in the 5'CpG promoter region of p16(INK4a). Results: In HCCs compared with the adjacent non-cancerous liver tissues, the expression of p21(waf1) and p16(INK4a) was reduced. Indeed, p21(waf1) was not detected in 36% (8/22) of HCCs in spite of the presence of p21(waf1) mRNA: among them, mutations of p53 gene were found in 50%, whereas a lack of p16(INK4a) expression in all of them. p21(waf1) and p16(INK4a) were reduced in proportion to the degree of methylation in p16(INK4a) gene. p73 did not mutated, and p63 did not expressed in HCCs. Conclusion: Methylation status of p16(INK4a) gene will play a part for reducing constitutive expression of p16(INK4a) and of p21(waf1) coordinately in HCCs.

B型肝硬変5例 (男性3例, 女性2例, 年齢28-53, 中央値44歳) に対してラミブジンを投与し, その有用性について検討した. ラミブジン投与開始時と投与9カ月目の比較では, 総ビリルビンが0.7-6.2中央値3.1mg/dlから0.8-3.1中央値0.8mg/dlへ, ALTが50-268中央値74IU/lから19-44中央値30IU/lへ, TTTが20.1-33.1中央値29.9 Kunkel から5.5-19.8中央値14.3 Kunkel へ, アルブミンが3.1-4.8中央値3.5g/dlから3.1-5.1中央値3.9g/dlへ, コリンエステラーゼが79-211中央値94IU/lから118-315中央値206IU/lへ, HBV DNAが108.00-108.92中央値108.32copies/mlから102.00未満-105.30中央値102.00copies/ml未満へと改善した. 肝硬変症例に対するラミブジン投与は, 肝炎を鎮静化するだけでなく, 肝予備能の改善をも期待し得る治療法であると考えられた.

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

OBJECTIVE We evaluated the measurements of serum alpha-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. Youden's 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 (Youden's index = 0.422) and 60 mAU/ml for PIVKA-II (Youden's 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 (Youden's 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.

We employed a PCR assay system TaKaRa Ex Taq™ (heat-resistant DNA polymerase), which has 3′→5′ exonuclease activity to increase the sensitivity for TT virus (TTV) DNA detection. Sera obtained from 95 hepatitis B virus carriers without hepatitis C virus coinfection were tested for TTV DNA and the sensitivity of this assay system was compared with the PCR systems reported previously. Of the 95 individuals, TTV DNA was identified in 14 (14.7%) with the PCR reported by Nishizawa et al., in 66 (69.5%) with the PCR reported by Okamoto et al., in 80 (84.2%) by our assay system, and in 86 (90.5%) with the PCR reported by Takahashi et al. Phylogenetic analysis of nucleotide sequences amplified by the PCR revealed that genotypes 1a, 1b, 2, 3, 4, 5 were amplified efficiently by our assay system, while only a part of TTV DNA clone of genotype 1a was amplified by the PCR reported by Nishizawa et al. The prevalence of circulating TTV DNA became higher with age. These results indicate that our assay system with TaKaRa Ex Taq™ has confirmed high prevalence of TTV infection and that at least five genotypes prevail in Japan. In addition, acute TTV infection is supposed to cause long-standing viremia.

The route of hepatitis B virus (HBV) infection and subsequent clinical course vary widely. It is not clear if the prevalence of HBV genotypes differs in the different clinical features of HBV carriers. The genotype of HBV was determined by direct sequencing of HBV DNA amplified with a PCR method from 310 Japanese HBV carriers (189 male and 121 female, aged from 14 to 82 years). Genotype A was detected in eight (2.6%) of 310 HBV carriers, genotype B was detected in 92 (29.7%) and genotype C was detected in 210 (67.7%). None of them had genotype D, E or F. Among the eight patients infected with genotype A, one patient had been infected with HBV in his adulthood. Furthermore, both of one married couple also had genotype A. On the other hand, a HBeAg-negative state and HBeAg-negative healthy carrier state were significantly more common in patients infected with genotype B than those with genotype C by univariate analysis (P

Alterations in carbohydrate metabolism associated with liver cirrhosis are characterized by a high serum insulin level and prolonged hyperglycemia on oral glucose tolerance test (OGTT). We measured plasma glucose, immunoreactive insulin (IRI), and C-peptide immunoreactivity (CPR) levels during a 75-g OGTT before and after varices obliteration in 10 cirrhotic patients with gastric varices. After obliteration, the indocyanine green retention rate was decreased and the portal flow velocity was increased. A significant decline in plasma glucose and IRI levels was also noted on OGTT. Moreover, the plasma glucose and IRI levels declined at 90 and 120 min in OGTT while they increased progressively by 120 min before obliteration. The levels of CPR were similar before and after treatment. These results indicate that decreased portal flow due to extrahepatic shunt and consequent impairment of insulin metabolism play a role in glucose intolerance observed in cirrhotic patients and that shunt occlusion improves glucose metabolism.

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.

Aims/Background: Transcatheter arterial chemoembolization (TAE) of hepatocellular carcinoma (HCC) causes anoxia. Escape of cancer cells from anoxic injury may be enhanced by induction of proteins which provide resistance to apoptosis. Methods: We examined HCCs immuno-histochemically for Bcl-2, vascular endothelial growth factor (VEGF), p53, and Ki67. The staining intensity for VEGF, a protein induced by anoxia, was assessed morphometrically with a computer-assisted image-analyzer. Results: The frequency of Bcl-2 positive cells was higher in HCCs that had undergone TAE (TAE HCC) than that in HCCs that had not undergone TAE (41.75 ± 15.06 vs. 1.01 ± 0.79 cells/1000 cells, p=0.0173). The frequency of p53- or Ki67-positive cells was not increased after TAE. Of 12 TAE HCCs, 7 had Bcl-2 positive HCC cells and 6 had clusters of Bcl-2 positive cells. In contrast, 2 of 11 HCCs that had not undergone TAE had only a few, sporadically distributed, Bcl-2-positive cells. The staining intensity for VEGF was higher in Bcl-2 positive than in Bcl-2 negative areas (1.208 ± 0.091 vs. 1.071 ± 0.017, p=0.0222). Furthermore, the VEGF staining intensity in Bcl-2 positive areas of TAE HCCs was higher than in Bcl-2 negative areas (1.296 ± 0.126 vs. 1.066 ± 0.024, p= 0.0186), while in HCCs that had not undergone TAE the staining intensity was similar. Conclusions: TAE of HCC can induce Bcl-2 expression, possibly through anoxic stress.

Helper T cells (Th) are classified as type 1 (Th1) and type 2 (Th2) according to the cytokines they produce; interferon-gamma is produced by Th1, and interleukin-4 by Th2. We counted the circulating CD4-positive Th cells that produce interferon-gamma or interleukin-4 with an enzyme-linked immunospot assay. CD4-positive T cells isolated from patients with chronic hepatitis B (n = 10), chronic hepatitis C (n = 16), and healthy subjects (n = 10) were stimulated with anti-CD3 antibody in vitro. The number of interferon-gamma-producing Th cells was significantly lower in patients with chronic hepatitis C than in healthy subjects (P = 0.0024), whereas in patients with chronic hepatitis B, the number was similar to that in healthy subjects (P = 0.8530). The number of interleukin-4-producing Th cells was significantly higher in patients with chronic hepatitis C (P = 0.0010) and chronic hepatitis B (P = 0.0089) than in healthy subjects. In chronic hepatitis C, the number of interferon-gamma-producing Th cells was increased after incubation of the cells with interferon-alpha (P = 0.008) or with recombinant interferon-gammala (P = 0.024), but not with interferon-beta (P = 0.051). The number of interleukin-4-producing Th cells was decreased after incubation with interferon-alpha (P = 0.0004), with interferon-beta (P = 0.003), and with recombinant interferon-gammala (P = 0.0004). Changes in the numbers of interferon-gamma- or interleukin-4-producing Th cells in vitro were more evident in sustained responders to interferon therapy than in non-responders. These results suggest that Th2 cells are the predominant cell type in chronic hepatitis C, and that their activity may be suppressed by the administration of interferon.

Purpose: To assess the arterial supply to hepatocellular carcinomas (HCCs) by inferior phrenic arteries (IPA). Material and Methods: A total of 126 consecutive cases of HCC were studied by contrast-enhanced CT and conventional arteriography. Blood supply from an IPA was suspected when the size of the HCC mass as seen on contrast-enhanced CT did not match the size of the tumor mass as seen on hepatic arteriography. Inferior phrenic arteriography was employed to confirm these findings. HCCs fed by the IPA were analyzed in terms of size, location, and history of prior treatment. Results: In 14 (11%) of the 126 cases, the tumor was found to have a blood supply from an IPA. Eleven of these tumors were located in segments 2 and 7. Three tumors, which had not been treated previously, had a blood supply from an IPA. Six tumors were almost exclusively fed by an IPA and were located in segments 7, 1, and 4. Conclusion: HCCs located in segments which form the bare area of the liver (S1, S2, S7) can be supplied by an IPA. This should be suspected when a lesion or part of a lesion is identified on contrast-enhanced CT but not on hepatic arteriography.

A recently discovered non-A non-B hepatitis virus has been designated hepatitis G virus (HGV). Blood contamination has been proposed as its mode of transmission. We studied the genoprevalence of HGV in Japanese people at high risk. HGV was identified in serum by a reverse-transcription polymerase chain reaction. HGV was detected in 16.0% of intravenous drug users (IDUs) (n = 25), 16.2% of those with tattoos (n = 37), 10.9% of IDUs with tattoos (n = 55), 5.7% of chronic hepatitis (CH)-C patients (n = 87), and in none of the CH-B (n = 50) or CH non-B non-C (n = 46) patients. Serum alanine aminotransferase (ALT) levels of those infected with HGV alone (n = 3) were all within normal range. In the patients with CH-C, serum ALT levels of those coinfected with HGV were similar to serum ALT levels of those without HGV infection. A phylogenetic tree of isolated HGV clones showed that the HGVs of these subjects bore only a distant-resemblance to clones reported from Africa and North America, and that variation in the phylogenetic index of HGV clones was small. These results suggest that HGV clones from different areas have genetic heterogeneity and that HGV causes no or mild hepatitis.

Tumor necrosis factor alpha (TNF-alpha), which is primarily produced by macrophages, is a cytokine with various biological activities. Macrophage infiltration often accompanies experimental cholangitis in rats, and chronic cholangitis in humans. The pathophysiologic significance of TNF-alpha in cholangitis is not known. We used cultured, polarized intrahepatic bile duct epithelial cells (IBDECs) from rat liver to determine whether TNF-alpha directly affects the organization of IBDEC monolayers. The addition of recombinant TNF-alpha (rTNF-alpha) to culture media at concentrations from 10 to 200 U/mL lacked cytotoxicity to the IBDECs as judged by trypan blue exclusion and lactate dehydrogenase (LDH) release. rTNF-alpha transiently reduced transepithelial electrical resistance in a dose-dependent manner. During this decrease in resistance, the cellular tight junctions became leaky, allowing horseradish peroxidase (HRP) penetration. rTNF-alpha, at concentrations up to 200 U/mL, did not detach IBDECs from Matrigel, an artificial basement membrane. Electron microscopy and immunohistochemistry for F-actin showed a well-preserved cell structure and organization of IBDECs. Results suggest that TNF-alpha is nontoxic to IBDECs, and that it increases the permeability of tight junctions. TNF-alpha may thus disturb the barrier function of the bile duct. (Hepatology 1996 Jun;23(6):1602-7)

The culture of fully differentiated intrahepatic bile duct epithelial cells (IBDECs) to use as a model for the in vivo intrahepatic biliary tract has not been established. IBDECs from normal rat livers were grown on a collagen-coated permeable filter and formed a confluent monolayer 7 days after being plated. Positive reactions for cytokeratin-19 and retained gamma-glutamyl transpeptidase (GGTP) activity were shown. The transepithelial electrical resistance between the apical and the basolateral compartment culture chambers increased with the culture age and plateaued after the 7th day. The resulting cultured cells displayed a number of characteristics. (1) The cells formed a thin, continuous monolayer and displayed microvilli on the apical surface and junctional complexes between the cells, consistent with in vivo IBDECs. (2) Cells cultured for more than 7 days prevented the passage of horseradish peroxidase (HRP) and ruthenium red through paracellular pathways. (3) Seven-day-old cultures displayed a mean transepithelial electrical resistance of 137.3 omega-cm2, which decreased by 27.1% from its initial level after cell treatment with ethylenediamineteraacetic acid (EDTA). These results indicate that confluent IBDEC monolayers are well differentiated and polarized with tight junctions (TJs) between the cells. These cell monolayers can provide a useful and relevant model for the in vitro study of various in vivo bile duct phenomena.

Rationale and Objectives. Currently available treatments for hepatocellular carcinoma are not satisfactory in terms of recurrence rates. In this study, we injected ethanolamine oleate (EO) into a portal branch in an attempt to cause necrosis of a liver segment in which hepatocellular carcinoma might be located. Methods. Nine dogs received EO injections via a balloon catheter into a segmental portal branch of the liver. Results. Immediately after injection, 80–100% of the liver cells in the EO-injected segment underwent coagulative necrosis. After 1 week, the EO-injected segment had become completely necrotic in two dogs. Only a few viable hepatocytes were still observed around the arteries and beneath the liver capsule in another dog. No pathologic changes were observed in the lungs, kidneys, or heart of any dog. There was a correlation between the EO dosages and the volume of the EO-injected liver tissue. Conclusion. EO injection into a portal branch results in the pharmacologic destruction of the corresponding liver segment. This procedure may be beneficial in the treatment of hepatic malignancies.

BACKGROUND: HBcAg-specific regulatory T (T(reg)) cells play an important role in the pathogenesis of chronic hepatitis B. Soluble heat shock proteins, especially soluble heat shock protein 60 (sHSP60), could affect the function of T(reg) cells via Toll-like receptor. METHODS: We analyzed the relationship between soluble heat shock protein production and hepatitis B virus (HBV) replication with both clinical samples from HBeAg-positive patients with chronic hepatitis B (n= 24) and HBeAb-positive patients with chronic hepatitis B (n= 24) and in vitro HBV-replicating hepatocytes. Thereafter, we examined the biological effects of sHSP60 with isolated T(reg) cells. RESULTS: The serum levels of sHSP60 in patients with chronic hepatitis B were statistically significantly higher than those in patients with chronic hepatitis C (P

To elucidate how actin filaments participate in bile formation, polarity of actin filaments in the pericanalicular cytoplasm was determined with myosin subfragment 1 by transmission electron microscopy of ultrathin sections and deep-etching replicas. Densely concentrated actin filaments were identified around the bile canaliculi in the forms of microvillous core filaments, pericanalicular web filaments, and filaments on the functional complex. They bound subfragment 1 to form double-helical strands on the deep-etching replica or typical arrowheads on the ultrathin section. All microvillous core filaments showed their arrowheads pointing basally, suggesting the molecular growth occurring at their apical ends. In contrast, filaments of the pericanalicular web, running in parallel to the cell surface, showed unfixed polarities as indicated by their arrowheads. Furthermore, neighboring filament pairs often showed opposite polarities, an alignment necessary for filament sliding. The junctional complex had filaments with arrowheads pointed mostly at the cell center with a small number in opposite direction. In addition, a group of sporadic filaments appeared to be installed to link to both the canalicular membrane and coated vesicles. Such regionally specialized actin filaments are considered inclusively to form a cytoskeletal system that is in charge of (a) maintenance of length of the microvilli, (b) contraction of the canalicular walls, and (c) translocation of coated vesicles in the pericanalicular cytoplasm.

Objective: To report on a patient with Hashimoto’s encephalopathy induced by lithium. Patient and interventions: A 61-year-old woman with a type II bipolar disorder and a history of lithium-induced thyrotoxicosis associated with silent thyroiditis was hospitalized to treat a severe major depressive episode. Given long-term treatment with levothyroxine for hypothyroidism that had resulted from silent thyroiditis, endogenous hormone in thyroid follicles was assumed to be minimized by the negative feedback, decreasing risk of recurrent thyrotoxicosis if lithium were restarted. Results: Lithium clearly relieved the patient’s depressive symptoms, but after 40 days encephalopathy developed. Thyrotoxicosis was ruled out, and serum antithyroid antibody titers were elevated. In the cerebrospinal fluid, protein content was substantially elevated and antithyroid antibodies were detected. Encephalopathy resolved dramatically after course of intravenous pulse therapy with methylprednisolone. Conclusions: We believe that autoantibodies against antigens shared by the thyroid gland and the brain were induced by exposure to lithium, causing the patient to develop Hashimoto’s encephalopathy.

It was recently shown that intrahepatic bile duct epithelial cells in situ or after isolation from rat liver have coated pits and vesicles, suggesting that they participate in receptor-mediated endocytosis. Therefore, using a morphologic approach and epidermal growth factor coupled to horseradish peroxidase or colloidal gold as probes, we studied freshly isolated or short-term cultured intrahepatic bile duct epithelial cells prepared from normal rat liver to determine if they participate in receptor-mediated endocytosis. Immunoelectron microscopy using a monoclonal antibody against the epidermal growth factor receptor was also used to examine for the presence of the growth factor receptor on the cells. Immediately after isolation, the cells did not internalize either epidermal growth factor-horseradish peroxidase or epidermal growth factor-colloidal gold; no growth factor receptor could be shown on these cells by immunocytochemistry, either. In contrast, cells cultured for 24 h bound and internalized both epidermal growth factor-horseradish peroxidase and epidermal growth factor-colloidal gold at 37 degrees C and showed growth factor receptors diffusely distributed on the plasma membrane. When cultured cells exposed to epidermal growth factor-colloidal gold were fixed with glutaraldehyde containing saponin and tannic acid, colloidal gold particles were observed in coated pits and in coated and uncoated vesicles. Preincubation of cultured cells with native epidermal growth factor completely blocked the internalization of both epidermal growth factor-horseradish peroxidase and epidermal growth factor-colloidal gold. When rat liver was stained in situ for epidermal growth factor receptor, reaction product was observed by immunoelectron microscopy exclusively on the basal surface of the plasma membrane of the intrahepatic bile duct epithelial cells. These results indicate that bile duct epithelial cells internalize epidermal growth factor by endocytosis via coated pits containing receptors localized in situ exclusively to the basal domain of their plasma membranes. The data demonstrate for the first time that intrahepatic bile duct epithelial cells participate in receptor-mediated endocytosis and raise the possibility that they are a target for epidermal growth factor.

Although recent data from our laboratory have established the occurrence of receptor-mediated endocytosis in intrahepatic bile duct epithelial cells (IBDEC) isolated from normal rat liver, no studies have assessed the role of isolated IBDEC in fluid-phase endocytosis. Therefore, to determine if IBDEC participate in fluid-phase endocytosis, we incubated morphologically polar doublets of IBDEC isolated from normal rat liver with horseradish peroxidase (HRP, 5 mg/ml), a protein internalized by fluid-phase endocytosis, and determined its intracellular distribution by electron microscopic cytochemistry. Pulse-chase studies using quantitative morphometry were also performed to assess the fate of HRP after internalization. After incubation at 37 degrees C, IBDEC internalized HRP exclusively at the apical (i.e., luminal) domain of their plasma membrane; internalization was completely blocked at 4 degrees C. After internalization, HRP was seen in acid phosphatase-negative vesicles and in acid phosphatase-positive multivesicular bodies (i.e., secondary lysosomes). Small acid phosphatase-negative vesicles containing HRP moved progressively from the apical to the basal domain of IBDEC. Pulse-chase studies showed that HRP was then discharged by exocytosis at the basolateral cell surface. These results demonstrate that IBDEC prepared from normal rat liver participate in fluid-phase endocytosis. After internalization, HRP either is routed to secondary lysosomes or undergoes exocytosis after transcytosis from the luminal to the basolateral cell surface. Our results suggest that IBDEC modify the composition of bile by internalizing both biliary proteins and fluid via endocytic mechanisms.

The entire nucleotide sequences were determined for hepatitis B virus (HBV) genotype B (HBV/B) genomes extracted from five patients in the Philippines and designated GenBank AB219426, AB219427, AB219428, AB219429 and AB219430. The serotype of the first four isolates was ayw and that of GenBank AB219430 was adw. Divergences of entire sequences were 1·0–2·0 % between the first four isolates and 3·8–4·2 % between these four and GenBank AB219430. Phylogenetic-tree analysis revealed that, worldwide, HBV/B comprises five subgenotypes: B1, B2, B3, B4 and the new Philippines group, designated B5. Divergences of the entire genome sequences between four isolates in subgenotype B5 and isolates from other countries (subgenotypes) were 4·4–4·8 % with Vietnam (B4), 2·9–3·5 % with Indonesia (B3), 4·7–5·1 % with China (B2) and 5·4–6·0 % with Japan (B1). Similarly, GenBank AB219430 showed the lowest divergences: 3·4 % with the isolate from Indonesia (B3), 5·0 % with Vietnam (B4), 5·4 % with China (B2) and 6·1 % with Japan (B1). This is the first report of entire nucleotide sequences of HBV/B from the Philippines and the results show that these sequences belong to a new subgenotype, B5. The present study identified that HBV/B isolates throughout the world are divided genetically into five subgenotypes, the relationships between geographical distances and the genetic distances of HBV/B being well-correlated.

Ribavirin and interferon-alpha induce Th1 polarization of human CD4+ T cells. The study was conducted to investigate the whether cellular immune response during ribavirin/interferon-alpha therapy is associated with viral eradication by examining mRNA expression of molecules relevant to Th1 and Th2 polarization in CD4+ cells of 13 patients with chronic hepatitis C (seven patients with sustained viral response and six with transient response). Peripheral CD4+ T lymphocytes at 0, 4 and 24 weeks of treatment were tested. There were no significant differences in the mRNA levels at each point of time of the treatment between patients with sustained viral response and those with transient response. The percent increase in mRNA level of the IL-12R beta2 chain from the baseline to the end of the treatment was significantly higher in patients with sustained viral response (15.3+/-6.1%) than in those with transient response (-1.6+/-4.7%, p0.05). There was no significant difference in percent changes in level of IL-12R beta1 chain mRNA between the two groups. In conclusion, the results of this study indicate that the increase of Th1 response is related to the inflammatory activity in the liver and possibly to ribavirin and interferon-alpha therapy. It is also suggested that the measurement of Th1 response has the potential to distinguish patients with relapse from those with sustained virus response.

In primary biliary cirrhosis (PBC), cytokines from CD4+ T lymphocytes were suggested to contribute to the intralobular bile duct damage together with cellular immunity by CD8+ T lymphocytes. Recently, we reported that immunolocalization of 7H6--a tight junction (TJ)-associated protein--was significantly diminished in cholangiocytes in the PBC liver. In this study, we examined the direct effects of several cytokines--tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-2 and 4 (IL-2 and 4)--on TJ in immortalized mouse cholangiocytes. Moreover, we examined the inhibitory effect of ursodeoxycholic acid (UDCA) on cytokine-induced changes in paracellular permeability.Barrier function of TJ was evaluated by measuring transepithelial electrical resistance (TER) and 3H-inulin flux. We also performed immunostaining and immunoblotting for TJ-associated proteins--claudin-1 and -3, occludin, zonula occluden-1 (ZO-1) and 7H6.TNF-alpha and IFN-gamma, but neither IL-2 nor IL-4, significantly decreased TER (P0.005). 3H-inulin flux studies confirmed IFN-alpha-induced increases in paracellular permeability of cholangiocytes (P0.001). In immunostaining and immunoblotting studies, TJ-associated proteins were well preserved in TNF-alpha- or IFN-gamma-treated cells. Ursodeoxycholic acid has been found to have no inhibitory effect on increased paracellular permeability induced by TNF-alpha or IFN-gamma.These findings show that TNF-alpha and IFN-gamma disrupt barrier function of TJ in cholangiocytes without major structural changes to TJ and suggest that disruption of TJ function and subsequent leakage of the bile constituents may influence the aggravation of cholestasis in PBC.

Entire nucleotide sequences of the HBV genome typical for various stages of HBV carriers are currently unknown. Comparison between conserved sequences in HBeAg-positive asymptomatic carriers (HBeAg ASCs) and mutations characteristic for HBeAb-positive asymptomatic carriers (HBeAb ASCs) are of clinical importance. In this study, we determined the entire nucleotide sequences of the HBV genome of patients infected with genotype C (HBeAg ASCs, 11 cases; HBeAb ASCs, seven cases; patients with liver cirrhosis (LC), five cases). Mutations in the entire nucleotide sequences were found more frequently in HBeAb ASCs than in HBeAg ASCs. In the precore/core (preC/C) region, amino acid mutations were more frequent in HBeAb ASCs (3.03%) than in HBeAg ASCs (0.00%) and patients with LC (0.69%). It was suggested that the mutations in the preC/C region had a close relationship with clinical status of HBV carriers. Mutations of leucine to isoleucine at a.a. 100 of the core region and of threonine to serine at a.a. 340 of the polymerase region were found frequently in HBeAb ASCs. In patients with LC, it was suggested that defective interfering particles (DI particles) play a role in the progression of stages. We conclude that attention should be given to mutations at a.a. 340 of the polymerase protein in addition to core protein.

Up-regulation of CD11a expression on CD4+ T lymphocytes is considered to be one of the mechanisms involved in the initiation of the Th-1-mediated immune response. In this study, peripheral blood mononuclear cells from patients with primary biliary cirrhosis (PBC) were evaluated for CD11a(high) CD2(low) T cells and populations of type 1 (Th-1) and type 2 (Th-2) helper T cells. CD11a(high) CD2(low) T cells were found in PBC (7/15) and in active rheumatoid arthritis (4/4), but not in chronic hepatitis C (0/5) or in healthy subjects (0/6). The population of Th-1 had a positive correlation with that of CD4+ CD11a(high) CD2+ cells in patients with PBC (P = 0.034). The serum levels of interferon-gamma also had a weak correlation with the population of CD4+ CD11a(high) CD2(low) cells (P = 0.050). There was no statistically significant correlation of Th-2 population (P = 0.295) or serum interleukin-4 level (P = 0.685) with the population of CD4+ CD11a(high) CD2(low) cells. These results suggest that CD4+ CD11a(high) cells play a role in Th-1-predominance and in the autoimmune process of PBC.

Objective: a part of patients with primary biliary cirrhosis (PBC) has anti-human carbonic anhydrase II (CA II) autoantibodies, although several contradictional reports followed. Since immunization of mice with CA II results in cholangitis in a susceptible strain, PBC with anti-CA II antibody may have distinct clinical features. Thus, we tested the sera of patients with PBC for anti-CA II antibodies and compared clinical characteristics of PBC patients with and without anti-CA II antibodies in Japanese patients. Methods: anti-CA II antibodies were detected in nine of 50 (18%) PBC patients by immunoblotting. The evaluation of these patients included various clinical parameters, autoantibodies, and immunological backgrounds. Results: the levels of serum liver tests and the prevalence of serum anti-mitochondrial antibody (77.8 vs. 92.7%) were not different between the patients with and without anti-CA II antibody. However, the prevalence of anti-nuclear antibody (ANA) was significantly higher in the patients with anti-CA II antibody than that in the patients without anti-CA II antibody (66.7 vs. 25.6%, P=0.044), although their mean titers were not statistically different. Association of Sjogren's syndrome tended to be more frequent in the patients with anti-CA II antibody than those without it (33.3 vs. 14.6%, P=0.327). Studies of HLA class I allotype revealed that three of five (60.0%) patients with anti-CA II antibodies and one patients from 34 (3.0%) patients without anti-CA II antibodies had HLA B51 allotype; the difference in the prevalence of this allotype was significant (P=0.004, Pc=0.01), and the prevalence of other HLA class I and HLA DR allotypes was similar between the patients with and those without anti-CA II antibody. Administration of ursodeoxycholic acid (600 mg per day) was accompanied by change in liver tests in a similar way between the two patient groups. Conclusions: These results suggest that, although clinical features are not distinctive, PBC patients with anti-CA II antibody may have a genetic background, which may contribute to a susceptibility to immune-mediated cholangitis.

We investigated the responses of peripheral blood mononuclear cells (PBMCs) to hepatitis C virus core protein in ten patients with chronic hepatitis C during interferon (IFN)-beta treatment to determine if the modulation of the immune reaction to hepatitis C virus by IFN treatment is associated with the viral clearance. Interleukin-2, interleukin-4, interleukin-10, and interferon-gamma in the supernatant of the patients' PBMC co-cultured with the HCV core antigen-presenting autologous PBMCs were measured by ELISA. Serum levels of soluble CD (sCD) 8 and sCD30 in these patients were also measured by ELISA. The production of interleukin-2 and interferon-gamma by PBMCs of sustained responders (SRs) increased after IFN-treatment, although it did not reach a significant level. Interleukin-10 was detected only in non-responders (NRs) at 0 and 4 weeks after the start of IFN treatment. Serum sCD8 level increased significantly in SR with IFN treatment. A close correlation between the serum sCD8 levels and interferon-gamma levels in the supernatant at week 8 was observed in SR. These results suggest that IFN treatment potentiates the cellular immune reaction against HCV core protein more efficiently in SR than in NR.

Background/aims Cholangitis in rats induced by N-formyl L-methionine L-leucine L-tyrosine (fMLT) is characterized by infiltration of mononuclear cells around bile ducts in portal tracts. Methods We investigated the initial process in fMLT-induced cholangitis histochemically. Results Administration of fMLT into the colons of adult male Wistar rats with acetate-induced colitis resulted in an infiltration of mostly macrophages and granulocytes into the portal tracts on day 1. Abnormal peroxidation as demonstrated by the nitro blue tetrazolium (NBT) reaction occurred in bile duct cells as well, although no apparent necrosis of the bile duct cells was observed. On day 4, the majority of the inflammatory cells in the portal tracts were CD4+ or CD8+ T lymphocytes. The oxidative products of the NBT reaction also disappeared from the bile duct cells. Administration of carrageenan, a potent inhibitor of macrophage function, resulted in a significant decrease in lymphocyte infiltration into the portal tracts. On day 8, portal inflammation subsided. Conclusions In formyl peptide-induced cholangitis, macrophages and granulocytes may injure bile ducts transiently. Further, macrophages are necessary for the subsequent migration of T lymphocytes around the bile ducts.

Aims/Background: Expression of urokinase-type plasminogen activator receptor (uPAR) was studied in 25 hepatocellular carcinomas (HCCs) and seven cholangiocellular carcinomas (CCCs) by immunohistochemistry. Methods and Results: uPAR was expressed mostly by host cells distributed along the tumour-host interface in all cases of HCC and CCC, and its expression was higher in CCC. These uPAR-positive cells were identified as macrophages by observation of serial sections stained for CD68, a marker for macrophages. Cancer cells were positive for uPAR in only one case of poorly differentiated HCC with sarcomatous changes and in three cases of CCC. Hepatocellular carcinomas were classified into two types: those with a fibrous capsule (expansive type) and those without a fibrous capsule (invasive type). Invasive-type HCCs showed more prominent expression of uPAR by macrophages than expansive HCCs (p

OBJECTIVE: 1,25(OH)2 vitamin D3 can affect immune cells. However, the mechanism responsible for the favorable effects of 1(OH) vitamin D3, which becomes 1,25(OH)2 vitamin D3 in the liver, is not clear. The aim of this study is to analyze the immunological response of 1(OH) vitamin D3 supplementation in CH-C patients. DESIGN: Forty-two CH-C patients were treated with 1(OH) vitamin D3/Peg-IFNα/RBV. Forty-two case-matched controls were treated with Peg-IFNα/RBV. The expression of Interferon-stimulated genes (ISGs)-mRNA in the liver biopsy samples and JFH-1 replicating Huh-7 cells were quantified by real-time PCR. Ten kinds of cytokines in the plasma were quantified during treatment by using a suspension beads array. A trans-well co-culture system with peripheral blood mononuclear cells (PBMCs) and Huh-7 cells was used to analyze the effect of 1(OH) vitamin D3. The activities of the Th1 response were compared between subjects treated with 1(OH) vitamin D3/Peg-IFN/RBV and those treated with Peg-IFN/RBV therapy alone. RESULTS: 1(OH) vitamin D3/Peg-IFN/RBV treatment could induce rapid viral reduction, especially in IL28B T/T polymorphism. Several kinds of cytokines including IP-10 were significantly decreased after 4 weeks of 1(OH) vitamin D3 treatment (p

While recent data in intrahepatic bile duct epithelial cells (IBDECs) isolated from normal rat liver have established their ability to undergo endocytosis, few studies have assessed endocytosis in IBDECs in situ. Thus, to clarify the activity of IBDECs in situ on macro-molecules in bile and blood, we injected horseradish peroxidase (HRP) into either the common bile duct or the portal vein, and determined its intracellular distribution by electron microscopic cytochemistry. Successful retrograde injection into the common bile duct was achieved by resection of the liver surface so as to avoid HRP leakage from the bile duct on injection. IBDECs internalized HRP through both the apical and basolateral plasma membranes. By quantitative analysis, counting the number of HRP-positive vesicles in the cells, apical endocytosis was found more active than basolateral. HRP internalized through the apical membrane was either routed to the acid phosphatase-positive lysosomes for degradation or to extracellular space for transcytosis. HRP through the basolateral membrane was transferred to the organelles having lipid inclusion, which were expected to be lysosomes negative for acid phosphatase. Our results suggest that IBDECs in situ are actively engaged in endocytosis for degradation of macromolecules in bile and blood, and possibly engaged in the excretion of macromolecules into extracellular space.

Concanavalin A (Con A) can induce an immune-mediated hepatitis. Since direct evidence of immune mechanism for this hepatitis is lacking, we employed adoptive transfer to study the mechanism of Con A-induced hepatitis. Intravenous administration of Con A (20 mg/kg) to Balb/c mice was accompanied by elevations of serum alanine aminotransferase (ALT) levels and midzonal necrosis with lymphocyte infiltration in the liver. None of the Balb/c nu/nu mice showed biochemical or pathologic hepatic abnormalities with the same dose of Con A. In the area of midzonal necrosis, CD4-positive T lymphocytes appeared at 24 hr after injection, and then both CD4-positive and CD8-positive T lymphocytes were found at the margin of zonal necrosis at 48 hr. Pretreatment with carrageenan, a potent inhibitor of macrophages, prevented these biochemical and pathologic changes. Mononuclear cells infiltrating in the liver of Balb/c mice 24 hr after priming with Con A were harvested and injected into Balb/c nu/nu mice injected with Con A 24 hr previously. Serum ALT levels elevated and the same pathologic changes observed in Con A-treated Balb/c mice were observed. These changes were not observed when the splenic cells from Con A-treated Balb/c mice were transferred to Con A-treated nude mice. These results suggest that Con A-induced hepatic injury is mediated by macrophages and T lymphocytes sensitized by Con A or its metabolites.

Two hundred and seventy-three serum specimens from hepatitis B virus (HBV) carriers were examined for the presence of a characteristic one point mutation at nucleotide (nt) 1896 from the EcoRI site of the HBV genome in the precore region (the preC mutant) using restriction fragment length polymorphism (RFLP) analysis. This assay approach could detect preC mutants or wild-type sequences when either form constituted more than 10% of the total sample. Overall, 65.5% (76/116) of HBeAg-positive carriers had only the preC wild-type. All HBeAg-positive asymptomatic carriers (n = 14) had only the preC wild-type. In patients with chronic hepatitis B and in anti-HBe-positive asymptomatic carriers, increased prevalence of the preC mutant was associated with the development of anti-HBe antibodies and normalization of the serum alanine aminotransferase concentration. Furthermore, 27 (29.0%) of 93 HBeAg-negative carriers had unexpectedly preC wild-type sequences only. Direct sequencing of the HBV precore region of HBV specimens from 24 patients revealed no mutation at nt 1896, supporting the specificity of the RFLP analysis. These results suggest that RFLP analysis was accurate for the detection of the preC mutation and that the absence of serum HBeAg cannot be explained solely by the dominance of the preC mutant. © 1995 Wiley-Liss, Inc.

Antibodies against hepatitis B surface antigen and against anti-hepatitis B surface antigen idiotype were assayed after immunization with hepatitis B vaccine both in sera, by enzyme-linked immunosorbent assay (ELISA), and in peripheral blood mononuclear cells, by enzyme-linked immunospot (ELISPOT) assay. After vaccination of 19 subjects, antibody to the idiotype of anti-hepatitis B surface antigen was detected in none of the sera tested with ELISA, but anti-idiotypic antibody-secreting cells were detected by ELISPOT assay in 4 (36.4%) of the 11 vaccine recipients who were positive for anti-hepatitis B surface antigen with ELISPOT assay. On the other hand, these cells were detected in none of those who remained seronegative for anti-hepatitis B surface antigen, or in the 7 normal subjects or the 2 chronic hepatitis type C patients. These results suggest that anti-idiotypic antibody production is more sensitively detected by ELISPOT assay than by ELISA, and anti-idiotypic antibodies to anti-hepatitis B surface antigen may be present in those with anti-hepatitis B surface antigen.

Primary sclerosing cholangitis (PSC) frequently accompanies inflammatory bowel diseases. In an attempt to increase our understanding of the pathogenesis of PSC, we studied bile duct changes in rats with colitis which had been given N-formyl L-methionine L-leucine L-tyrosine (fMLT) rectally; fMLT is one of the chemotactic peptides produced by Escherichia coli, and is secreted into the bile by hepatocytes after it enters the portal blood. Transrectal administration of fMLT induced a marked inflammation in the portal triad and mild hepatocyte necrosis on the 4th day. The infiltrating leukocytes in the portal tract were mostly mononuclear cells, which densely infiltrated around the bile ducts. These mononuclear cells appeared to attach to bile duct epithelial cells, and they were more numerous in the smaller bile ducts. Electron microscopy revealed that lymphocytes were in direct contact with bile duct lining cells and that some epithelial cells had degenerated or collapsed. These results suggest that this E. coli-derived peptide may induce cholangitis in the small bile duct through cell-mediated mechanisms. Since these pathologic changes resemble those of the bile duct observed in the early stage of PSC, it can be concluded that bacterial chemotactic peptides may play a role in the pathogenesis of small-duct PSC.

SAITOH, Y., MIURA, M., NIITSUMA, H., NUMATA, N., OHORI, H., ISHII, M. and TOYOTA, T. Changes of Serum Hepatitis C Virus Levels in Patients with Chronic Hepatitis C Treated with Two Courses of Interferon Administration. Tohoku J. Exp. Med., 1994, 173 (4), 361-369-To assess the efficacy of repeated interferon (IFN) administration in patients with chronic hepatitis C unresponsive to initial therapy, serum hepatitis C virus (HCV)-RNA levels were measured in 12 patients who had failed prior IFN therapy. Serum HCV-RNA was assayed by measuring DNA complementary to HCV-RNA using a quantitative reverse transcription-polymerase chain reaction assay. The mean total dose of IFN was 227.8 megaunits for first treatment and 270.7 mega-units for second treatment. Five responders with a normal alanine aminotransferase (ALT) concentration (less than 40IU/liter) at the end of the first treatment also had a normal ALT concentration at the end of the second treatment. By contrast, all nonresponders with an elevated ALT concentration during the first treatment likewise had an elevated ALT concentration at the end of the second treatment. HCV-RNA levels before the first treatment varied from 106 to 108 copies/μl. The serum HCV-RNA levels fell in 9 out of 10 patients after the first treatment and in 11 out of 12 patients after the second treatment. One patient had unchanged normal serum ALT levels after two courses of IFN treatment. These results suggested that the outcome of a second course of IFN treatment was similar biochemically and virologically to a first course, and that patients who did not respond initially seldom respond to additional IFN therapy. Therefore, readministration of IFN should be restricted to patients who respond biochemically and virologically to initial treatment. The optimal second dose shall be determined with further studies.

SATO, S., YAMADA, M., MIYAZAKI, Y., MATSUDA, K., KANNO, A., ISHII, M., OHORI, H. and TOYOTA, T. Relationship between Molecular State of Serum HBV-DNA and Clinical Features of Hepatitis B Virus Carriers. Tohoku J. Exp. Med., 1993, 171(4), 309-317-Molecular species of serum hepatitis B virus (HBV)-DNA in HBV carriers were classified by Southern blot hybridization into three types; type I with two bands of 4.0kb and 3.2kb, type II with the two bands of type I plus the smear between 4.0kb and 3.2kb, and type III with a broad band below 4.0kb. A total of 51 HBV carriers were classified into three groups (group I, n=19 with type I; group II, n=12 with type II; and group III, n=20 with type III). Serum aminotransferase levels of group I were significantly lower than those of groups II and III. Liver pathology revealed that 18 of the 19 (94.7%) group I patients showed nonspecific reactive hepatitis (NSRH), while 11 of the 12 (91.7%) group II patients and 19 of the 20 (95.0%) group III patients showed chronic persistent hepatitis (CPH) or chronic active hepatitis (CAH). Immunohistochemical study showed that hepatitis B core antigen (HBcAg) was localized in the nucleus of hepatocytes in most of patients with type I while it was localized in both the nucleus and cytoplasm in those with types II and III. Since the smear between 4.0kb and 3.2kb is specifically found in groups II and III, HBV-DNA with this smear may be related to active hepatitis.

The molecular species of hepatitis B virus (HBV)-DNA of 44 sera taken from 25 HBV carriers were examined by Southern blot hybridization with a biotin-labeled HBV-DNA probe and classified into three patterns. Type I consisted of two distinct bands of 4.0 kb and 3.2 kb. Type II consisted of the 4.0 kb and 3.2 kb bands and a smear between these two bands. Type III showed a broad band below 4.0 kb. With endogenous HBV-DNA polymerase reaction, the smear disappeared and 4.0 kb and 3.2 kb bands appeared. After EcoRI digestion, the 4.0 kb band disappeared to form a single band of 3.2 kb. These results suggest that the 4.0 kb and 3.2 kb forms are a relaxed circular, fully double-stranded DNA and a linear, fully double-stranded DNA, respectively, and that the smear between 4.0 kb and 3.2 kb is formed from the sum of relaxed circular, partially double-stranded DNAs with various length of plus strands. Comparison between histological diagnosis and Southern blot hybridization patterns of 25 HBV carriers indicates that these three patterns are closely related to the degree of hepatitis.