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BACKGROUND: Interferon (IFN) therapy is effective in 20-40% of patients with chronic hepatitis C, but the relationship between histological changes and the response to interferon is still unclear. We investigated the long-term histological prognosis and the changes of serum fibrosis markers after interferon therapy relation to the response. METHODS AND RESULTS: One hundred and eighteen patients with chronic hepatitis C who received interferon therapy were divided into four groups based on the detection of viremia and the serum alanine aminotransferase (ALT) level after treatment. A histological examination was performed by using the histological activity index and the criteria of the METAVIR score. Serum fibrosis markers were used to measure the levels of hyaluronic acid and type IV collagen 7s. Responders, whose serum ALT levels became normal after treatment, demonstrated histological improvement. Histological improvement was more rapid in sustained virological responders with hepatitis C virus (HCV) RNA seronegativity than in biochemical responders with HCV-RNA seropositivity. Only sustained virological responders exhibited histological cure. In partial responders, whose serum ALT levels decreased to less than twice the upper of normal, and non-responders whose serum ALT levels were not reduced, liver fibrosis was unchanged or showed progression. Serum fibrosis markers increased with progression of the histological stage and varied depending on the response to interferon. CONCLUSION: Normalization of serum ALT levels after interferon therapy led to a histological improvement, and that with viral clearance achieved histological cure. Serum fibrosis markers were useful indicators for long-term according to the response of IFN therapy.

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TT virus (TTV) can replicate not only in the liver but also in hematopoietic cells. To investigate the possible influence of TTV infection on the hematological parameters of patients with chronic liver disease, serum samples from 581 patients with chronic, viral or non-viral liver disease were tested for TTV DNA by polymerase chain reaction (PCR). PCR was performed by two distinct methods using N22 primer pairs (N22 PCR), which detect primarily the four major TTV genotypes (1-4), and using genotype 1-specific primers (Genotype 1-PCR). Thirteen hematological parameters measured in routine laboratory tests were compared among the patients who were positive or negative for TTV infection. The platelet count was significantly lower in the N22 PCR-positive group than in the N22 PCR-negative group among the 189 patients with chronic hepatitis C (CH-C) and among the 40 patients with chronic hepatitis without serological markers of ongoing hepatitis B virus or hepatitis C virus infection (CH-NBNC) (P<0.0001, P<0.005, respectively). Similarly, among the patients with CH-C or CH-NBNC, the platelet count was significantly lower in the TTV genotype 1-positive group than in the TTV genotype 1-negative group (P<0.001, P<0.05, respectively). The reduced platelet count of the TTV DNA-positive group was observed in every fibrotic stage of chronic hepatitis (F0-F3). These results suggest that infection of TTV of certain genotypes including genotype 1 may worsen the thrombocytopenia of patients with CH-C or CH-NBNC, irrespective of the degree of hepatic fibrosis.

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The sera of 38 patients with nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH), were tested for TT virus (TTV) DNA by polymerase chain reaction (PCR) using three different primer pairs (UTR PCR, N22 PCR and genotype-1 PCR), and various histological features of the liver biopsy specimens were compared among those who were positive or negative for TTV infection. By UTR PCR which detects all TTV genotypes, TTV DNA was detected in 37 (97%) of the 38 patients. In contrast, N22 PCR which detects primarily TTV genotypes 1-4, detected TTV DNA in 18 patients (47%). In the liver biopsy specimens, moderate to many acidophilic bodies, moderate to marked focal/spotty necrosis of hepatocytes and marked stellate, pericellular or perivenular fibrosis were observed significantly more frequently among those who were positive for TTV DNA by N22 PCR, than among those who were negative by N22 PCR. Twelve patients (32%) were positive for TTV genotype 1. Moderate to marked vacuolation of nuclei, moderate to many acidophilic bodies, and moderate to marked focal/spotty necrosis as well as marked stellate, pericellular or perivenular fibrosis were found significantly more frequently in the TTV genotype 1-positive group than in the TTV genotype 1-negative group. These results suggest that certain TTV genotypes including genotype 1 influence the necrosis and inflammation of hepatocytes and liver fibrosis in NAFLD patients.

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The aim of this study was to investigate the effect of various medications other than interferon (IFN) on liver fibrosis in chronic hepatitis C (CH-C) patients, and the results were compared with those obtained in CH-C patients without therapy. Fifty CH-C patients (32 men and 18 women; mean age 58.5 years) without previous IFN therapy, who randomly received medicines other than IFN such as Stronger Neo-Minophagen C, Ursodeoxycholic acid and a herbal medicine, Sho-saiko-to (TJ-9) (Group I), and as a control group, 45 CH-C patients (27 men and 18 women; mean age 56.6 years) without therapy (Group II) were examined. All patients had persistent alanine aminotransferase (ALT) elevation more than 6 months before this study and were also subdivided into three subgroups according to different pattern of ALT during the observation period, i.e. (a): persistently ALT<60 IU/l (below about twice the upper limit of normal range); (b): persistently ALT>/=60 IU/l; and (c) other than (a) and (b). All patients were biopsied twice before starting this study and during observation period and the liver fibrosis was compared between them by staging in each case. When the fibrosis stage was the same between two specimens, we determined whether the degree of fibrosis had improved or worsened by computed image analysis. Blood tests for fibrosis marker, serum aminoterminal peptide of type III procollagen (P III P) and liver enzyme such as albumin (Alb) and zinc turbidity test (ZTT) levels, and platelet (Plt) counts were also examined on the two times of liver biopsy. As a result, there were no significant differences in fibrotic improvement rate when assessed by both staging only and staging together with fibrotic ratio, determined by computed image analysis and also in yearly change of P III P (P/Y) and fibrosis (F/Y), the changed rate of Alb, ZTT levels and Plt counts between Group I and Group II, except for P/Y in subgroup (a) which was rather higher in Group I than in Group II. There were also no significant relationship between the changes of histological activity and fibrosis staging in both groups. In conclusion, other medications than IFN could not significantly improve both liver fibrosis and its associated laboratory data irrespective of ALT levels in CH-C patients as compared to the control group during average 3 years' follow-up period.

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The sera of 43 patients with alcoholic liver disease (ALD) who abstained from alcohol for 4 weeks, were tested for TT virus (TTV) DNA by polymerase chain reaction (PCR) using three different primer pairs (UTR PCR, N22 PCR and genotype-1 PCR). The clinical course of the TTV DNA-positive and -negative groups was compared. By UTR PCR which detects all TTV genotypes, TTV DNA was detected in 40 patients (93%). N22 PCR which detects primarily TTV genotypes 1-6, detected TTV DNA in 17 patients (40%). The alanine aminotransferase (ALT) level 4 weeks after the start of abstinence was significantly higher and the rate of change in ALT {[(ALT on admission-ALT 4 weeks after abstinence)/(ALT on admission)]x100} was lower in the patients who were positive by N22 PCR, than in those who were negative by N22 PCR. Twelve patients (28%) were positive for TTV genotype 1. In the TTV genotype 1-positive group, the ALT 4 weeks after the start of abstinence was significantly higher, and the improvement rates of ALT, gamma-glutamyl transpeptidase and alkaline phosphatase levels were lower than those in the TTV genotype 1-negative group. These results suggest that certain genotypes of TTV may interfere with the improvement of liver function following the start of abstinence in ALD patients.

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Four of 107 samples obtained from hepatitis C virus (HCV) carriers showed lower HCV core antigen levels in a fluorescence enzyme immunoassay (FEIA) than expected from corresponding HCV RNA levels. Nucleotide sequencing revealed a mutation in the HCV core region (Thr49Pro) that appears to have reduced the FEIA sensitivity.

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The aim of this study was to evaluate the antifibrotic effect of interferon (IFN)-alpha in chronic hepatitis C (CH-C) patients with no response to IFN-alpha therapy. We studied 76 patients (46 men, 30 women; mean age, 55.6 years) who received IFN-alpha intramuscularly, at a total close of 480 to 880MU for 6 months (group A). As a control group, we studied 50 patients (32 men and 18 women; mean age, 58.5 years) with CH-C who received medication other than IFN (ie, Strong-Neo-Minophagen C, ursodeoxycholic acid, and a herbal medicine, Sho-saiko-to [TJ-9]) and who had persistent alanine aminotransferase (ALT) elevation (group B). All patients were subdivided into three subgroups according to different patterns of ALT changes during the observation period, ie, (a) persistent ALT level < 60IU/ 1 (below about twice the upper limit of the normal range), (b) persistent ALT level > or = 60IU/1, (c) ALT levels other than (a) and (b). Liver biopsy was performed within 6 months prior to IFN therapy and more than 6 months after IFN therapy, while two liver biopsies were performed during therapy in group B. Liver fibrosis was compared between two specimens by staging. When the fibrosis stage was the same in the two specimens, we determined whether the fibrosis had improved or worsened by comparing the fibrotic ratio, ie, the ratio of the area of fibrosis to the area of the entire liver tissue specimen, calculated using computed graphic software. Serum aminoterminal peptide of type III procollagen (PIIIP) levels were measured on the day of the liver biopsy and their mean yearly changes were compared between the two groups. Improvement of liver fibrosis was found in 12% to 30% of patients in each ALT subgroup and in 24% of all patients in group A and there were no significant differences in liver fibrosis in comparison with findings in of group B when assessed by staging alone. However, these percentages rose to 59% to 75% and 66%, respectively, when liver fibrosis was assessed by the fibrotic ratio together with staging, resulting in a significant difference in fibrosis between groups A and B in total (P < 0.01). The mean yearly changes in serum PIIIP levels in each subgroup and in all patients in group A were below zero, indicating a tendency to improvement of fibrosis after IFN therapy, while these changes in group B were all above zero, except for subgroup (c). Improvement of fibrosis after IFN therapy was found in 15 of 24 patients (64%) whose ALT changes had the same pattern before and after IFN therapy, although no significant difference was noted between improved and worsened patients. These results suggest that IFN-alpha may have an antifibrotic effect even in CH-C patients with no overt response to IFN-alpha therapy, compared with the effect of medications other than IFN.

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BACKGROUND/AIMS: Chronic hepatitis C virus carriers may have repeatedly normal alanine aminotransferase activity despite detectable viremia and histological hepatitis. We aimed to evaluate the effect of interferon treatment in these cases. METHODOLOGY: Twelve patients with persistently normal alanine aminotransferase levels at least 6 months before therapy were treated with recombinant interferon (IFN)alpha-2b for 6 months, totaling 840 MU in amount. Alanine aminotransferase levels were measured monthly during treatment and after treatment withdrawal, and HCV-RNA levels were measured by polymerase chain reaction before treatment, and 6 and 12 months after treatment withdrawal. RESULTS: At treatment withdrawal, HCV-RNA levels had significantly decreased and HCV-RNA disappeared in 9 of the 12 patients by polymerase chain reaction. At 6 months after treatment withdrawal, HCV-RNA reappeared in 6 of the 9 patients whose HCV-RNA was negative at treatment withdrawal. Over all, only 4 of the 12 patients (33%) were sustained virological responders (HCV-RNA is negative more than 6 months after treatment withdrawal). Pre-treatment HCV-RNA levels in a sustained virological responder was significantly lower than that of transient and non-responders (4.9 +/- 1.6 vs. 7.7 +/- 1.6 log10[copies/ml], p < 0.05). Of 8 patients who did not achieved sustained virological response, alanine aminotransferase levels had transiently increased above normal during treatment in one patient and after treatment withdrawal in 6 patients; however, in the remaining one patient abnormal values have continued from 8 months after treatment withdrawal till now for 24 months. CONCLUSIONS: In patients with chronic hepatitis C with normal alanine aminotransferase levels, the response to interferon therapy was by no means satisfactory. However, if it would be used in cases with the lower pre-treatment HCV-RNA levels with careful attention to a transient alanine aminotransferase elevation, the more a sustained virological response might be expected.

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A new cell line, Yumoto, derived from a squamous cell carcinoma of the uterine cervix, was established from serially transplanted tumor tissues in nude mice. Monolayer cultured cells were polygonal and formed pavement-like sheet. They showed a piling-up tendency and were devoid of contact inhibition. Electron micrographs demonstrated the presence of microvilli on the cell surface, abundant tonofilaments in the cytoplasm, and the connection with desmosomes. These electron micrographical characteristics of Yumoto cells were consistent with those of squamous cell origin. Yumoto cells were highly tumorigenic in BALB/c nude mice and produced a well-differentiated squamous cell carcinoma of keratinizing type which closely resembled to the original tumor tissues in nude mice. The presence of HPV DNA was examined using polymerase chain reaction and Southern blot analysis, but no known types of HPV DNA could be detected. Exons 2 through 11 of the p53 gene were analyzed by direct DNA sequencing, revealing a homozygous mutation at codon 281 in exon 8, GAC to CAC (Asp-->His). Furthermore, physical p53-gene deletion was demonstrated by dual-color fluorescence in situ hybridization. This cell line is useful for studying the carcinogenesis of cervical carcinoma and for investigating the biological characteristics of a HPV-negative and mutated p53 squamous cell carcinoma of the uterine cervix.

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We have proposed that hepatitis C virus should be classified into eleven genetic groups (types) which further divide into more than 80 genotypes (subtypes). However, only eight genetic groups (1-6, 10 and 11) have been defined on the basis of the full-length sequence. Hence, the entire nucleotide sequences of three HCV isolates in genetic groups 7-9 have now been determined. Phylogenetic analysis over the full-length sequences of these three isolates, along with 30 more in the other eight genetic groups, indicated that genetic groups 6-9 and 11 have bifurcated from a common branch and groups 3 and 10 from another. In the former branch groups 7 and 11, and groups 8 and 9, are closely related. Consequently, HCV can be classified into either eleven (1-11) or six groups (1; 2; 3 and 10; 4; 5; 6-9 and 11), allowing a clear separation of group and genotype similarity within the NS5b region or a subregion of 1093 nt. When pairwise comparison of 1093 nt in the NS5b sequence was performed on 106 HCV isolates of 36 genotypes in eleven genetic groups, they were classified into either eleven (1-11) or six (1; 2; 3 and 10; 4; 5; 6-9 and 11) genetic groups. However, group and genotype similarities were not clearly separable in either classification. The overlapping range was smaller using the classification into eleven genetic groups as compared to six genetic groups (2.7 vs 4-7%). These results indicate that HCV might not have evolved in the two-tiered fashion, at least in a strict sense.

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It has been well documented that caspase-1 (interleukin-1beta-converting enzyme, ICE) and its related cysteine proteinases such as caspase-3 (CPP32, apopain) and caspase-2 (ICH-1L) play important roles in apoptosis. In the present study, these genes were inserted into an inducible eukaryotic expression vector, pMSG, and transfected into NIH 3T3 mouse fibroblasts. The expression of caspases-1 and -3 was effectively induced by treatment with dexamethasone (Dex). The expression of caspase-2 was elevated in the transfected cells without treatment with Dex but was not further stimulated by Dex. High expression of these proteases alone induced neither apoptosis-like cell death nor any morphological change. However, the expression of caspase-1 but not of caspase-2 or -3 enhanced chemosensitivity toward cytotoxic anticancer drugs such as aclarubicin, epirubicin, adriamycin, nimustine and ifosfamide. It is thus concluded that caspase-1 mediates cytotoxic effects of these drugs.

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Immunocompromised or malnutritional hosts are high risk group of pulmonary tuberculosis. Chronic liver disease especially decompensated cirrhosis of the liver is one of the risk group for this infection. When ascites or pleural effusion developed in patient with hepatic cirrhosis, complication of pulmonary tuberculosis must be considered. In such condition, drug metabolism was impaired so that anti-tuberculous drugs should be used carefully, but in almost cases except decompensated cirrhotic patients are tolerable for standard anti-tuberculous combination therapy and they could be cured. Hepatitis C virus infection is common in patients with old pulmonary tuberculosis because many of them were infected Hepatitis C virus at the time of blood transfusion for pulmonary resection or thoracoplasty. In such condition recurrence of pulmonary tuberculosis is rare but probability of recurrence must be considered when they developed decompensated cirrhosis.

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The effects of anticancer drugs on cell growth have been investigated by MTT colorimetric assay using mouse and rat cultured cells transformed by various oncogenes and tumor viruses. Aclarubicin, mitomycin C and 1-hexylcarbamoyl-5-fluorouracil showed higher growth-inhibitory activities toward transformed cells than those toward the normal counterparts. Nimustine, bleomycin and 5-fluorouracil also showed selective growth-suppressive activities toward transformed cells except for a few cell lines. ras-oncogene-transformed cells were more sensitive toward 5-fluorouracil and 1-hexylcarbamoyl-5-fluorouracil than the normal parent cells and other transformed cells. These drugs would thus be effective in the chemotherapy of ras-induced cancer.

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A second-generation method of genotyping hepatitis C virus (HCV) was developed by the polymerase chain reaction (PCR) with sense as well as antisense primers deduced from the core gene. HCV RNA specimens extracted from sera were reverse-transcribed and amplified with universal primers in the first round of PCR to obtain fragments of 433 base pairs representing nucleotides 319-751. In the second round of PCR, portions of PCR products were amplified separately with sense and antisense primers specific for each of the five common genotypes prevailing across the world, i.e., I/1a, II/1b, III/2a, IV/2b and V/3a. The specificity of the method was verified by a panel of 177 HCV isolates of various genotypes in the genetic groups 1-9. It allowed clear differentiation of genotype I/1a from II/1b which was not always accomplished by the previous method. When 501 sera from blood donors and hepatitis patients with HCV viremia from various countries were genotyped by the second-generation method, 478 (95.4%) were classified into the five genotypes. HCV RNA samples from 23 (4.6%) sera were not classifiable into any of the five common genotypes and, by sequence analysis, 22 were found to be of four genotypes in group 4 and one of genotype 1c in Simmond's classification.

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Hepatitis C virus (HCV) isolates from 126 hepatitis patients in Jakarta, Indonesia were genotyped by PCR with genotype-specific primers deduced from the HCV core gene. Fifty-five isolates (44%) were classified as genotype II/1b, 15 (12%) as 1c, 33 (26%) as III/2a, and 1 (1%) as V/3a, while the remaining 22 (17%) were not classifiable into any of the five common genotypes (I/1a, II/1b, III/2a, IV/2b and V/3a) or 1c. Sequences of a part of the NS5b region [1093 bp (nucleotides 8279-9371)] of the 22 isolates of unclassifiable genotype were subjected to pair-wise comparison and phylogenetic analysis along with those of 62 isolates of 25 genotypes in nine genetic groups. Seven of the isolates were classified into 2e and two into 2f, representing novel genotypes in genetic group 2, while ten and three were classified into two new genetic groups, 10 and 11, respectively, and their genotypes were provisionally designated 10a and 11a. The isolates of genotype 10a (JK049) and 11a (JK046) were sequenced in full. Comparison of 24 HCV genomes including those of JK049 and JK046, over the entire genome and subgenomic regions, supported the classification of HCV into 11 genetic groups.

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Interleukin-6 (IL-6) is a pleiotropic cytokine that is not only a mediator in major immunologic reactions but also a growth factor of keratinocytes. We studied the IL-6 secretion in vitro of 15 human cell lines derived from both squamous cell carcinoma (SCC) and adenocarcinoma of the uterine cervix. Four of the eight well differentiated SCC secreted a large amount (> 1500 pg/48 h/10(6) cells) of IL-6 in nude mice. In contrast, poorly differentiated SCC cell lines and all of the 7 adenocarcinoma cell lines secreted a small amount (< 500 pg/48 h/10(6) cells of IL-6). The expression of IL-6 mRNA of the cell lines correlated well with their IL-6 secretion potential. However, the expression of IL-6 receptor did not correlate with the IL-6 secretory potential. We also studied the IL-6 secretion of freshly isolated normal squamous epithelium and of dysplastic epithelium. In culture, two normal squamous epithelia secreted a large amount (> 2000 pg/48 h/10(6) cells), whereas 8 dysplasia epithelia secreted an extremely small amount (< 10 pg/48 h/10(6) cells). About one-third of patients with SCC had a raised serum IL-6 value. IL-6 production may help to differentiate between SCC and adenocarcinoma of the uterine cervix. IL-6 regulation seems to change in the course of SCC carcinogenesis.

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We sequenced the entire genome of an Italian isolate of hepatitis C virus: the first full-length sequence for the genotype 2c. We report hereby its characteristics and differential detection of 2c isolates using PCR.

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The effects of a synthetic serine protease inhibitor, FOY-305, on the proliferation of normal and transformed mouse fibroblasts were investigated in vitro by MTT colorimetric assay. FOY-305 inhibited the growth of normal NIH3T3 cells and their src- and erbB2-transformed cells with a half maximum inhibitory concentration (IC50) of 1-1.2 mg/ml whereas it suppressed the growth of ras-transformed cells more effectively (IC50 was 0.5-0.6 mg/ml). Flow cytometric analysis using synchronized NIH3T3 cells has shown that the growth-inhibitory activity of FOY-305 was due to the suppression of G(1)/S transition. The synergistic effects between FOY-305 and traditional anticancer drugs were also investigated by the MTT assay and the results showed that FOY-305 significantly enhanced the antiproliferative activities of 5-fluorouracil (5-FU), 1-hexylcarbamoyl-5-fluorouracil (HCFU), 7-ethyl-1-hydroxy-7-ethyl-10-[4-(1-piperidino)-1-piperidino] camptothecin (SN-38), pirarubicin (THP) and 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU).

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Malignant fibrous histiocytoma is the most frequent soft tissue sarcoma. However, the pathogenesis still remains unclear, because there are very few human malignant fibrous histiocytoma cell lines available for precise cellular study. In this study, a human malignant fibrous histiocytoma cell line (MMF-1) was established from the pulmonary metastatic lesion of a 55-year-old man with malignant fibrous histiocytoma. Human cell line MMF-1 and its heterotransplanted tumor had almost the same characteristics as the original tumor morphologically and immunohistochemically. This cell line is expected to be a useful for studying the pathogenesis of malignant fibrous histiocytoma. The cloned cell lines (MMF-2 and MMF-3) also consisted of spindle-shaped, polygonal, and multinucleated giant cells, meaning that the fibroblast-like cells, histiocyte-like cells, and multinucleated giant cells seen in malignant fibrous histiocytoma were derived from a single tumor cell. Human cell line MMF-1 produced inflammatory cytokines, such as tumor necrosis factor-alpha, macrophage colony-stimulating factor, interleukin-8, and monocyte chemotactic and activating factor, that might be involved in the morphogenesis of malignant fibrous histiocytoma. Furthermore, the results of the analysis of human cell line MMF-1 suggested that malignant fibrous histiocytoma originated from a poorly differentiated fibroblast.

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A human tumor xenograft model for cancer cachexia was established by growing a uterine cervical carcinoma, Yumoto, in nude mice. The tumor transplanted into the mice induced severe body weight loss (30% of body weight) when the tumor weight was only 1 g. In addition, other indicators for cachexia, such as adipose tissue and muscle wasting and hypoglycemia, were also observed in the tumor-bearing mice, suggesting that this is a proper model for experimental cachexia induced by a human tumor. We then examined the association of this model with various cytokines, such as tumor necrosis factor alpha, interleukin (IL)-1alpha, IL-1beta, IFN-gamma, IL-6, and leukemia inhibitory factor, and identified human IL-6, which was produced by the tumor cells, as a mediator of cachexia. A neutralizing antibody against hIL-6 administered to the mice after the development of cachexia symptoms significantly improved body weight loss, adipose tissue wasting, hypoglycemia, acute phase reaction, and leukocytosis, although it did not suppress the tumor growth. These results demonstrate that the hIL-6 produced by the tumor cells is an essential mediator of the cachexia induction in this model.

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