Akira Hayasaka

Immunohistochemical metallothionein expression in hepatocellular carcinoma: relation to tumor progression and chemoresistance to platinum agents.

BackgroundMetallothionein (MT), which is known to detoxify heavy metal ions, is considered to serve as a mechanism of resistance to platinum complex compounds. In the present study, MT expression in hepatocellular carcinoma (HCC) was immunohistologically investigated to clarify its relationship to clinical background factors and responsiveness to anticancer drugs.MethodsSpecimens from 117 patients with HCC were immunohistologically studied, using a monoclonal anti-MT antibody. the percentage of MT-positive HCC (MT ratio) cells was determined, to evaluate the extent of staining with anti-MT antibody. Staining with an MT ratio of more than 50% was categorized as diffusely positive; an MT ratio of 5% to less than 50% was focally positive; and an MT ratio of less than 5% was negative. Twenty-two patients received repeated arterial infusion chemotherapy with carboplatin (CBDCA), a platinum-containing compound, and the MT expression was analyzed in relation to their chemotherapeutic response.ResultsThe ratio of MT-positive cells in HCC decreased with the degree of histological differentiation and also decreased with higher tumor stage. In patients treated with CBDCA, the ratio of MT-positive cells in responders was significantly lower than that in non-responders.ConclusionsMT expression decreases with the degree of histological differentiation and decreases with increasing tumor stage in HCC. In addition, MT expression may lower the antitumor effect of CBDCA.

Immunohistochemical study of transforming growth factor-beta 1, matrix metalloproteinase-2, 9, tissue inhibitors of metalloproteinase-1, 2, and basement membrane components at pancreatic ducts in chronic pancreatitis

Little is known about the pathogenesis of fibrosis in chronic pancreatitis. To reach a better understanding of this problem, we investigated the immunolocalizations of type IV collagen (Col-IV) and laminin around pancreatic ducts, and those of matrix metalloproteinase-2.9 (MMP-2,9), tissue inhibitors of metalloproteinase- 1,2 (TIMP- 1,2), and transforming growth factor-β1 (TGFβ1) at the ductal epithelia in chronic pancreatitis. This study included 20 surgical specimens of fibrotic pancreas from patients with chronic pancreatitis and five normal samples from autopsy cases. Immunostaining was performed by the streptavidin-biotin method after antigen retrieval. We evaluated the staining patterns and the percentage of positive cells of each antigen. In chronic pancreatitis, the immunostainings of Col-IV and laminin along the basement membrane (BM) of pancreatic ducts were disrupted in 11 (55%) of 20 and eight (40%) of 20, respectively, whereas no disruption was detected in normal pancreas. Positive immunostainings for MMP-2, MMP-9, TIMP-I, and TIMP-2 in ductal epithelia were 15 (75%) of 20, five (25%) of 20, four (20%) of 20, and 10 (50%) of 20, respectively, whereas no immunostaining was seen in normal pancreas. The staining intensity of MMP-2 in ductal epithelia was associated with the staining intensity of Col-IV around the pancreatic ducts. Also, the staining intensity of MMP-2 was progressively increased in proportion to the staining intensity of TGFβ1. These findings suggest that TGFβ1 induced in pancreatic duct cells also induced MMP-2 in an autocrine or paracrine manner, and that this MMP-2 decomposed Col-IV of the BM of pancreatic ducts in chronic pancreatitis.

Serum concentrations of the carboxyterminal cross-linking domain of procollagen type IV (NC1) and the aminoterminal propetide of procollagen type III (PIIIP) in chronic liver disease

Serum concentrations of both the carboxyterminal cross-linking domain (NC1) of procollagen type IV and the aminoterminal propeptide of procollagen type III (PIIIP) were measured by specific radioimmunoassays in 60 patients with chronic liver disease and 50 healthy controls. Compared with controls (5.3 +/- 1.3 ng/ml, mean +/- S.D.), NC1 concentrations were significantly elevated in patients with chronic active hepatitis (10.2 +/- 2.0 ng/ml) and liver cirrhosis (13.5 +/- 3.0 ng/ml), but not in chronic persistent hepatitis (6.0 +/- 0.9 ng/ml). The concentrations in patients with active liver cirrhosis were significantly higher than those in patients with inactive cirrhosis. Serum concentrations of PIIIP in controls, parients with chronic persistent hepatitis, chronic active hepatitis and cirrhosis were 5.8 (4.3-7.9), 5.3 (3.5-7.9), 17.5 (10.6-28.9), 16.7 (10.4-26.7) ng/ml, respectively (logarithmic mean and range of mean +/- S.D. after retransformation). Patients with liver cirrhosis had significantly higher concentrations of NC1 in serum than those with chronic active hepatitis, but there was no difference in serum PIIIP concentrations between the two groups. These data suggest an alteration of type IV collagen metabolism in chronic liver disease. In liver cirrhosis, the metabolism of collagen IV is apparently different from that of collagen type III; serum NC1 determinations may therefore provide additional information on chronic liver disease, particularly in patients with cirrhosis with a normal level of serum PIIIP. Further follow-up studies as well as investigations related to the basic mechanism of the elevation of these peptides in serum are needed in order to understand their clinical significance fully.

Serum Markers as Tools to Monitor Liver Fibrosis

Reliable diagnostic tests are necessary for monitoring hepatic fibrogenesis in patients with chronic liver disease. Until recently, liver fibrosis has been believed to be irreversible, but recent studies suggest that liver fibrosis and cirrhosis are potentially reversible in response to therapy, even in man [1]. Studies are ongoing to find novel, effective anti-fibrotic agents for patients with liver fibrosis [2]. We must diagnose cirrhosis at an early, compensated stage with accuracy and precision, but also be able to determine the various degrees of liver fibrosis at the precirrhotic stage of chronic liver disease. Once reliable information has been obtained about the amount of fibrotic tissue deposited in the liver, we then need to know if the fibrotic liver disease is going to progress, and how fast this progression is (as determined by the balance and speed of liver fibrogenesis and fibrolysis). Clear, noninvasive markers of these dynamics would become clues to predict the subsequent clinical course of the patient, to estimate the likely and to determine the actual response to the antifibrotic therapy. Liver biopsy is regarded as the gold standard for the determination of the stage of liver fibrosis, but cannot be performed on a day-to-day basis. Also, it only provides static in contrast to dynamic information about the fibrotic process. The favored tests should be easy and safe to perform, to be done repeatedly and at short time intervals. Serum biochemical markers are ideal candidates for this purpose. In this summary we describe some general aspects, potential clinical applications, and discuss open questions as to the serum markers for liver fibrosis.

The serum concentrations of the aminoterminal propeptide of procollagen type III and the hepatic content of mRNA for the α1 chain of procollagen type III in carbon tetrachloride-induced rat liver fibrogenesis

Serum concentrations of the aminoterminal propeptide of procollagen type III (PIIIP) are elevated in fibrogenic diseases of the liver, but the mechanism of elevation is not fully understood. To investigate the mechanism, we compared serum concentrations of PIIIP with total liver content of mRNA for the pro alpha 1 (III) chain, in rats with carbon tetrachloride (CCl4)-induced liver fibrosis. Adult male rats received CCl4 in mineral oil twice weekly for 8 weeks and were compared with age-matched controls. Serum concentrations of PIIIP were measured by a specific radioimmunoassay; molecular sizes of PIIIP in serum were also determined. Pro alpha 1 (III) mRNA content in the liver was quantitated by RNA slot-blot hybridization and chemical measurement of total hepatic RNA content. Total collagen content of the liver was estimated by hydroxyproline measurement. All CCl4-treated animals had septal fibrosis after 4 weeks, and evidence of cirrhosis (regenerative nodules, ascites) was seen after 7 weeks of treatment. Serum concentrations of PIIIP and pro alpha 1 (III) mRNA content in the liver were correlated well until cirrhosis has established. They increased simultaneously after 3 weeks of treatment, 1 week before any elevation of hepatic hydroxyproline could be detected. After cirrhosis has established, pro alpha 1 (III) mRNA content in the liver decreased markedly, but serum PIIIP levels continued to be elevated. Hepatic hydroxyproline plateaued after 5 weeks. The molecular sizes of serum PIIIP indicate the release of intact native procollagen peptide during the development of cirrhosis. In conclusion, at least in CCl4-induced liver fibrosis in the rats, serum PIIIP levels can be used as a fibrogenic marker for the period progressing to cirrhosis. But the use of the serum PIIIP levels in cirrhosis seems to be limited by factors other than liver fibrogenesis.

Pyridinoline collagen cross-links in patients with chronic viral hepatitis and cirrhosis

BACKGROUND/AIMS The mature form of collagen cross-linking increases the resistance of collagen to degradative enzymes, and thus renders the protein in the fibrotic lesions extremely stable and the fibrosis virtually irreversible. It is crucial to elucidate the extent of cross-linking in fibrotic and cirrhotic livers if we are to control the subsequent removal of the excessive deposited collagen, whether by natural enzymes or induced by therapy. We aimed to quantitate pyridinoline, a mature form of the cross-linking, in normal control livers, viral fibrotic livers with various degrees of fibrosis and viral cirrhotic livers. METHODS Needle liver biopsy samples from 75 patients with chronic viral hepatitis and 13 patients with viral liver cirrhosis, and six normal control livers were analyzed. Collagen and pyridinoline contents were determined by high-performance liquid chromatography. RESULTS Significantly higher levels of pyridinoline cross-links per collagen molecule were found in the viral cirrhotic livers (0.60 [0.46, 0.65] pmol/pmol of collagen; median [25%, 75%]) compared with those in normal livers (0.39 [0.24, 0.43] pmol/pmol of collagen, p = 0.03491). But no differences were found in levels between cirrhosis and chronic hepatitis with various degrees of fibrosis. These data suggest that liver collagen may be susceptible to degradation to a similar degree in viral cirrhosis and in chronic viral hepatitis. CONCLUSION The extent of the pyridinoline cross-linking of hepatic collagen does not seem to be responsible for the irreversibility of viral liver fibrosis.

High sustained virologic response rate after interferon monotherapy in Japanese hepatitis C patients with a low HCV RNA titer and/or HCV genotype 2. A prospective study.

Objective: Hepatitis C virus (HCV) RNA titer and HCV genotype are considered to be major determinants of the outcome of interferon monotherapy. To clarify whether interferon monotherapy is really effective in patients with the appropriate viral parameters, we prospectively examined these parameters and treated the patients with interferon monotherapy. Methods: Sixty-four patients with an HCV RNA titer <100 kIU/ml and/or HCV genotype 2 were enrolled in the study. Eighteen patients with an HCV RNA titer >100 kIU/ml and genotype 1 were also enrolled as controls. All patients were treated with 10 megaunits of interferon-α2b every day for 2 weeks and then 3 times a week for 24 weeks. Results: Of the 64 patients with either HCV RNA <100 kIU/ml and/or genotype 2, seven dropped out from the study. Of the remaining 57 who completed the treatment, 48 (84%) showed a virologic sustained response. In contrast, only 4 of the 18 patients (22%) with HCV RNA >100 kIU/ml and genotype 1 were virologic sustained responders (p < 0.001). Conclusion: Our current study showed that the patients with HCV RNA <100 kIU/ml and/or HCV genotype 2 are good candidates for interferon monotherapy.

The effect of interferon alpha and prednisolone on the serum concentrations of the aminoterminal domain of procollagen type III(PIIIP) and type IV (7-S collagen) in patients with chronic hepatitis B

Since interferons are reported to inhibit collagen synthesis in vivo and in vitro, they are possible anti-fibrogenic drugs. However, little is known about the effect of interferons on connective tissue metabolism in liver disease. To investigate the effect, serum concentrations of the aminoterminal domain of procollagen type III and type IV (7-S collagen) were measured in 16 patients with chronic hepatitis B during alpha interferon treatment. The levels were also determined during prednisolone withdrawal treatment to investigate the effect on hepatic collagen turnover. In patients with chronic hepatitis B, the serum levels of both markers were significantly elevated before any treatment (procollagen type III: 1.10 +/- 0.39 U/ml, 7-S collagen: 6.69 +/- 3.71 ng/ml) compared with levels in 41 healthy volunteers (pro-collagen type III: 0.53 +/- 0.13 U/ml, P < 0.001; 7-S collagen: 4.72 +/- 0.57 ng/ml, P < 0.001). Both levels were even more elevated during interferon therapy (procollagen type III: 1.36 +/- 0.58 U/ml, P < 0.05; 7-S collagen: 8.77 +/- 4.68 ng/ml, P < 0.01). On the other hand, serum procollagen type III and 7-S collagen levels were both decreased during prednisolone treatment (procollagen type III: 0.62 +/- 0.13 U/ml, P < 0.001; 7-S collagen: 4.66 +/- 1.69 ng/ml, P < 0.01). These preliminary data suggest that interferon alpha enhanced, but prednisolone inhibited the turnover of procollagen type III and IV in the liver. Further study is required to interpret this observation in relation to anti-fibrotic therapy.