Differences in background characteristics of patients with chronic hepatitis C who achieved sustained virologic response with interferon-free versus interferon-based therapy and the risk of developing hepatocellular carcinoma after eradication of hepatitis C virus in Japan.

We compared the background characteristics of patients with chronic hepatitis C who achieved eradication of hepatitis C virus (HCV), that is sustained virologic response (SVR), with interferon (IFN)-based versus IFN-free antiviral therapy in Japan. In addition, we used a previously reported risk assessment model to compare the incidence of hepatocellular carcinoma (HCC) after SVR by treatment type. Pretreatment characteristics of 1533 patients who achieved SVR with IFN-based therapy and 1086 patients with IFN-free therapy from five institutions across Japan were compared. The risk of HCC after SVR was assessed based on pretreatment characteristics, and the incidence of HCC after SVR was estimated in both groups. Age and serum alpha-fetoprotein levels were higher, platelet count was lower, and liver fibrosis was more advanced in patients who achieved SVR with IFN-free therapy compared with IFN-based therapy. The incidence of HCC after SVR in the IFN-free group was estimated to be more than twofold higher than in the IFN-based therapy group (7.29% vs. 3.09%, and 6.23% vs. 3.01% when excluding patients who have underwent curative treatment for HCC). There are large differences in pretreatment characteristics between patients who achieved SVR with IFN-based and IFN-free therapies in Japan, which are associated with differential risk of HCC after SVR. These differences can influence the incidence of HCC after SVR and should be taken into consideration when comparing IFN-based and IFN-free therapies in terms of hepatocarcinogenesis suppression with HCV eradication.

Mechanism of restoration of immune responses of patients with chronic hepatitis B during lamivudine therapy: increased antigen processing and presentation by dendritic cells.

Restoration of host immunity has been reported in patients with chronic hepatitis B (CHB) after treatment with lamivudine; however, the underlying mechanisms of this treatment have not been determined. This study examined the role of antigen-presenting dendritic cells (DC) in restoration of host immunity. Circulating DC were isolated from peripheral blood of 23 patients with CHB before and 1, 3, and 12 months after starting lamivudine therapy. The non-antigen-specific proliferation of DC was assessed in allogenic mixed leucocyte reaction. Dendritic cells were cultured with hepatitis B surface antigen (HBsAg) to prepare HBsAg-pulsed DC. Proliferative capacity and production of interleukin (IL)-12 and interferon (IFN)-γ of HBsAg-pulsed DC were evaluated. Circulating unpulsed DC and HBsAg-pulsed DC showed significantly higher levels of T-cell proliferation capacities 1 month after lamivudine therapy compared to proliferation levels before therapy (P<0.05). HBsAg-pulsed DC also produced significantly higher levels of IL-12 and IFN-γ with lamivudine therapy compared to levels before therapy (P<0.05). HBsAg-pulsed DC from lamivudine-treated patients induced proliferation of T cells of patients with CHB in an antigen-specific manner (P<0.05). However, T-cell stimulatory capacity of DC did not increase significantly 3 and 12 months after lamivudine therapy compared to 1 month after lamivudine therapy. Immune restoration as a result of lamivudine therapy is regulated at least in part by activation of DC. However, progressive activation of DC was not seen as treatment duration progressed, indicating the limitations of this mechanism of viral clearance.

Absence of CD83-positive mature and activated dendritic cells at cancer nodules from patients with hepatocellular carcinoma: relevance to hepatocarcinogenesis.

Mature and activated dendritic cells (CD83-positive DCs) are essential for the recruitment and survival of activated tumor-specific lymphocytes during carcinogenesis. The frequencies of CD83 positive DCs were almost same in peripheral blood from patients with hepatocellular carcinoma (HCC) and cirrhosis of liver (LC). However, the numbers of CD83 positive DCs in liver tissues were significant lower in HCC compared with LC (6.6+/-10.9 vs. 33.3+/-24 DCs/specimen, P<0.05). Most importantly, there were no CD83-positive DCs at cancer nodules in HCC. A role of infiltration of activated DCs in liver during hepatocarcinogenesis is shown.

Macrophage migration inhibitory factor in hepatocellular carcinoma and liver cirrhosis; relevance to pathogenesis.

The levels of macrophage migration inhibitory factor (MIF), a proinflammatory and carcinogenic cytokine, were significantly higher in the sera from patients with hepatocellular carcinoma (HCC; 25.6+/-15.3 ng/ml, n=55) and liver cirrhosis (LC; 18.9+/-10.7 ng/ml, n=26) compared with sera from patients with gastrointestinal cancer (6.8+/-7.5 ng/ml, n=29) and normal controls (5.6+/-1.2 ng/ml, n=45; P<0.01). Hepatocytes from patients with LC and HCC, but not from chronic hepatitis, expressed very high levels of MIF. A possible association between overexpression of MIF and hepatocarcinogenesis is suggested.

Changes in gastrointestinal lymph and blood vessels in patients with cirrhotic portal hypertension.

BACKGROUND: The aim of this study was to characterize the lymph vessels in different parts of the gastrointestinal tract and also to evaluate morphometric changes in these vessels during cirrhotic portal hypertension. METHODS: Sixteen patients with cirrhotic portal hypertension and 18 control subjects without portal hypertension were enrolled in the study. Tissue specimens were collected at autopsy or surgery, and were stained enzyme histochemically, using 5'-nucleotidase and alkaline phosphatase to distinguish lymph vessels and blood vessels, respectively. The numbers of vessels and their luminal areas were estimated using computer graphics software (National Institutes of Health [NIH] image program). RESULTS: The numbers and luminal areas of the lymph vessels varied considerably among the different organs of the gastrointestinal tract, both in controls and in the patients with cirrhotic portal hypertension. There was no significant difference in the numbers of lymph vessels between controls and patients with cirrhotic portal hypertension. However, the luminal area of the lymph vessels in the esophagus and stomach was significantly greater in the patients with cirrhotic portal hypertension than in the controls. These differences in lymph vessels were not seen in the small intestine and colon. CONCLUSIONS: These data indicate that dilatation of lymph vessels may be related to the absorption of excess interstitial fluid, resulting from congestion, in cirrhotic portal hypertension.

Three patients with autoimmune cholangiopathy treated with prednisolone.

We describe three patients with autoimmune cholangiopathy, i.e., anti-mitochondrial antibody-negative and anti-nuclear antibody-positive primary biliary cirrhosis, who were treated with prednisolone. Serum anti-mitochondrial antibody and anti-pyruvate dehydrogenase-E2 component antibody were determined by immunofluorescence of frozen sections and enzyme-linked immunosorbent assay, respectively. Immunoblotting using mitochondria prepared from rat liver was performed to analyze anti-mitochondrial antibody in detail. Serum from one patient reacted with a 48-kilodalton protein, but sera from the other two patients failed to react with the mitochondrial proteins. There was a marked improvement in liver function test results after prednisolone treatment. Before treatment, liver biopsy in all three patients showed histological features of primary biliary cirrhosis with hepatocellular necrosis. Repeat biopsy during treatment showed marked amelioration of hepatocellular damage in all three patients, although bile duct involvement persisted in two patients. These findings suggest that prednisolone is an effective treatment for hepatocellular damage in patients with autoimmune cholangiopathy, but has little impact on the bile duct involvement.

Immunohistochemical localization of metallothionein in hepatocellular carcinoma: preferential expression in non-cancerous cirrhotic nodules.

Metallothionein (MT), an oncofocal gene product was strongly expressed in 35%-95% of hepatocytes in hepatocellular carcinoma (HCC) and MT-positive hepatocytes were localized mainly in the non-cancerous cirrhotic nodules but not in malignant hepatocytes. On the other hand, <10% hepatocytes showed weak staining for MT in chronic hepatitis and cirrhosis of liver. Strong expressions of MT in non-cancerous cirrhotic nodule in HCC and low expressions in liver cirrhosis without HCC indicate a relationship between malignant transformation of hepatocytes and the expression of MT.

The effectiveness of interferon therapy on occurrence of hepatocellular carcinoma in chronic hepatitis C.

To clarify the effect of interferon (IFN) therapy for chronic hepatitis C (CHC) on the occurrence of hepatocellular carcinoma (HCC), 149 patients who were observed over a period of five years (mean: 7.6 years) were studied. The C-1 group, 33 patients with complete response to IFN; the C-2 group, 55 patients with no response to IFN; and the C-3 group, 61 patients who did not receive IFN. The occurrence rate of HCC was 0.9%/year/person. In the C-1, C-2 and C-3 groups, the rates were 0%, 0.3%, and 1.6%, respectively. The rate in C-1 + C-2 groups was significantly lower than that of the C-3 group (P<0.05). These data suggest IFN may suppress the occurrence of HCC in CHC.

Antigen-presenting cells at the liver tissue in patients with chronic viral liver diseases: CD83-positive mature dendritic cells at the vicinity of focal and confluent necrosis.

Although defective functions of peripheral blood or splenic antigen-presenting cells (APCs) are implicated in the pathogenesis of persistent infection in murine and human hepatitis B virus (HBV) and hepatitis C virus (HCV)-carriers, little is known regarding liver-infiltrating APCs in patients with chronic liver diseases. Using immunohistochemical methodology and antigen retrieval technique, we have detected APCs such as HLA DR-positive cells, interdigitating cells (IDCs) and CD83-positive mature and activated dendritic cells (DCs) at the liver specimens from 39 patients with chronic hepatitis (CH) and 10 patients with liver cirrhosis (LC). All 3 types of APCs were detected at the portal areas in both CH and LC, the most abundant being the HLA DR-positive APCs. CD83-positive, mature and activated DCs were detected in patients with CH around the areas of focal and confluent necrosis at the liver parenchyma in close association with T cells. The localization of CD83-positive mature and activated DCs at the liver tissues from patients with CH warrants further study about the role of these DCs in the induction of hepatocellular damage. This may also help to design DC-based therapy for patients with chronic liver diseases.

TT virus infection among renal transplant recipients.

To clarify the prevalence of TT virus (TTV) infection in renal transplant recipients and to estimate the role of TTV in patients with post-transplant liver function abnormalities, TTV-DNA of 47 renal transplant recipients was screened by polymerase chain reaction (PCR) according to a method described by Okamoto et al. before and after the renal transplantation. One of them was positive for hepatitis B surface antigen (HBsAg), one was positive for anti-hepatitis C virus (HCV) and other 45 were negative for both HBsAg and anti-HCV. TTV-DNA was detected in 22 of 47 patients before renal transplantation, and nine became positive after transplantation. All 47 patients showed a normal level of ALT before transplantation. Three of nine (33%) who became positive for TTV-DNA after transplantation and three of 16 (19%) who were negative for TTV-DNA before and after transplantation showed transient elevation of ALT. These results indicate that TTV was highly prevalent among renal transplant recipients, but a clear association between TTV and post-transplant liver function abnormality was not found.

Immunohistochemical localization of antigen presenting cells in liver from patients with primary biliary cirrhosis; highly restricted distribution of CD83-positive activated dendritic cells.

In order to have insights into the abnormal immune regulation in primary biliary cirrhosis (PBC), different types of antigen presenting cells (APC) were localized immunohistochemically in liver specimens from 26 patients with PBC and compared with the distributions of APC from 11 and 10 patients with chronic hepatitis C (CH-C) and large bile duct obstruction, respectively. In all diagnostic conditions, 30-90% of the infiltrating cells were positive for HLA DR. In PBC, the numbers of interdigitating cells (IDC) were significantly higher than the numbers of CD83-positive dendritic cells (DC) (34.0 +/- 38.8 vs. 5.5 +/- 7.1/specimen, mean +/- SD, p < 0.05). On the other hand, the numbers of IDC (14.2 +/- 20.0/specimen) and CD83-positive DC (7.9 +/- 8.7/specimen) were almost similar in CH-C (p > 0.05). Positive stainings for IDC and CD83-positive DC were rarely seen in large bile duct obstruction. This is the first report on the existence of activated CD83-positive DC in PBC. The significantly increased numbers of IDC and the highly restricted distributions of CD83-positive DC in PBC indicate that activated DC may play a role in the abnormal immune pathogenesis of PBC.

Anti-hepatitis C virus immunoglobulin M antibody in patients with chronic hepatitis type C.

Anti-hepatitis C virus immunoglobulin M antibody titers were measured by enzyme-linked immunosorbent assay in 28 patients with chronic hepatitis C. Nine patients were treated with alpha-interferon at a dosage of 3 MIU/day for 4 weeks. Anti-HCV IgM was positive in 12 of the 28 patients, and these 12 patients also had significantly higher anti-HCV antibody titers than those patients who were negative for anti-HCV IgM. Among the 9 patients treated with alpha-interferon, anti-HCV IgM positivity was significantly lower in 4 good responders than in 2 non-responders and 3 transient responders at 12 months after the completion of treatment (p < 0.01). These results suggest that anti-HCV IgM titers might be useful for monitoring the antiviral effect of interferon treatment in CH C.

An autopsy case of primary biliary cirrhosis with severe interstitial pneumonia.

A 74-year-old woman was admitted to our hospital because of interstitial pneumonia. She had a 14-year history of primary biliary cirrhosis (PBC) diagnosed histologically, with a positive test for anti-mitochondrial antibodies and elevated biliary enzyme activity. She also had a 7-year history of rheumatoid arthritis and a 26-year history of Sjögren's syndrome. Though the symptoms of these complications improved, the interstitial pneumonia deteriorated very quickly and the patient died of respiratory failure due to acute exacerbation of interstitial pneumonia when the activity of PBC decreased. We report this case because it is relatively rare for PBC to be complicated by severe interstitial pneumonia, and it may offer insight into the etiology of these diseases.

Serum IL-8 levels and localization of IL-8 in liver from patients with chronic viral hepatitis.

BACKGROUND/AIMS: Interleukin 8 is known as a chemotactic factor for neutrophils and T cells. Such inflammatory cells are observed in the liver tissue in chronic viral hepatitis. However, it is not known whether interleukin 8 relates to hepatic injury in patients with chronic viral hepatitis. Therefore, we determined serum interleukin 8 levels and identified the cells related to interleukin 8 production in liver tissue. METHODOLOGY: Studies were performed on 29 patients with chronic viral hepatitis and 20 normal controls. Serum interleukin 8 levels were assayed using a sandwich ELISA. Immunohistochemical examination was performed to identify the cells related to interleukin 8 production by using a rabbit polyvalent antibody to human interleukin 8. RESULTS: Serum interleukin 8 levels were found to be increased significantly (p<0.05) in patients with chronic viral hepatitis compared with normal controls. They were also increased significantly in patients with chronic active hepatitis compared with chronic persistent hepatitis (p<0.05), and during exacerbation stages compared with remission stages (p<0.05). Out of the 10 patients examined immunohistochemically, interleukin 8 positive cells in the liver tissue were visualized in 7 patients and observed mainly along sinusoids. There was significant correlation between serum interleukin 8 levels and intensity of staining for interleukin 8 in the liver tissue (p<0.05, r=0.869). CONCLUSIONS: Interleukin 8 plays a role in hepatic injury in patients with chronic viral hepatitis, and is mainly produced by nonparenchymal cells in the liver.

The seroepidemiology of hepatitis A and B in a Japanese town.

Sera collected from 1,118 healthy children and adults aged between four years and 90 years during the period 1989 to 1990, were tested for serological markers of hepatitis A virus (HAV) [antibody to HAV (anti-HAV)] and hepatitis B virus (HBV) [hepatitis B surface antigen (HBsAg) and antibody to hepatitis B surface antigen (anti-HBsAb)]. The overall prevalence rates of anti-HAV, HBsAg, and anti-HBV were 20.2%, 0.36%, and 5.1%, respectively. No body was found to be positive for anti-HAV below 30 years of age but more than 70% of the adults aged 50 years or over were positive for anti-HAV. The level of exposure of HAV infection is declining in Japan and paradoxically at the same time a vast majority of people are becoming susceptible to more severe illness. The fall in prevalence of HBsAg possibly represents the positive impact of ongoing vaccination programs and other preventive measures against HBV.

[An epidemiological study of hepatitis C virus infection in a local district in Japan].

Mass screening of anti-HCV in Iyo city, Japan was performed. Sera from 136 subjects were tested for anti-HCV by ELISA (Ortho Diagnostic Inc.). Anti-HCV was positive in 13 subjects (9.6%). The familial study for anti-HCV revealed 2 persons positive for anti-HCV in 2 families each, while only 1 person was positive in each of the 8 other families. Two females were positive for both anti-HCV and HBsAg, and all of their 3 children were negative for anti-HCV and positive for HBsAg. Positive rate of anti-HCV was higher in subjects who had a history of blood transfusion or "Chinese acupuncture" than that in those who did not. These results indicate that not only blood transfusion but also "Chinese acupuncture" and intrafamilial transmission are possible routes of HCV infection. The rate of vertical transmission of HCV is definitely lower than that of HBV.

[A staining method of PIVKA-II by an indirect immunoenzyme technic].

The localization of PIVKA-II in liver tissue was studied by the immuno-staining method using monoclonal antibody. The cell lines derived from hepatoblastoma (huH-6), hepatocellular carcinoma (PLC/PFR/5), and normal liver (Chang) were used as materials for staining. We succeeded to stain PIVKA-II, when the materials were fixed in a formalin solution and frozen. In patients with hepatocellular carcinoma, PIVKA-II was stained in the cytoplasm of both hepatoma and non-hepatoma cells.

[Detection of Ki-67 in liver biopsy specimens using monoclonal antibody to Mib-1].

Ki-67 antigen was visualized in formalin-fixed, paraffin-embedded liver biopsy specimens using monoclonal antibody to Mib-1 to identify the proliferating hepatocytes. Thirty liver specimens obtained from 10 patients with chronic hepatitis (CH) or liver cirrhosis (LC) and 10 patients with hepatocellular carcinoma were studied. Liver specimens were treated with a pepsin solution or heated with autoclave or treated with microwave as a part of antigen retrieval system; then stained with an immunoperoxidase method using a monoclonal antibody to Ki-67 (Mib-1). Stable stainings were obtained in the sections treated with autoclave. Ki-67 was detected in the nuclei of hepatocytes, bile duct epithelium, fibroblast and infiltrating mononuclear cells. In patients with CH and LC, the numbers of hepatocytes positive for Ki-67 has a good co-relation with serum GPT level (p < 0.01), while has no relationship with the degree of fibrosis. The number of hepatocytes positive for Ki-67 has a good co-relation with the degree of the differentiation of hepatocellular carcinoma. Detection of proliferating hepatocytes using Mib-1 is useful to understand the degree of proliferation.

[A case of hemophagocytic syndrome with severe liver injury manifestating adult Still's disease].

In April 1988, a 23 year-old woman developed high fever, arthralgia, eruptions and splenomegaly. She was treated with non Steroid anti-inflammatory drugs, and the symptoms disappeared. In June 1991, she was diagnosed as adult Still's disease and treated with prednisolone. In July 1994, she was treated with pulse therapy methylprednisolone due to high fever, eruptions, arthralgia and the high levels of ferritin. However, due to the marked increase of serum transaminase and bilirubin levels, she was referred to University hospital. She developed hepatic failure after admission Bone-marrow puncture revealed hemophagocytosis. She died ten days after admission. She was diagnosed as hemophagocytic syndrome combined with acute hepatic failure.

[A cell kinetic study of liver cells in various liver diseases by the detection of DNA polymerase alpha].

DNA polymerase alpha (DNA-P alpha) in the nuclei of hepatocytes was visualized by the immunoperoxidase method to study the number of liver cells which were at the stage of G1, S, and G2 stage in the cell cycle. Seven liver specimens from patients with acute hepatitis (AH), 17 with chronic persistent hepatitis (CPH), 32 with chronic active hepatitis (CAH), 6 with liver cirrhosis (LC), 4 with hepatocellular carcinoma (HCC) and 4 with hospital controls were studied. The number of DNA-P alpha-positive hepatocytes in 1000 hepatocytes were as follows: 19.1 +/- 18.0 in AH, 8.8 +/- 6.1 in CPH, 27.3 +/- 23.8 in CAH, 21.8 +/- 14.3 in LC, 545.3 +/- 184.0 in HCC and 1.1 +/- 1.1 in hospital controls. The number of DNA-P alpha-positive hepatocytes in HCC were significantly increased compared with other liver diseases. Likewise, those in CAH and LC were higher than those in CPH and hospital controls. The liver cell necrosis was thought to be one of the secondary stimulators for cell division of hepatocytes.