Safety and immunogenicity of hepatitis B surface antigen-pulsed dendritic cells in patients with chronic hepatitis B.

The immune modulator capacity of antigen-pulsed dendritic cells (DC) has been documented in patients with cancers and in animal models of chronic viral infections. Cancer antigen-pulsed DC are now used for treating patients with cancer. But viral antigen-pulsed DC are not used in chronic viral-infected patients because safety of antigen-pulsed DC has not been evaluated in these patients. DC were isolated from human peripheral blood mononuclear cells by culturing with human-grade granulocyte-macrophage colony stimulating factor and interleukin-4. Human blood DC were cultured with hepatitis B surface antigen (HBsAg) for 8h to prepare HBsAg-pulsed DC. After immunogenicity assessment of HBsAg-pulsed DC in vitro, five million HBsAg-pulsed DC were administered intradermally to five patients with chronic hepatitis B (CHB) 1-3 times. HBsAg-pulsed DC were immunogenic in nature because they produced significantly higher levels of interleukin-12 and interferon-γ compared to unpulsed DC (P<0.05). Also, HBsAg-pulsed DC induced proliferation of HBsAg-specific T lymphocytes in vitro. CHB patients injected with HBsAg-pulsed DC did not exhibit generalized inflammation, exacerbation of liver damage, abnormal kidney function, or features of autoimmunity. Administration of HBsAg-pulsed DC induced anti-HBs in two patients and HBsAg-specific cellular immunity in 1 patient. This is the first study about preparation of antigen-pulsed DC using human consumable materials for treating patients with CHB. Because HBsAg-pulsed DC were safe for all patients with CHB and had immune modulation capacity in some patients, phase I and phase II clinical trials with antigen-pulsed DC in CHB and other chronic infections are warranted.

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.

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.

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.

Increased serum levels of macrophage migration inhibitory factor in alcoholic liver diseases and their expression in liver tissues.

OBJECTIVES: To study the role of macrophage migration inhibitory factor (MIF) in the pathogenesis of alcoholic liver diseases. DESIGN AND METHODS: The levels of MIF in the sera were estimated by an enzyme-linked immunosorbent assay in 13 patients with alcoholic hepatitis (ALH), 9 patients with alcoholic cirrhosis (ALC) and 26 normal controls. MIF was localized in the liver specimens by immunohistochemistry. RESULTS: The mean levels of MIF in the sera were significantly higher in ALH and ALC compared with the normal controls (p < 0.05). Serial observations revealed a relationship between serum MIF levels and the serum transaminase levels. MIF was expressed by the hepatocytes and by the infiltrating cells around the site of accumulation of neutrophils and ballooned hepatocytes in ALH. CONCLUSIONS: This is the first report on MIF in human alcoholic liver diseases, and the data suggest that MIF may be related to abnormal cytokine homeostasis in ALH.

Soluble intercellular adhesion molecule-1 in serum in chronic hepatitis B and C.

Intercellular adhesion molecule-1 (ICAM-1) is expressed on the hepatocyte membrane in patients with chronic hepatitis B and C. We assayed levels of the soluble form of this molecule (sICAM-1) in serum by an enzyme-linked immunosorbent assay method; we then analyzed the results in relation to hepatitis activity. Fifty-one patients with chronic hepatitis (22 with type B and 29 with type C) and 10 normal controls were examined. The serum levels of sICAM-1 in hepatitis B and C were significantly higher (P < 0.01) than in normal controls. The serum level of sICAM-1 was correlated with serum glutamic-pyruvic transaminase level (P < 0.01), and the level of sICAM-1 in patients in whom exacerbation was seen was significantly higher (P < 0.05) than that in patients in a remission. There was no relationship between the serum level of sICAM-1 and the degree of ICAM-1 expression on the hepatocyte membrane. These results suggest that the serum level of sICAM-1 reflects the degree of activity of chronic hepatitis B and C.

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.

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.

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.

Proatlantal intersegmental artery with absence of bilateral vertebral arteries. Case report.

A case of proatlantal intersegmental artery with absence of bilateral vertebral arteries is presented. This rare anomaly was accidentally encountered on angiography in a patient with a ruptured aneurysm of the anterior communicating artery.

The CD81 expression in liver in hepatocellular carcinoma.

The expression of CD81, a cell receptor for hepatitis C virus, was examined on cancer cells in hepatocellular carcinoma (HCC) C (n=29) to clarify its clinical role. CD81 was expressed on hepatocyte membrane in non-tumor portions in all patients, however, this was not stained on the cancer cell membranes in 6 of 29. Reverse transcriptase-PCR revealed that CD81 gene expression was not detected in the tumorous portions in 3 of 7 samples. The loss of CD81 was prominent in poorly differentiated HCC (5 of 8) and extrahepatic metastasis patients (6 of 8). The loss of CD81 is associated with differentiation and metastasis of HCC.

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.

Identification of intrahepatic lymphocyte subpopulations in patients with fulminant hepatitis by the immunoenzymatic technique, using monoclonal antibodies.

Intrahepatic lymphocyte subpopulations were studied in 11 patients with fulminant hepatitis (FH) (of whom 9 cases died) by the immunoenzymatic technique, using monoclonal T cell specific antibodies and other reagents. In peripheral blood, the OKT4 positive cells: OKT8 positive cells ratio in FH was higher than that in normal controls. In liver tissue, OKT8 positive cells were the predominant type of T cells, and these cells were in broad contact with the surface of the hepatocyte. Leu7 positive, OKT4/4a positive and Leu 15 positive cells were scarce and did not contact the hepatocyte. These results suggest that cytotoxic T cells may play an important role in hepatocyte necrosis in patients with FH.

Serum levels of soluble CD4 and CD8 in patients with chronic viral hepatitis.

The blood levels of soluble CD8 (sCD8) and soluble CD4 (sCD4) were measured in patients with various liver diseases, and their significance was studied. The levels of sCD8 were significantly higher in patients with chronic active hepatitis (CAH), autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), acute hepatitis (AH), fulminant hepatitis (FH) and liver cirrhosis (LC) as compared with the normal controls (NC), and correlated positively with those of GPT (r = 0.67, p < 0.001). In addition, a comparison of the exacerbation of CAH with remission showed that the sCD8 levels were significantly higher in the former. On the other hand, there was no significant rise in the level of sCD4 in patients with any liver disease, except FH, no definite relationship between sCD4 and sGPT, and no consistant tendency in sCD4 levels between exacerbation and remission. The reason for an insignificant elevation of the sCD4 level is the fact that in hepatitis the CD8-positive cells, which are cytotoxic T cells, are directly involved in hepatocyte damage; therefore the CD8-positive cells are predominantly activated, while the activity of the CD4-positive cells is considered to be lower. Instead of determining the number of CD4-positive cells and CD8-positive cells in the mononuclear cells of peripheral blood, serum sCD4 and sCD8 levels can be measured simply and inexpensively. Thus, these levels may be useful immune markers.

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.

Intrahepatic lymphocyte subpopulations and HLA class I antigen expression by hepatocytes in chronic hepatitis C.

The lymphocyte subpopulations in the peripheral blood and liver were studied in 17 patients with chronic active hepatitis type C (CAH C) by immunoenzymatic and immunofluorescence techniques, using mono-specific T cell antibodies and other reagents. The peripheral blood subsets showed no significant differences between the patients with CAH C and normal controls. In the liver, CD8-positive cells were predominant over CD4- and Leu 7-positive cells. Leu 7-, CD4-, and CD11-positive lymphocytes were all few in number. HLA class I antigen was expressed diffusely on the cell surfaces of the hepatocytes. Serum levels of soluble interleukin 2 receptor (sIL2R) in the CAH C patients were significantly higher than in normal controls (p less than 0.01). In CAH C, sIL2R levels were higher during exacerbations than during remissions (p less than 0.01). These results suggest that cytotoxic T cells (CD8- positive, CD4-negative, and CD11-negative) may play an important role in the pathogenesis of hepatocyte injury in patients with CAH C.

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.