Shiro Murashima

A decrease in AFP level related to administration of interferon in patients with chronic hepatitis C and a high level of AFP

It is known that there is a very high incidence of hepatocellular carcinoma (HCC) among patients with type C chronic hepatitis and cirrhosis, and α -fetoprotein (AFP) has been widely used as a diagnostic marker for HCC. However, there are some patients showing continuous high AFP values but no evidence of HCC, and some studies have defined such patients as a high-risk group for HCC. In vitro study has shown that interferon (IFN) inhibits cell proliferation and enhances apoptosis as well as specific cytotoxic T lymphocytes against HCC, resulting in direct anticancer actions. In this study, we investigated the effect of IFN on AFP changes in chronic hepatitis C patients. Of 40 patients with chronic hepatitis C in whom diagnostic imaging confirmed the absence of HCC, 24 patients showed high pretreatment AFP values (high AFP group: AFP level > 10 ng/dl; mean ± SD, 46.3 ± 41.5 ng/dl) and 16 showed low pretreatment AFP values (low AFP group: pretreatment AFP level ≤ 10 ng/dl; mean ± SD, 5.3 ± 2.2 ng/dl). Pretreatment clinical parameters were statistically evaluated in relation to the AFP value. In the high AFP group, the platelet count, albumin level, and prothrombin (%) were significantly lower (P = 0.047, P = 0.0002, and P = 0.044, respectively), suggesting that AFP value increases with advancing liver disease. Subsequently 27 patients were administered IFN (IFN group), and the remaining 13 patients were administered Stronger Neo-minophagen C (SNMC), a glycyrrhizin preparation (SNMC group), as a control group receiving liver-protective therapy. Alanine aminotransferase was reduced in both the IFN and the SNMC group (mean, 132.56 to 60.07 mg/ml [P < 0001] and 147.85 to 56.23 mg/ml [P = 0.0240], respectively). AFP was significantly reduced in the IFN group (mean, 30.03 to 12.65 ng/ml; P = 0.0034), but there was no significant change in AFP in the SNMC group (mean, 29.70 to 39.17 ng/ml). AFP is useful for diagnosing HCC; however, some patients show a persistently high AFP level in the absence of HCC, and these patients have been described as a high-risk group for HCC. In this study, we found that IFN therapy but not SNMC universally reduced the AFP baseline. Since AFP is a significant predictor for HCC, therapeutic strategies for hepatitis C, e.g., long-term low-dose IFN treatment, may reduce hepatocarcinogenesis.

A real-time quantitative polymerase chain reaction method for hepatitis B virus in patients with chronic hepatitis B treated with lamivudine

OBJECTIVES:During treatment of chronic hepatitis B with lamivudine, changes in the level of hepatitis B virus (HBV) DNA were investigated using a real-time polymerase chain reaction (PCR) method with a detection limit of 1.7 log copies/ml (50 copies/ml) to clarify its clinical significance, particularly the association between HBV DNA levels and the emergence of tyrosine-methionine-aspartate-aspartate (YMDD) mutants.METHODS:Twenty-four patients who had received lamivudine therapy for >1 yr were studied. HBV DNA levels were determined using transcription-mediated amplification for sera with >3.7 log genome equivalents/ml, the Roche Monitor kit for sera with ≥2.6 log copies/ml, and real-time PCR for sera with < 2.6 log copies/ml (the detection limit was 1.7 log copies/ml). Patients were classified into three groups according to the minimal HBV DNA level attained during lamivudine therapy: the <1.7 log copies/ml group (eight patients), the 1.7–2.5 log copies/ml group (five patients), and the ≥2.6 log copies/ml group (11 patients).RESULTS:Pretreatment HBV DNA levels were significantly lower in the <1.7 copies/ml group than in the other two groups (p < 0.05). Neither the emergence of YMDD mutants nor a virological breakthrough of serum HBV DNA was observed in any of the eight patients in the <1.7 copies/ml group. In contrast, in the 1.7–2.5 copies/ml and ≥2.6 copies/ml groups, virological breakthroughs resulting from the emergence of YMDD mutants were observed in two of five patients and in all 11 patients, respectively (p < 0.001). Virological breakthroughs were observed at a mean of 49.6 ± 18.4 wk in 11 the patients in the ≥2.6 copies/ml group and at wk 107 and 115 in two patients in the 1.7–2.5 copies/ml group.CONCLUSION:The real-time PCR method is useful for predicting the emergence of YMDD mutants and the estimated time of their emergence.

Effect of interferon treatment on serum 2',5'-oligoadenylate synthetase levels in hepatitis C-infected patients.

Interferon (IFN) is widely used for patients with hepatitis C. Less than half of treated patients respond to IFN therapy, however, and increased resistance to IFN is particularly observed in genotype 1b patients. Recently, genotype 1b patients with the wild type sequence in the NS5A gene were shown to be resistant to therapy, suggesting that the NS5A protein may be involved to IFN resistance. Thus, we investigated the serum 2′,5′‐oligoadenylate synthetase (2′,5′‐OAS) levels before and during IFN treatment. In addition, other biochemical markers and NS5A mutations were also examined in 30 HCV genotype 1b‐positive patients. Before IFN treatment, 2′,5′‐OAS activity in sera was significantly lower in wild type patients than in mutant type patients. All patients were subsequently enrolled in IFN therapy, and 2′,5′‐OAS activity was elevated both in wild and mutant type patients, irrespective of the number of mutations in NS5A. Logistic regression analysis revealed that clearance of serum HCV RNA was independently related to the pretreatment viral load and NS5A mutations, but not to serum 2′,5′‐OAS activity. We concluded that the NS5A protein, that is associated with the outcome of IFN therapy, affects the kinetics of IFN‐induced molecules, such as 2′,5′‐OAS. 2′,5′‐OAS activity does not, however, seem to be related to long‐term virological response to IFN therapy. J. Med. Virol. 62:185–190, 2000.

Mutations in the NS5A Gene Predict Response to Interferon Therapy in Japanese Patients with Chronic Hepatitis C and Cirrhosis

The virus genotype, serum HCV-RNA level and liver histology are reported to be important factors in the response to interferon therapy. Recent studies have revealed that HCV NS5A 2209-2248 amino acid changes affect the response to interferon therapy of genotype 1b chronic hepatitis C. In contrast, some studies done in western countries have reported no such correlation. In the present study, interferon therapy was given to 58 Japanese patients, including 15 liver cirrhosis patients. NS5A 2209-2248 changes, the serum HCV level, ALT level, age and histology were examined in relation to the interferon effect. Twenty-four of the 58 patients (41%) showed a sustained virological response to the therapy. The responses to interferon therapy were significantly correlated with NS5A 2209-2248 changes (p < 0.0001), the HCV-RNA level (p < 0.0001) and histology (p < 0.0060). Among 15 liver cirrhosis patients, 3 of 6 mutant type patients showed a sustained virological response; 5 intermediate and 4 with wild type virus infected patients showed no responses. In conclusion, NS5A 2209-2248 changes may be a useful predictive marker of response to interferon therapy in addition to the serum HCV RNA level even in histologically advanced patients.

Interferon-γ brings additive anti-viral environment when combined with interferon-α in patients with chronic hepatitis C

Abstract T-cell hyporesponsiveness may lead to chronicity of hepatitis C virus (HCV) infection. We evaluated whether interferon (IFN)-γ injection can bring a Th1-dominant environment to patients with chronic hepatitis C. Seventeen patients with genotype 1b received natural IFN-α 5MU daily for the first 2 weeks and three times a week for the next 22 weeks followed by natural IFN-γ 1 MU daily for 2 weeks. In 4 of 17 patients (23.5%), alanine aminotransferase (ALT) was normalized and 3 of these 4 patients (75.0%) cleared HCV RNA. β2 microglobulin (BMG), neopterin and soluble (s) Fas increased with IFN-α and increased more with IFN-γ. Serum interleukin (IL)-12, CD4 and CD8 remained unchanged with IFN-α but increased after IFN-α was replaced by IFN-γ. IL-10 was not changed either with IFN-α or γ. Productions of IL-2, IFN-γ and tumor necrosis factor (TNF)-α by peripheral blood mononuclear cells did not change by IFN-α therapy, however, they were enhanced at the end of IFN-γ therapy. Productions of IL-2 and 4 were unaffected. These results show that some immune parameters become Th1-dominant by additional IFN-γ in patients with chronic hepatitis C. Combination of these two IFNs should be explored.

Short term and two-step interferon therapy for chronic hepatitis C patients with low HCV RNA levels.

Objective: Chronic hepatitis C patients with low HCV RNA levels were treated with short term therapy and for patients in whom the virus was not eliminated in this short therapy, we designed a two-step IFN therapy. Subjects: There were 31 patients with chronic hepatitis C whose HCV RNA level before IFN therapy was <1.0 Meq/ml or 100 kcopies/ml. The HCV serotypes were serotype 1 in nine patients, serotype 2 in 21 and mixed type in one. A 9--10 MU per day of natural IFN was administered daily for 2 weeks, then three times weekly for 8 weeks (the first therapy), then the therapy was ended. For patients in whom the virus was not eliminated, an additional IFN therapy was administered during the decreasing phase of HCV RNA (second therapy). The historical control group consisted of 57 patients who fulfilled the above criteria and underwent IFN therapy for 24 weeks. Complete responders were defined as patients in whom HCV RNA was not detected in their serum for at least 6 months after the end of treatment. Results: Nineteen (61%) of 31 patients who completed the first therapy showed complete response to the first therapy. Eight patients underwent the second therapy and the complete response rates were 0/4 and 2/4 (50%) in the serotype 1 and 2 groups, respectively. The overall complete response rate was 44% (4/9) in the serotype 1 group, 76% (16/21) in the serotype 2 group and 100% (1/1) in the mixed type. The complete response rate in the historical control group was 70% (40/57), showing no difference in efficacy. However, the IFN dosage was significantly lower in the patients than in the control group (P<0.001). Conclusions: In patients with low HCV RNA levels and serotype 2, short term therapy is sufficiently effective. Furthermore, 50% of the complete response was obtained by an additional second therapy. This therapy design showed high efficacy and was cost-effective in these patients.

Sequence Variation of the Hypervariable Region in HCV Carriers with Normal ALT Levels: A Comparison with Symptomatic Carriers

The clinical significance of the hypervariable region (HVR) in the N‐terminus of the E2/NS1 region, which encodes the putative envelope glycoprotein (gp 70) of HCV, has not yet been elucidated. We studied the relation between HVR changes and elevation of the alanine aminotransaminase (ALT) level due to liver cell injury as well as the persistence of HCV infection. Three patients (carrier group) who were HCV RNA positive and had normal ALT levels for as long as five years and three patients with high ALT levels were studied. None of the six patients had a history of treatment. HCV RNA was extracted from serum obtained from each patient in 1990 and 1995. The E2/NS1 region, including HVR‐1 and HVR‐2, was amplified using the RT‐PCR method. PCR products were cloned and nucleotide sequences were determined using the dideoxynucleotide chain termination method. No clear correlation was found between the ALT levels and the number of nucleotide substitutions in HVR‐1. The number of nucleotide substitutions in HVR‐1 during the five years was greater than in other regions. Furthermore, more nucleotide substitutions occurred in the 1st and 2nd codon positions of HVR‐1 than in the control region, even in the carrier group. In conclusion, HVR‐1 changes are probably a more important factor in persistent viral infection than liver cell injury.

Change in cerebral blood flow in an acute hepatitis C patient with depressive state while receiving interferon

Abstract Regional cerebral blood flow (rCBF) was measured with single photon emission computed tomography (SPECT) using N-isopropyl-p-[123I] iodoamphetamine (123I-IMP) in a patient with acute hepatitis C who showed depressive state during interferon therapy. During his depressive state the rCBF was decreased markedly in the inferior frontal cortex. This case suggests that psychotic symptoms during IFN therapy may result from decreased rCBF.