Biochemical response to interferon therapy correlates with interferon sensitivity-determining region in hepatitis C virus genotype 1b infection.

Biochemical responders maintain normal alanine aminotransferase levels after interferon (IFN) therapy despite persistent presence of hepatitis C virus (HCV) RNA in their sera. There have been few reports on predictive factors for biochemical response. A region associated with sensitivity to IFN was identified in the nonstructural protein 5 A of genotype 1b [aa 2209-2248; IFN sensitivity-determining region (ISDR)]. The substitutions in ISDR correlate with sustained response to IFN. In this report, we assessed the association of ISDR with biochemical response. The sequences of ISDR were determined in 62 patients with HCV genotype 1b treated by IFN in two randomized controlled trials. 30 patients had wild ISDR (identical to HCV-J), 20 intermediate ISDR (1-3 amino acid substitutions compared with HCV-J), and 12 mutant ISDR (four or more amino acid substitutions). All 12 patients with mutant ISDR had a sustained response, while only one of those with wild or intermediate ISDR had a sustained response (P < 0.0001). In the 49 patients other than sustained responders, the patients with intermediate ISDR obtained biochemical response significantly more frequently (52.6%, 10/19) than those with wild-type ISDR (20.0%, 6/30) (P < 0.05). Multivariate analysis indicated the number of substitutions in ISDR as the most important predictor for biochemical response (discriminant coefficient=1.08, P < 0.05) and sustained response (discriminant coefficient=6.13, P < 0.0001). In phylogenetic analysis, clustering of sustained responders and biochemical responders was observed. These results demonstrate that the substitutions in ISDR are the most important predictor for biochemical response to IFN in patients infected with genotype 1b as well as for sustained response.

Randomized controlled trial of twice-a-day administration of natural interferon beta for chronic hepatitis C.

We conducted a randomized controlled trial to assess the efficacy of twice-a-day administration of natural interferon beta (IFNbeta) as an induction of IFN therapy for chronic hepatitis C. Seventy-one patients with chronic hepatitis C were enrolled into the trial and randomly assigned into three treatment groups. Six million units (MU) of IFNbeta were administered once-a-day for the first 4 weeks, and then thrice weekly for 12 weeks in 20 patients (once-a-day group). Three milion units of IFNbeta were administered twice-a-day for the first 2 weeks, 6 MU once-a-day for the next 2 weeks, and then thrice weekly for 12 weeks in 23 patients (twice-a-day+beta group), or 6 MU of lymphoblastoid IFNalpha were administered thrice weekly for the last 12 weeks instead of IFNbeta in 28 patients (twice-a-day+alpha group). Four patients in once-a-day group (20%), 9 in twice-a-day+beta group (39%), and 12 in twice-a-day+alpha group (43%) obtained sustained response. Sustained response rate in twice-a-day groups was higher than in once-a-day group, although there was no statistical significance. The present study suggested the possible superiority of twice-a-day administration of IFNbeta as an induction therapy to once-a-day administration, but further studies are needed to confirm this regimen.

Randomized controlled trial of lymphoblastoid interferon alpha for chronic hepatitis C (comparison of 9-MU and 6-MU doses). IFN Treatment Group of Affiliated Hospitals of the Third Department of Internal Medicine at Nagoya University School of Medicine.

OBJECTIVE: We conducted a randomized controlled trial to compare the efficacy of two different dosages of lymphoblastoid interferon alpha (IFN) for the treatment of chronic hepatitis C. METHODS: Eighty-four patients with chronic hepatitis C were enrolled and randomly assigned into the two groups; group A was treated with 6 million units (MU) and group B with 9 MU daily for the first 2 wk, and then thrice weekly for an additional 14 or 22 wk. RESULTS: Eighty patients were evaluated (39 patients in group A and 41 in group B); 14 patients in group A (35.9%) and 15 in group B (36.6%) obtained sustained response. The percentages of patients who became negative for HCV RNA at the end of the second wk differed slightly between the groups, without statistical significance (56.4% and 68.3%). When assessed in detail, patients with genotype 1 and < 1 Meq/ml of viral load became negative for HCV RNA significantly more frequently in group B (eight of eight) than in group A (three of seven) (p < 0.05) at the end of the second week, whereas the sustained response rate was similar between the groups (five of eight and four of seven). Predictors of sustained response by multivariate analysis were low viral load (< 1.0 Meq/ml) and negativity of HCV RNA at the end of the second wk of IFN. CONCLUSIONS: The results indicated that there was no difference in sustained response rate between the 6-MU and 9-MU doses. The earlier disappearance of HCV RNA, at the end of the second wk or at least by the end of the fourth week, is an essential condition for sustained response.

Biochemical improvement of chronic hepatitis C after gastrointestinal bleeding.

Although chronic hepatitis C is frequently complicated by iron overload, it remains unclear whether iron cytotoxicity is involved in the disease process. Five patients with chronic hepatitis C showed rapid reduction of serum aminotransferase activity after gastrointestinal bleeding. Posthemorrhagic reduction of liver enzyme levels lasted for more than one week. Anemia was associated with a reduction of serum ferritin concentration. Considering the short half-lives of circulating liver enzymes, reduced release of enzymes, that is inactivation of cell lysis, is the likely cause of the improved biochemical indices. Reactive iron, which is cytotoxic for patients infected by HCV, may be rapidly incorporated into hemoglobin when erythropoiesis is stimulated. Our observation also suggests that intensive iron removal by phlebotomy is a safe, economic treatment for patients with chronic hepatitis C.

Hepatic copper accumulation in patients with primary biliary cirrhosis.

Liver biopsy specimens from 18 patients with primary biliary cirrhosis were examined histochemically and by energy-dispersive x-ray microanalysis. Using two indices, we classified hepatic copper accumulation into three stages based on the Cu x-ray intensity of cuproproteins that had accumulated in hepatocyte lysosomes and on the binding ratio of postulated copper transfer proteins between the cytosol and lysosomes. Eight patients were in stage 1 with an initial accumulation of lysosomal cuproproteins, mediated by transfer proteins not saturated with copper. Two patients were in stage 2, in which transfer proteins were saturated with copper. The first two stages gave negative results for histochemical copper. The remaining eight patients were in stage 3, in which copper accumulation detected by histochemical included transfer proteins saturated with copper and large amounts of lysosomal cuproproteins. Five patients (one each in stages 1 and 2, and three in stage 3) underwent a second liver biopsy after treatment with 600 mg of ursodeoxycholic acid daily for 14 to 39 months. Results of blood chemistry tests improved, but liver histologic findings and copper accumulation were unchanged in all five patients. It seems likely that ursodeoxycholic acid does not affect the copper accumulation in hepatocyte lysosomes that reflects the state of cholestasis in patients with primary biliary cirrhosis.

Increased peripheral blood Ia positive T cells and their effect on autologous mixed lymphocyte reaction in chronic active liver disease.

We measured Ia antigen bearing peripheral blood T cells, as an index of immunological stimulation, of patients with chronic active liver diseases (CALD) by the rosette assay method. We also examined the role of Ia antigen which represents the products of the genes of the major histocompatibility complex on the autologous mixed lymphocyte reaction (AMLR) since this reaction may reflect self regulation of immune responses. The percentages of Ia positive T cells of 29 patients with CALD (17.1 +/- 4.3%, P less than 0.001) and of 12 patients with other liver diseases (12.9 +/- 2.4%, P less than 0.05) were increased when compared with that of normal individuals (10.7 +/- 2.0%). However, levels of Ia positive T cells activated by phytohaemagglutinin-P in patients with CALD and other liver diseases did not differ from normal subjects. Ia positive cells in OKT8 positive cells were markedly elevated (P less than 0.001), whereas those in OKT4 positive cells were decreased (P less than 0.01) in CALD. The impaired values for the AMLR correlated inversely (P less than 0.01) with the increased percentages of Ia positive T cells in patients with CALD. Further analysis showed that there was no suppression of the proliferation of Ia and OKT4 positive cells by Ia and OKT8 positive cells although the culture of increasing numbers of Ia and OKT8 positive cells and decreasing numbers of Ia and OKT4 positive cells gave a lesser AMLR value. These data suggest that the increase in Ia positive T cells and the alteration of Ia positive cells in the T cell subsets reflect an activation of immune system and provide further evidence in favour of an abnormality of the immunoregulatory system in CALD.

The specificity of human liver membrane lipoprotein: studies with monoclonal antibodies.

Hybrid cell lines which secreted antibodies to liver-specific membrane lipoprotein (LSP) were obtained by immunizing SMA and BALB/c mice with human LSP and fusing their splenocytes with the myeloma cell line P3-NSI/1-Ag4-1. The secretion of antibody to LSP (anti-LSP) was monitored by binding to a human hepatocellular carcinoma cell line, SK-Hep-1, which possesses surface membrane LSP, and to 125I-antimouse F(ab')2 antibody in radiobinding assay, and by reacting with 125I-LSP in double-antibody radioimmunoassay. From four separate cell fusions, seven secreting hybrids were cloned by dilutional techniques. Of these, four cell lines produced antibodies reacting with a wide variety of cells. The culture supernatants of the remaining three (6D6, 6G3 and 8F10) demonstrated the strongest binding activities against SK-Hep-1 among the various kinds of cell lines tested. However, binding with other cell lines, including renal cancer cells (SK-RC-6) and myeloid cell (HL-60) also occurred. Absorption test of ascitic fluids derived from 6D6 showed that ascitic fluids lost their capacity to bind to target SK-Hep-1 cells when absorbed with SK-Hep-1. Similarly absorption by SK-RC-6 and HL-60 removed almost all of the binding activity of ascitic fluids. Moreover, the binding activities of the ascitic fluids to SK-RC-6 and HL-60 were eliminated when absorbed with SK-RC-6, HL-60 and SK-Hep-1. The present study indicates that our LSP preparation contains nonspecific organ antigens, and although LSP exists on liver cell membrane, it is also found on the cell membrane of other organs albeit in less quantity.

B lymphocyte function stimulated by staphylococcus aureus Cowan I in chronic liver disease.

Peripheral blood B cell response to staphylococcus aureus (SpA) Cowan I was evaluated in 8 healthy subjects, 8 patients with liver cirrhosis (LC), and 2 patients with chronic active hepatitis (CAH) with hypergammaglobulinemia (greater than 2 g/dl). In control studies it was shown that stimulation by SpA Cowan I was much less T cell-dependent than that induced by pokeweed mitogen (PWM) or con-canavalin A (ConA) when the amounts of immunoglobulins (Ig) secreted into culture supernatants were measured by radioimmunoassay (RIA) and the blastogenic response was measured by incorporation of tritiated thymidine. B cells from only 2 patients revealed increased Ig synthesis by SpA Cowan I stimulation. In the study of blastogenic response, increased DNA synthesis of B cells by SpA Cowan I stimulation was observed in 2 patients and decreased DNA synthesis in 3 patients. The remaining patients demonstrated normal range response. There was no correlation between B cell response to SpA Cowan I and clinical data such as gammaglobulin level in the patients studied. These studies indicate that B cell function remains intact in many patients with chronic liver disease with hypergammaglobulinemia.

Activation of suppressor function of human peripheral blood T cells by (+)cyanidanol-3: its application to chronic active liver diseases.

(+)Cyanidanol-3, a substance belonging to the flavonoids group, has been used in acute viral hepatitis (AVH) and chronic active liver disease (CALD). Studies were undertaken to determine if (+)cyanidanol-3 could affect a function of mitogen stimulated peripheral blood lymphocytes (PBL) in humans. First, (+)cyanidanol-3 was added directly to pokeweed mitogen (PWM) stimulated PBL or co-culture of B and T cells resulting in severe suppression of immunoglobulin (Ig) production. This suppression was mediated by radiosensitive T cells. Secondly, when normal T cells pre-incubated with 25 micrograms/ml of (+)cyanidanol-3 for 48 h were cultured with freshly prepared autologous or allogeneic normal PBL in the presence of PWM, Ig production was markedly suppressed. Similarly, there was a consistent suppression of blast transformation of concanavalin A (Con A) stimulated autologous or allogeneic responding PBL by (+)cyanidanol-3 pre-treated normal T cells. On the other hand, (+)cyanidanol-3-induced suppressor cell activity of T cells from patients with CALD was significantly decreased (P less than 0.001) when compared with that of normal individuals. These studies may explain the therapeutic effect of (+)cyanidanol-3 on certain types of liver disease.

Experimental autoimmune hepatitis in mice after immunization with syngeneic liver proteins together with the polysaccharide of Klebsiella pneumoniae.

Experimental autoimmune hepatitis could be produced in SMA mice by monthly injections of syngeneic liver homogenate or liver-specific lipoprotein together with the polysaccharide of Klebsiella pneumoniae 03:K1 as a powerful adjuvant. Using a gel-diffusion technique, antibodies reacting with liver-specific lipoprotein and liver-specific membrane lipoprotein were detected in approximately 50% of sera from the immunized mice after an 8-mo period. After a full immunization schedule, 60%-80% of the livers of the sensitized mice developed infiltration of mono-nuclear cells consisting mainly of lymphocytes and plasma cells in portal areas, frequently associated with focal necrosis of hepatocytes. Moderate-to-severe piecemeal necrosis of hepatocytes appeared in 10 of 59 animals. However, a gradual decrease in the morphologic severity was observed 3-6 mo after cessation of injections. The transfer of splenic cells from animals with the damaged liver led to a hepatitis in recipients that was characterized by portal infiltration with mononuclear cells and by necrosis of liver parenchymal cells seen on day 14 after cell transfer. The suppressor cell activity determined by the ability of concanavalin A-activated cells to suppress blast transformation of splenic cells of normal SMA mice was significantly decreased (p less than 0.05) in mice immunized with a mixture of liver-specific lipoprotein and the polysaccaride of Klebsiella pneumoniae compared with mice immunized with the polysaccaride of Klebsiella pneumoniae alone or the polysaccaride of Klebsiella pneumoniae plus kidney lipoprotein.

Loss of suppressor T cell function and circulating immune complexes in chronic active liver diseases.

Suppressor T cell function is decreased in patients with chronic active liver diseases (CALD). To account for the alterations, we examined the effect of sera of patients with various liver diseases on concanavalin A (Con A) induced suppressor T cell activity of normal individuals. The suppressor T cell activity was inhibited by heat-inactivated serum pretreatment in 13 of 27 cases of patients with CALD and in five of 11 cases of patients with acute viral hepatitis, whereas only two sera of 18 patients with other liver diseases affected suppressor cell activity. Using a 125I-C1q-binding test, a significant correlation (P less than 0.01) was detected between the degree of inhibition in the development of suppressor T cells and the level of circulating immune complexes in the sera of CALD patients. The blocking effect of patients' sera disappeared when the immune complexes were removed with polyethylene glycol. These data suggest that circulating immune complexes modulate cellular immunity in patients with CALD by influencing the suppressor T cell function.

Antibody dependent cellular cytotoxicity in chronic active liver diseases.

Antibody-dependent cellular cytotoxicity (ADCC) of peripheral blood lymphocytes for Chang cells coated with antibody to liver specific membrane lipoprotein was studied in patients with chronic active liver diseases (CALD) to investigate the pathogenesis of liver cell injury. Of 20 CALD patients, a reduced ADCC was found in 5 cases, whereas an increased ADCC was noted in 8 cases. The remaining 7 cases showed a normal ADCC. There was a significant correlation (p less than 0.01) between a decreased ADCC and an increased level of immune complexes in the sera of CALD patients when a 125I-Clq binding test was used. When sera from ADCC patients, which had a high level of circulating immune complexes, or aggregated human IgG was added, ADCC of normal lymphocytes was significantly inhibited (p less than 0.01) compared with that of lymphocytes alone. These results suggest that circulating immune complexes play a role on the pathogenesis of liver damage in CALD.