Masafumi Naito

Circulating matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 as serum markers of fibrosis in patients with chronic hepatitis C : relationship to interferon response

BACKGROUND/AIMS/METHODS The imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) is considered to be an important determinant of extracellular matrix deposition and breakdown. We measured serum MMP-1, MMP-2, TIMP-1 and TIMP-2 levels using the respective one-step sandwich enzyme immunoassays in 98 patients with chronic hepatitis C treated with interferon beta to examine their clinical significance for assessment of liver histology and to determine whether they can be useful as predictors of the interferon response. RESULTS Serum TIMP-1 levels showed a positive correlation with the degree of fibrosis (r(s)=0.30, p= 0.004). Serum MMP-2 levels revealed positive relationships with the degree of periportal necrosis (r(s)= 0.32, p=0.002), the degree of fibrosis (r(s)=0.26, p= 0.01) and total score of histological activity index (r(s)=0.24, p=0.02). Serum MMP-2 levels were significantly higher in patients with no response than in those with sustained and transient response (p<0.01 and p<0.05, respectively), while serum MMP-1 levels did not differ among the three groups. Compared with the levels in sustained responders, the total amounts of serum TIMP-1 were significantly lower in transient responders and non-responders (p<0.01 and p<0.001, respectively). As for serum TIMP-2 levels, a significant decrease was found in transient responders and non-responders (p<0.01). The ratios of serum MMP-2 to TIMP-1 levels were significantly higher in transient responders and non-responders than in sustained responders (p<0.001, respectively) even when HCV RNA levels were low in patients with HCV genome subtype 1b or when the HCV genome subtype was 2a or 2b. Sustained response was never found in type 1b patients with ratios of serum MMP-2 to TIMP-1 levels of over 6.0. In logistic multivariate regression analysis, the ratios of serum MMP-2 to TIMP-1 level (p=0.0001), HCV genome subtype (p=0.005) and serum TIMP-2 level (p=0.03) were the independent predictors for sustained response, while serum MMP-2 level (p=0.0006) was the only predictor for no response. CONCLUSIONS Serum MMP-2 and TIMP-1 levels might be useful for estimating the degree of liver fibrosis. The ratio of serum MMP-2 to TIMP-1 levels may serve as a new predictor of interferon response in patients with chronic hepatitis C.

Evaluation of the effects of combination therapy with branched-chain amino acid and zinc supplements on nitrogen metabolism in liver cirrhosis

Aim:  Disorders of protein metabolism in liver cirrhosis can affect prognosis or cause complications. Treatment with branched‐chain amino acid (BCAA) and zinc supplements has been shown to be effective against abnormal nitrogen metabolism in liver cirrhosis. There are, however, few studies on the effects of combining these supplements. In this study, the effect of combining BCAA and zinc treatment in cirrhosis was investigated.

Serial quantitative analysis of serum hepatitis C virus RNA level in patients with acute and chronic hepatitis C

To examine changes in the serum level of hepatitis C virus RNA in acute and chronic phases of hepatitis C virus infection, we tested serial serum samples of six patients with acute hepatitis C (posttransfusion: three; sporadic: three) and 11 patients with chronic hepatitis C using a competitive reverse transcription and polymerase chain reaction assay. The internal standard consisted of known amounts of synthetic mutated RNA. No patient with acute hepatitis showed resolution during the follow-up period (24-57 weeks). In posttransfusion cases, titers of hepatitis C virus RNA (log10[hepatitis C virus RNA copies/ml serum]) rose to a high level (7.5-9.5) in the early phase of infection (4-12 weeks after the transfusions) in association with the first serum alanine aminotransferase peaks. Titers of hepatitis C virus RNA then decreased, while serum alanine aminotransferase levels fluctuated with multiple peaks. In sporadic cases, titers of hepatitis C virus RNA had already reached a high level (7.0-7.5) at the first alanine aminotransferase peaks 2-3 weeks after the clinical onset. In chronic hepatitis C virus infection, titers of hepatitis C virus RNA remained high for follow-up periods of 6-12 years in patients with chronic active hepatitis. These results indicate that the replication of hepatitis C virus rose to a high level in the early phase of infection and that a high replicative level of hepatitis C virus might be related with progression of liver disease in the chronic phase of infection.

Splenectomy and antiviral treatment for thrombocytopenic patients with chronic hepatitis C virus infection.

Summary.  Thrombocytopenic patients with chronic hepatitis C virus (HCV) infection are poor candidates for antiviral treatment with interferon (IFN), but no standard treatment for thrombocytopenia has yet been established. We evaluated the safety of splenectomy and its efficacy for the initiation and continuation of antiviral therapy. From March 2003 to April 2006, 10 patients (mean age 62.5 years) with HCV‐related cirrhosis, low platelet count (≦106 000/mm3) and splenomegaly (spleen size ≧10 cm) underwent splenectomy. Platelet counts significantly increased at 4–8 weeks after splenectomy [pre: 64 200 ± 6900/mm3vs post 209 000 ± 40 600/mm3 (P = 0.004)]. No severe operative complications were observed. All patients subsequently received antiviral therapy. Of the eight patients who were infected with HCV genotype 1 and had a high viral load (≧100 KIU/mL), four received combination therapy with pegylated IFNα‐2b plus ribavirin, and the other four received standard IFNα‐2b plus ribavirin. One patient infected with HCV genotype 2 and another with HCV genotype 1 and a low viral load (<100 KIU/mL) were treated with pegylated IFNα‐2a. Six patients achieved sustained virologic response (SVR). Among four patients who failed to achieve SVR, one was given retreatment with pegylated IFN plus ribavirin, and the other three received low‐dose long‐term IFN therapy. Although this study was small, the treatment results were similar to those for patients without thrombocytopenia and suggested that splenectomy would not reduce the antiviral efficacy of IFNα‐based treatment.

Immunological response to interferon-γ priming prior to interferon-α treatment in refractory chronic hepatitis C in relation to viral clearance

The aim of this study was to clarify the immunological and virological responses to pre‐administration of interferon‐γ prior to initiation of interferon‐α treatment in patients with refractory chronic hepatitis C. Twenty‐two nonresponders to 6‐months of IFN‐α treatment were enrolled. The hepatitis C virus (HCV) genotype was Ib in all. Natural IFN‐γ (1 MIU/day) was administered daily for 14 days followed by natural IFN‐α (5 MIU/day) daily for 14 days and then three times weekly for 22 weeks. Serum immunological parameters (IL‐10, neopterin, BMG, sCD8, sCD4, IL‐6, IL‐12) were measured as were the levels of several cytokines (IFN‐γ, TNF‐α, IL‐2, IL‐4, IL‐5, IL‐6, IL‐10). Three patients dropped out; two because of the occurrence of other diseases and one because of an adverse effect. At the end of the period of IFN‐α treatment, HCV‐RNA had become negative in six of 19 patients (end‐of treatment response; ETR). Six months after the completion of IFN administration, a virological sustained response (SR) was seen in two of 19 patients. The mean serum levels of IL‐10 were significantly decreased 6 weeks after the start of treatment. Other immunological parameter levels increased significantly during the period of IFN‐γ administration, and tended to return to the pretreatment level after the start of IFN‐α administration. Univariate logistic regression analysis showed that the initial change in the levels of these parameters or the change in the ratios of Th1/Th2 parameter levels are useful factors indicative of the end of the treatment response. These findings suggest that priming with IFN‐γ prior to the initiation of IFN‐α treatment in patients with refractory chronic hepatitis C can modulate the host immune response and this might contribute to viral clearance.

Hepatitis C Virus Antibodies and Virus Replication in Asymptomatic Blood Donors

We assessed hepatitis C virus (HCV) infection in 99 asymptomatic blood donors positive using a first‐generation HCV antibody assay. When tested with second‐generation assays, 86 (87%) donors were reactive (group 1), 2 (2%) were indeterminate (group 2), and 11 (11%) were non‐reactive (group 3). Viraemia was revealed by polymerase chain reaction in all group 1 cases. The 2 group 2 cases and 6 (55%) group 3 cases were also viraemic. Viraemia was confirmed by a branched DNA assay in the 2 group 2 cases and 4 (36%) group 3 cases. Serum HCV RNA levels were further studied using a competitive reverse transcription‐polymerase chain reaction assay. All cases in groups 2 and 3 were low viraemic (range 104–105.5copies/ml) compared with the 9 group 1 cases examined (range 107–109copies/ml). No correlation was evident between viraemic levels and antibody cut‐off index in the first‐generation assay. These findings indicate the possibility that low levels of viraemia can occur in individuals non‐reactive in second‐generation HCV antibody assays.

Baseline quasispecies selection and novel mutations contribute to emerging resistance-associated substitutions in hepatitis C virus after direct-acting antiviral treatment

Resistance-associated substitutions (RASs) in hepatitis C virus (HCV) appear upon failure of treatment with direct-acting antivirals (DAAs). However, their origin has not been clarified in detail. Among 11 HCV genotype 1b patients who experienced virologic failure with asunaprevir (ASV)/daclatasvir (DCV), 10 had major NS5A L31M/V-Y93H variants after treatment. L31M/V-Y93H variants were detected as a minor clone before therapy in 6 patients and were the most closely related to the post-treatment variants by phylogenetic tree analysis in 4 patients. Next, to consider the involvement of a trace amount of pre-existing variants below the detection limit, we analysed human hepatocyte chimeric mice infected with DAA-naïve patient serum. L31V-Y93H variants emerged after treatment with ledipasvir (LDV)/GS-558093 (nucleotide NS5B inhibitor) and decreased under the detection limit, but these variants were dissimilar to the L31V-Y93H variants reappearing after ASV/DCV re-treatment. Finally, to develop an infection derived from a single HCV clone, we intrahepatically injected full-genome HCV RNA (engineered based on the wild-type genotype 1b sequence) into chimeric mice. A new Y93H mutation actually occurred in this model after LDV monotherapy failure. In conclusion, post-treatment RASs appear by 2 mechanisms: the selection of pre-existing substitutions among quasispecies and the generation of novel mutations during therapy.

Colorectal endoscopic submucosal dissection: Recent technical advances for safe and successful procedures

Endoscopic submucosal dissection (ESD) is very useful in en bloc resection of large superficial colorectal tumors but is a technically difficult procedure because the colonic wall is thin and endoscopic maneuverability is poor because of colonic flexure and extensibility. A high risk of perforation has been reported in colorectal ESD. To prevent complications such as perforation and unexpected bleeding, it is crucial to ensure good visualization of the submucosal layer by creating a mucosal flap, which is an exfoliated mucosa for inserting the tip of the endoscope under it. The creation of a mucosal flap is often technically difficult; however, various types of equipment, appropriate strategy, and novel procedures including our clip-flap method, appear to facilitate mucosal flap creation, improving the safety and success rate of ESD. Favorable treatment outcomes with colorectal ESD have already been reported in many advanced institutions, and appropriate understanding of techniques and development of training systems are required for world-wide standardization of colorectal ESD. Here, we describe recent technical advances for safe and successful colorectal ESD.

Diuretics aggravate zinc deficiency in patients with liver cirrhosis by increasing zinc excretion in urine.

Liver cirrhosis is often accompanied by zinc deficiency. The exact mechanisms underlying zinc deficiency remain unclear. This study was undertaken to clarify the influence of diuretics on blood zinc levels and zinc excretion in urine in liver cirrhosis.

Long-term biochemical and virological response to natural interferon-α in patients with chronic hepatitis C

To determine the long-term response to interferon-α, 134 patients with chronic hepatitis C were followed for more than one year after therapy. Follow-up was stopped for 14 patients and 43 patients received retreatment. The remaining 77 patients were followed for 26–46 months, and 39 of them achieved long-term sustained alanine aminotransferase (ALT) normality. This normality was achieved in 35/38 short-term sustained responders, which was significantly higher (P<0.001) than in the short-term relapsers (2/38) and the short-term nonresponders (2/44). Hepatitis C virus (HCV) RNA remained negative in 27 (73%) of 37 patients in the long-term sustained ALT normality group. However, only one (1.6%) of 63 long-term nonresponders was negative for HCV RNA. The results of HCV RNA testing for the long-term period agreed with those at three to six months after therapy. Serum ALT levels during the first six months after therapy and HCV RNA testing at three to six months after therapy are important for predicting long-term sustained ALT normality and HCV RNA negativity.