The efficacy and safety of thymosin alpha-1 in Japanese patients with chronic hepatitis B; results from a randomized clinical trial.

Thymalfasin (thymosin alpha-1; Talpha1) is a 28-amino acid polypeptide that has shown efficacy in the treatment of chronic hepatitis B virus (HBV) infection. The objective of this study was to evaluate the long-term, dose-related efficacy and safety of Talpha1 treatment in chronic hepatitis B patients with positive HBV-DNA and abnormally high alanine aminotransferase (ALT) levels. A total of 316 patients were randomized to receive either 0.8 or 1.6 mg of Talpha1 monotherapy for 24 weeks. At the end of the 72-week observation period (12 months after cessation of therapy), 36.4% of patients in the 1.6-mg treatment group achieved normalization of ALT, 30% achieved clearance of HBV-DNA by branched DNA vs 15% by transcription-mediated amplification, and 22.8% achieved clearance of HBe-antigen. Patients in the 0.8-mg treatment group achieved similar efficacy rates, although patients with advanced fibrosis demonstrated a significantly better response rate when treated with 1.6 mg of Talpha1 monotherapy vs 0.8 mg (as determined by intragroup analysis; patients were not stratified by liver biopsy). All adverse drug reactions were mild and most involved the fluctuation of liver enzymes, which was most likely related to the positive immune effects caused by the response to Talpha1 treatment. Adverse event incidence was similar in the 1.6- and 0.8-mg treatment groups. In conclusion, Talpha1 at doses of 0.8 and 1.6 mg exhibits long-term efficacy against hepatitis B with a good safety profile.

Interferon treatment improves survival in chronic hepatitis C patients showing biochemical as well as virological responses by preventing liver-related death.

Interferon therapy for chronic hepatitis C reduces the risk of hepatocellular carcinoma, especially among virological and biochemical responders. However, little is known about the effect of interferon therapy on mortality. We studied the long-term effect of interferon therapy on mortality in patients with chronic hepatitis C. For this retrospective cohort study, 2954 patients with chronic hepatitis C were recruited, of whom 2698 received interferon therapy and 256 did not. The effect of interferon therapy on survival was assessed by standardized mortality ratio (SMR) based on published mortality data for the general Japanese population and by risk ratio calculated by proportional hazard regression. Over 6.0 +/- 2.2 years follow-up, death from liver-related diseases was observed in 69 (68%) of 101 deaths among interferon-treated patients and in 42 (81%) of 52 deaths among untreated patients. Compared with the general population, overall mortality was high among untreated patients (SMR: 2.7; 95% CI: 2.0-3.6) but not among interferon-treated patients (SMR: 0.9; 95% CI: 0.7-1.1). Liver-related mortality was extremely high among untreated patients (SMR: 22.2; 95% CI: 16.0-30.0) and less among interferon-treated patients (SMR: 5.5; 95% CI: 4.3-6.9). The risk of death from all causes was lower for interferon-treated than untreated patients (risk ratio: 0.47; 95% CI: 0.261-0.836; P = 0.01). The risk of death from liver-related diseases was significantly lower for sustained virological responders (risk ratio: 0.04; 95% CI: 0.005-0.301; P = 0.002) compared with untreated patients, but not for nonsustained virological responders. Sustained biochemical responders (risk ratio: 0.03; 95% CI: 0.004-0.230; P < 0.001) and transient biochemical responders (risk ratio: 0.18; 95% CI: 0.063-0.532; P = 0.002) showed a significantly reduced risk of death from liver-related death, whereas biochemical nonresponders did not. Hence interferon treatment improved survival in chronic hepatitis C patients showing a biochemical as well as a virological response by preventing liver-related deaths.

Expression of fas/fas ligand (fasL) and its involvement in infiltrating lymphocytes in hepatocellular carcinoma (HCC).

BACKGROUND: This study was conducted to examine the expression of Fas/Fas ligand (FasL), to elucidate its relationship with tumor-infiltrating lymphocytes (TILs), and to detect possible gene mutation of Fas/FasL in patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). METHODS: Indirect immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections of liver biopsy and surgery specimens from five normal livers, and from the livers of 30 patients with HCC. Fas/FasL mRNA-expressing cells and apoptotic cells were detected by in situ hybridization and DNA nick end labeling (TUNEL), respectively. We also performed polymerase chain reaction (PCR)-amplifying and direct sequencing for the Fas/FasL gene. RESULTS: Fas/FasL and its mRNA were localized on the membrane or in the cytoplasm in some HCC cells, as well as hepatocytes. Their expression was enhanced in areas with infiltrating inflammatory cells in the noncancerous regions of liver tissue and on the margins of the cancerous tissue. The positivity rate for TUNEL was elevated along these margins. The labeling index of Fas/FasL was lower in the cancerous liver tissue than in the surrounding noncancerous region (P < 0.01), and tended to decrease in proportion to the malignancy of tumor cells; Fas/FasL expression was not found on poorly differentiated type cancer cells. Fas(-)/FasL(+), FasL-mRNA(+) HCC cells were seen in one specimen of moderately differentiated type. Some CD8+T lymphocytes were TUNEL-positive around the cancerous region. In this study, cancerous and noncancerous tissues in HCC revealed no genetic mutations in any exons of Fas/FasL. CONCLUSIONS: These findings suggest that Fas/FasL expression was decreased in proportion to the malignancy of tumor cells, and that infiltrating CD8+ T lymphocytes play a role in apoptosis in HCC. The apoptosis in HCC could be regulated by the suppression of Fas/FasL expression, or, sometimes, by the enhancement of FasL expression.

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.

[Combination therapy of IFN and ribavirin].

Ribavirin is a purine nucleoside analog that inhibits the replication of a wide range of DNA and RNA viruses, although ribavirin treatment dose not significantly decrease serum virus load in patients with chronic hepatitis C. Several lines of evidence from the randomized large-scale studies of USA and Europe indicate that combination therapy with ribavirin significantly enhances the sustained response rate of IFN therapy in major patient types with chronic hepatitis C. A recent trial in Japan also proved that the combination therapy is superior to IFN monotherapy. Thus, IFN-ribavirin combination is likely to become the antiviral therapy of choice for chronic hepatitis C.

Th1/Th2 cytokine profiles and their relationship to clinical features in patients with chronic hepatitis C virus infection.

PURPOSE: An imbalance in helper T-cell type 1 (Th1) and type 2 (Th2) cytokines is suggested to play an important role in the pathogenesis of chronic viral infections, but this issue is not resolved in patients with hepatitis C virus (HCV) infection. The aim of this study was to clarify the relationship between the balance of Th1 and Th2 cytokines and liver damage. METHODS: We investigated cytokine levels in the peripheral blood and liver tissue of patients with chronic HCV infection (n = 59) by three different methods; we used flow cytometry to detect intracellular cytokines, and we measured cytokine titers in sera and in the supernatants of lymphocyte cultures with enzyme-linked immunosorbent assays (ELISAs). RESULTS: In both CD4+ and CD8+ cells, interferon (IFN) gamma-producing cell populations increased, while there was no difference in interleukin (IL)-10 production, indicating a shift to a Th1 cytokine profile with the progression of liver disease. With respect to the ratio of IFN-gamma to IL-10, a correlation was found in CD4+ cells between peripheral blood and liver tissue (r = 0.98; P = 0.0011). Th1 cytokine was predominant in intrahepatic CD4+ cells, while it was predominant in peripheral blood CD8+ cells. CONCLUSIONS: These findings indicate a correlation between dominant Th1 response and disease activity and progression. In addition, we suggest that intrahepatic CD4+ T cells play a pathogenetic role in the hepatic injury of HCV infection.

A patient with chronic hepatitis C who obtained sustained response by retreatment of interferon after decrease of viral load and mutation in interferon sensitivity determining region.

A 44-year-old man with chronic hepatitis C received three courses of interferon (IFN) therapy. HCV genotype was 1b, viral load was 1,200 kcopies/ml and interferon sensitivity determining region (ISDR) was the intermediate type before the 1st IFN therapy. The 1st and 2nd IFN therapies resulted in failure to yield a sustained response. Seven years after from the 1st therapy, viral load had decreased to 15 kcopies/ml and ISDR had changed to mutant type. The 3rd IFN therapy yielded sustained response. Thus, we should consider retreatment with IFN when a decrease of the viral load and change of ISDR to mutant type are observed.

Central suppressive effect of octreotide on the hyperglycemic response to 2-deoxy-D-glucose injection or cold-swim stress in awake rats: possible mediation role of hypothalamic noradrenergic drive.

Somatostatin (SRIH) and its analog have been reported to act within the central nervous system to suppress the hyperglycemic response to a variety of neural stimuli. On the other hand, the hyperglycemic response to 2-deoxy-D-glucose (2-DG) injection or cold-swim stress is well demonstrated to be closely associated with an increase in hypothalamic noradrenergic neuronal activity (NNA). To evaluate whether the suppression of the hypothalamic NNA response could be involved in the central mechanism whereby a SRIH analog inhibits the hyperglycemic response, octreotide, a clinically used long-acting octapeptide SRIH analog, was administered into the third cerebral ventricle of awake rats prior to the intraperitoneal injection of 2-DG or cold-swim stress. Hypothalamic noradrenaline (NA) and its neuronal metabolite, 3,4-dihydroxyphenylethyleneglycol (DHPG), were analyzed, and the ratio of DHPG to NA was used as an index of NNA. Intracerebroventricular (i.c.v.) pretreatment with octreotide suppressed the 2-DG-induced increase in hypothalamic NNA, accompanied by the inhibition of the serum glucose, NA and adrenaline responses. This suppressive effect of octreotide was dose-dependent. Similarly, i.c.v. pretreatment with octreotide prevented the hypothalamic NNA response to cold-swim stress, accompanied by a blockade of the increases in serum glucose, NA and adrenaline. A close relationship between hypothalamic NNA and serum glucose emerged from these studies. Intraperitoneal pretreatment with octreotide had no significant effect on the hyperglycemic or hypothalamic NNA response to 2-DG injection. These findings suggest that the inhibitory effect of octreotide on the hypothalamic NNA response to 2-DG injection or cold-swim stress is associated with the simultaneous suppression of the hyperglycemic response. Supporting the concept that hypothalamic NNA contributes to the modulation of blood glucose in stressful conditions, it is suggested that the suppression of the hypothalamic NNA response is, at least in part, involved in the central mechanism by which octreotide inhibits the hyperglycemic response to 2-DG injection or cold-swim stress.

Mutation at codon 130 in hepatitis B virus (HBV) core region increases markedly during acute exacerbation of hepatitis in chronic HBV carriers.

Mutations within T-cell or B-cell epitopes are suggested to have some influence on the clinical course of chronic hepatitis B virus (HBV) infection. To investigate the relationship between liver cell injury and heterogeneity of the HBV core gene, we focused on the sequence of codon 130, which is located on both T- and B-cell epitopes, and serially analyzed the proportion of mutant virus (core130Thr) to wild-type virus (core130Pro) during the exacerbation of chronic hepatitis B. Sera obtained serially from five HBV carriers who had exacerbation of hepatitis, and three asymptomatic HBV carriers (ASCs) with persistently normal serum aminotransferase (ALT) values were studied, using the restriction fragment length polymorphism (RFLP) method. Core130Pro predominated in the sera in the remission state, but core130Thr increased markedly in parallel with ALT elevation and decreased again after the ALT peak, followed by the predominance of core130Pro, in all the five patients. In one patient, the ratio of core130Thr/core130Pro (Thr/Pro) was more than 70% at the ALT peak. On the other hand, in sera from the three ASCs core130Pro always predominated, and no divergence was identified in the ratio of Thr/Pro. Our data suggest that codon 130 is one of the most important immunogenic regions in the HBV core gene and that elevation of Thr/Pro could be the result of immune selection.

Real-time detection system for quantitation of hepatitis C virus RNA: a comparison with the other three methods.

Real-time detection (RTD) system for quantitation of hepatitis C virus (HCV) was developed. Its sensitivity and usefulness were compared with the other three commercially available methods for quantitation of HCV. The sera of 166 patients positive for serum HCV RNA by Amplicor HCV test were assessed. HCV was detected in 78.5% (128/163) by branched DNA assay, in 88.8% (111/125) by HCV core protein assay, in 94.5% (156/165) by Amplicor HCV Monitor test, and in 97.0% (161/166) by the RTD system. The values of viral load by the RTD system were significantly well correlated with those obtained by the other three methods. In the 50 patients treated by interferons (IFNs), the range which predicts the highest sustained response rate was less than 0.5 Meq/ml for branched DNA assay (sustained response rate: 57.9% (11/19)), less than 1 kcopies/ml for Amplicor HCV Monitor test (85.7% (6/7)), and less than 10(4) copies/ml for RTD system (100% (7/7)). None of the patients with greater than or equal to 2.8 Meq/ml by branched DNA assay (n=14), greater than or equal to 250 kcopies/ml by Amplicor HCV Monitor test (n=19), or greater than or equal to 2x10(6) copies/ml by RTD system (n=16) obtained sustained response. In conclusion, RTD system was demonstrated to be the most sensitive method for quantitation of HCV, and useful for the prediction of sustained response to IFN therapy.

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.

Clinical characteristics of patients with chronic hepatitis C showing biochemical remission, without hepatitis C virus eradication, as a result of interferon therapy. The Osaka Liver Disease Study Group.

During long-term follow-up of patients chronically infected with the hepatitis C virus (HCV) and treated with interferon (IFN), we identified some who had persistent normalization of serum alanine aminotransferase (ALT) but remained positive for HCV RNA. The aims of this study were to clarify the characteristics of these patients and to examine their clinical outcome after treatment. Nine hundred and ninety-eight patients treated with IFN were followed-up biochemically and virologically, and by liver ultrasound, for 13-95 months. A short-term biochemical sustained response, where ALT remained within the normal range for 6 months after the completion of IFN therapy, was found in 296 patients; in 240 of these patients serum HCV RNA remained undetectable during long-term follow-up. The rate of HCV RNA persistence was 7.09 times greater in short-term biochemical sustained responders with a high viral load than in those with a low viral load (P=0.0001, odds ratio [OR]=7.09), and 3. 70-fold lower in those treated with a large dose of IFN than in those treated with a small dose (P=0.02, OR=0.27). Thirty-three (59%) of 56 patients without HCV eradication showed continuous ALT normalization for 26-80 months after cessation of IFN therapy. Short-term biochemical sustained responders who were older (P=0.009, OR=10.43) and who were male (P=0.03, OR=6.98) had a significantly greater probability of maintaining a normal ALT level, even when serum HCV RNA was positive. When the incidence of HCC was investigated during long-term follow-up in patients without HCV eradication, it was found to be significantly lower in patients with persistently normal ALT levels than in those with abnormal ALT levels (P=0.03). Hence, when HCV is not eradicated as a result of IFN therapy, it may induce a long-term carrier state of HCV infection with normal ALT levels in older or male patients, in whom the cumulative incidence of HCC is markedly decreased.

Mutations in interferon sensitivity-determining region of hepatitis C virus: its relation to change in viral load.

OBJECTIVE: Interferon sensitivity-determining region (ISDR) in nonstructural region 5A of hepatitis C virus (HCV) genotype-1b has been reported to be associated with viral load. Viral load is usually small in the patients with mutant type (four or more amino acid substitutions, compared with HCV-J) and large in those with wild (identical to HCV-J) or intermediate type (from one to three amino acid substitutions). A possible correlation was investigated between mutations in ISDR and alterations of viral load during the course of disease. METHODS: The sequences of ISDR were determined in eight patients with significant changes of viral load and in 11 patients without changes. RESULTS: In two of the eight patients with significant alterations of viral load, ISDR sequences changed significantly. In one patient whose viral load increased after a course of interferon therapy, the number of substitutions, compared with HCV-J, decreased from five to zero or one; the type of ISDR converted from mutant type to wild or intermediate type. In one patient whose viral load decreased significantly after two courses of interferon therapy, the number of substitutions increased from one to six; ISDR changed from intermediate type to mutant type. In the remaining six patients with changes of viral load and in the other 11 patients without changes, the sequences of ISDR did not change significantly. CONCLUSIONS: The mutations in ISDR are one of the viral factors involved in the changes in viral load during the course of disease, although the majority of other factors involved are still unknown.

Effect of the lipoprotein lipase activator NO-1886 on adriamycin-induced nephrotic syndrome in rats.

Hyperlipidemia associated with nephrotic syndrome may play a role in the deterioration of renal function. Tsutsumi et al have previously reported that the novel compound NO-1886 increases lipoprotein lipase (LPL) activity, resulting in a reduction of plasma triglycerides and an elevation of high-density lipoprotein (HDL) cholesterol in normal rats. The aim of this study was to ascertain whether NO-1886 suppresses the renal injury by treatment of the hyperlipidemia in an Adriamycin (Kyowa Hakko Kogyo, Tokyo, Japan) induced nephrosis rat model fed a high-protein diet that induced renal dysfunction and tubulointerstitial injury. Administration of Adriamycin caused hyperlipidemia, proteinuria, and edema with ascites in rats in 4 weeks. Furthermore, a combination of Adriamycin and a high-protein diet increased plasma creatinine and blood urea nitrogen (BUN) and decreased plasma albumin. Histologically, in Adriamycin-treated rats, marked interstitial cellular infiltration, tubular lumen dilation, and tubular cast formation in the kidney were observed. NO-1886 decreased plasma triglyceride and increased HDL cholesterol in Adriamycin-induced nephrotic rats. NO-1886 treatment reduced plasma creatinine and BUN levels and increased plasma albumin in Adriamycin-treated rats; it also ameliorated the ascites and proteinuria. Histologically, NO-1886-treated rats showed a quantitatively significant preservation of tubulointerstitial lesions. These data suggest that NO-1886 may have a protective effect against Adriamycin-induced nephrosis with tubulointerstitial nephritis in rats by a modification of the plasma lipid disorder.

Decreased function of peripheral blood dendritic cells in patients with hepatocellular carcinoma with hepatitis B and C virus infection.

BACKGROUND: Tumour immunity does not seem to be induced effectively in tumour-bearing hosts, including in patients with hepatocellular carcinoma (HCC). One possible reason is that function of dendritic cells (DC) is decreased in such hosts. METHODS: We evaluated T cell stimulatory activity and interleukin (IL)-12 production of DC and interferon (IFN)-gamma and IL-10 production of T cells of peripheral blood from 12 control individuals and 21 patients with chronic hepatitis C virus (HCV) infection (six with chronic hepatitis (CH), eight with liver cirrhosis (LC) and 13 with HCC). Five hepatitis B virus (HBV)-infected patients with HCC were included as a disease control group. The DC were prepared by the culture of T cell-depleted populations of peripheral blood mononuclear cells in the presence of granulocyte-macrophage colony stimulating factor and IL-4 for a total of 11-12 days. The cytokine levels were assayed by ELISA. To test the stimulatory function of DC in T cell proliferation, mytomycin C-treated DC were cultured with allogeneic T cells from a control. RESULTS: When the T cell-stimulatory activity of DC was expressed as stimulation index value of [3H]-thymidine incorporation of T cells, the values were lower in HCV-infected HCC (2.6 +/- 1.8, P < 0.01) than in controls (5.5 +/- 2.0) and CH (5.0 +/- 1.3). Staphylococcus aureus Cowan 1-induced IL-12 production of DC was decreased in HCV-infected HCC (P < 0.001, P < 0.01 and P < 0.05, respectively) compared with controls, CH and LC, while similar amounts of IL-10 were produced in patients and controls. Interleukin-10 and IFN-gamma production of T cells in response to anti-CD3 antibody or IL-12 were equivalent between patient groups and controls, respectively. Similarly decreased DC function and normal T cell response were observed in HBV-infected HCC patients. CONCLUSIONS: These findings suggest that the depressed function of DC is associated with pathogenesis of HCC with HBV or HCV infection.