K. Kiyosawa

EFFECT OF HEPATITIS G VIRUS INFECTION ON CHRONIC HEPATITIS C

Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis throughout the world [1-3]. A new virus, tentatively called hepatitis G virus (HGV), was recently cloned and sequenced [4]. This virus is closely related to HCV in genomic structure; like HCV, HGV is transmitted through transfusion and may be associated with acute and chronic hepatitis [4-6]. We analyzed the role of HGV infection in patients with chronic hepatitis C, including patients treated with interferon-. Methods Patients We enrolled 189 patients with chronic hepatitis C who were seen at Shinshu University Hospital, Matsumoto, Japan (122 men and 67 women; mean age, 51.1 11.0 years). All patients were positive for antibody to HCV according to a second-generation assay and were negative for hepatitis B virus (HBV) surface antigen and antibody to human immunodeficiency virus. No patients had hepatocellular carcinoma or a history of alcohol intake exceeding 80 g/d. Of the 189 patients, 101 (66 men and 35 women; mean age, 50.0 11.2 years) had been retrospectively selected so that our sample would include all patients who had received a single course of interferon- therapy between October 1991 and December 1993. The remaining 88 (47%) patients (56 men and 32 women; mean age, 52.5 10.7 years) were consecutively selected from patients with chronic hepatitis C who had been followed for more than 1 year and had had liver biopsy within the same period but had not received antiviral therapy. Interferon- 2a had been administered at a dosage of 9 million U daily for 2 weeks, followed by 9 million U three times a week for 22 weeks (total dose, 720 million U). Treated patients had histologic examination within the 6 months before interferon- therapy was initiated and were followed for at least 6 months after therapy was completed. For all patients, serum samples were obtained at the time of liver biopsy and were stored at 70C until testing. For patients receiving interferon- therapy, serum samples were also collected just before therapy began, just after therapy was completed, and 6 months after therapy was completed. Serum alanine aminotransferase levels (normal range, 5 to 45 IU/L) were measured at liver biopsy and, in patients receiving interferon- therapy, at least once every 4 weeks during therapy and follow-up. The grade (extent of hepatic inflammation and hepatocellular destruction) and stage (degree of fibrosis) of liver histologic findings [7] were judged by three authors; the final diagnosis was established by consensus. Investigators involved in separate portions of the study were blinded to the results of other portions. Serologic Markers of and Molecular Assays for Hepatitis C Virus RNA Levels of HCV antibody, HBV surface antigen, and antibody to human immunodeficiency virus were measured by using commercially available second-generation enzyme-linked immunosorbent assays (Abbott Laboratories, North Chicago, Illinois). Serum levels of HCV RNA were measured by using nested reverse-transcription polymerase chain reaction (PCR) with primers in the 5 noncoding region [8] and were quantified by using the branched-DNA signal amplification assay [9, 10]. The detection limit of the branched-DNA assay was set at 105.7 equivalents/mL. Hepatitis C virus genotypes were tested by nested PCR using genotype-specific primers of core region [11] and were categorized according to the classification system of Simmonds and colleagues [12]. Serum Levels of Hepatitis G Virus RNA Levels of HGV RNA in serum were measured by using reverse-transcription PCR as described elsewhere [5]. Total nucleic acids were extracted from 50 mL of serum. After reverse transcription, 45 PCR cycles (for detection) or 35 PCR cycles (for quantitation) were done with primers in the putative NS5 region of the HGV RNA genome. The PCR products were analyzed by dot-blot hybridization with a 32P-labeled oligonucleotide probe. The sensitivity of this assay system was 20 RNA copies/mL of starting serum. Quantitative measurement of HGV RNA levels was done using standards of known HGV RNA levels. Statistical Analysis Statistical analyses were done using the Student t-test, the Mann-Whitney U test, the Wilcoxon rank-sum test, the chi-square test, the Fisher exact test, and the Somers D test. A P value of 0.05 or less indicated statistical significance. Results Clinical Features and Hepatitis C Virus Markers Twenty-one (11.1%) of the 189 enrolled patients were positive for HGV RNA. The rate of detection of HGV RNA was similar in the subgroup of 88 untreated patients (12.5%; 11 of 88) and the 101 patients who received interferon- therapy (9.9%; 10 of 101). Mean age and the number of men and women were also similar in the two subgroups. Thus, the clinical features of patients who had chronic hepatitis C could be compared with those of patients who had HCV and HGV infection without respect to interferon- therapy (Table 1). Patients with HGV infection were significantly younger than those without HGV infection. Other clinical features did not differ between HGV RNA-positive and HGV RNA-negative patients (Table 1). Table 1. Clinical Features in HGV RNA-Positive and HGV RNA-Negative Patients with Chronic Hepatitis C* Hepatitis C virus genotype and serum HCV RNA level were compared between the 21 patients with HGV RNA and the 52 patients without HGV RNA who were randomly selected from the 168 HGV-negative persons. The HCV genotypes and HCV RNA levels were distributed similarly in the two groups (Table 2). Table 2. Markers of HCV and Response of HCV to Interferon- in HGV RNA-Positive and HGV RNA-Negative Patients with Chronic Hepatitis C* Effect of Hepatitis G Virus Infection on Response of Hepatitis C Virus to Interferon- Therapy Of the 101 patients receiving interferon-, 36 had a sustained loss of HCV RNA and normalization of alanine aminotransferase levels; they were therefore considered to have responded to interferon-. The remaining 65 patients were positive for HCV RNA 6 months after completing therapy and thus were classified as nonresponders. The rate of HCV response to interferon- did not differ between patients with and those without HGV infection (Table 2). Response of Hepatitis G Virus to Interferon- Response of HGV to interferon- was analyzed in 9 of 10 patients with HGV infection. The serum HGV RNA level decreased during interferon- therapy in all 9 patients. The geometric mean titer of HGV RNA just after interferon- therapy (mean, 6.3 RNA copies/mL; range, 0.0 to 5000 RNA copies/mL) was significantly (P = 0.008; Wilcoxon rank-sum test) lower than the titer just before therapy (mean, 3200 RNA copies/mL; range, 20 to 1 000 000 RNA copies/mL). Two patients had a sustained loss of HGV RNA 6 months after discontinuation of therapy. In the remaining 7 patients, HGV RNA level increased after cessation of therapy to levels similar to those just before therapy. Thus, the sustained response rate of HGV (22%; 2 of 9 patients) was lower than but not significantly different from (P > 0.2; Fisher exact test) the sustained response rate of HCV (36%; 36 of 101 patients). However, a dichotomy was seen in the response to interferon-; two patients who had a sustained loss of HGV RNA did not clear HCV RNA, and three patients who had a sustained loss of HCV RNA did not clear HGV RNA. In the latter three HCV responders, alanine aminotransferase levels remained normal despite the reappearance of HGV viremia. Discussion Approximately 10% of patients with chronic hepatitis C are also infected with HGV [5, 6]. Although the precise routes of HGV transmission have not been established, this agent is parenterally transmitted through blood transfusion and exposure to shared needles during injection drug use [4]. In our study, the frequency of previous blood transfusion was similar in patients with HGV and HCV co-infection and patients with hepatitis C alone. The apparent link between HGV and HCV infections probably reflects common exposures and transmission patterns rather than a specific interdependence of the two agents. Patients with chronic hepatitis often harbor more than one hepatitis agent [4, 13-15], and important interactions between HBV and HCV have been documented [15]. The relation between HCV and HBV replication is reciprocal: Increasing replication of one agent can diminish the replication of the other. The key question underlying our study is whether coexistent HGV infection alters the level of viremia, clinical course, or treatment response of HCV infection. Although it is unclear whether our findings are applicable to non-Japanese persons, we found no evidence of such an effect on any of these variables. The following findings support this claim. First, the HCV RNA level in patients with HGV and HCV infection was the same as in patients with HCV infection alone. In addition, Nakatsuji and colleagues [5] reported that the serum level of HGV RNA did not differ between these two groups of patients. These two studies showed no evidence of unidirectional or bidirectional viral interference between HGV and HCV. Second, no evidence suggested that HGV infection increased the severity of hepatitis C. When patients with chronic hepatitis C were compared with those who had HCV and HGV infection, no differences were seen in the mean alanine aminotransferase level or liver histologic findings. Further, in five co-infected patients in whom HGV and HCV responses to interferon- therapy were dissociated, hepatic inflammation after discontinuation of therapy seemed to depend on HCV replication, not on HGV replication. These data suggest that HGV has limited pathogenicity compared with HCV, and they are consistent with previous observations that HGV-positive blood donors were no more likely to have elevated alanine aminotransferase than were HGV-negative donors [4]. Several viral and host factors have been reported to influence the response of HCV to interferon- [8, 16-18]. We found no effect of HGV on the HCV respo

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

Summary.  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.

Clinical evaluation of a new enzyme immunoassay for hepatitis B virus core‐related antigen; a marker distinct from viral DNA for monitoring lamivudine treatment

Summary. We aimed to assess the clinical performance of a newly developed chemiluminescence enzyme immunoassay (CLEIA) for the detection of hepatitis B virus (HBV) core‐related antigen (HBcrAg) in patients with chronic HBV infection. A total of 82 patients with chronic HBV infection and 167 HBV‐negative controls were studied. HBcrAg was measured by CLEIA with monoclonal antibodies to hepatitis B e antigen (HBeAg) and hepatitis B core antigen (HBcAg), and HBV DNA was measured by transcription‐mediated amplification assay (TMA) and in‐house real‐time detection polymerase chain reaction (RTD‐PCR). The HBcrAg assay detected viremia in 189 of 216 samples (88%) collected from 72 patients whilst the TMA assay detected viremia in 178 of the 216 samples (82%) (P = 0.019). The HBcrAg concentration correlated linearly with the HBV DNA concentration (P < 0.001) over a range which varied 100 000‐fold. The accuracy in the measurement of the patients’ HBV load obtained using the HBcrAg assay was not affected by the absence of hepatitis B e antigen from the serum or the presence of precore mutations in the HBV genome. In patients without anti‐viral drugs, changes in their serum HBcrAg concentration over time corresponded to their HBV DNA concentration. In six additional patients who were later treated with lamivudine, HBV DNA concentration declined more rapidly than their HBcrAg concentration. Three months after treatment commenced, the ratio of HBcrAg: HBV DNA had increased in all six patients (P = 0.031). The HBcrAg assay is a sensitive and useful test for the assessment of a patients HBV load. When monitoring the anti‐viral effect of lamivudine, HBcrAg provides a viral marker which is independent of HBV DNA.

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

Summary.  Thymalfasin (thymosin alpha‐1; Tα1) 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 Tα1 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 Tα1 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 Tα1 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 Tα1 treatment. Adverse event incidence was similar in the 1.6‐ and 0.8‐mg treatment groups. In conclusion, Tα1 at doses of 0.8 and 1.6 mg exhibits long‐term efficacy against hepatitis B with a good safety profile.

Serum collagen type IV for the assessment of fibrosis and resistance to interferon therapy in chronic hepatitis C

Sixty-nine patients with chronic hepatitis C (CH-C) were treated with interferon therapy, and serum collagen type IV (s-collagen IV) levels were measured by enzyme immunoassay to analyze the responsiveness to interferon therapy. Classified by the improved pattern of serum alanine aminotransferase levels after interferon administration, 23 patients were judged as sustained responders, 23 as transient responders, and 23 as non-responders. Fibrotic grades of the liver sample correlated statistically with the levels of s-collagen IV (P < 0.01). Pre-therapy s-collagen IV levels of sustained responders were significantly lower than those of the other responders, and only sustained responders showed a significant decrease of s-collagen IV levels after interferon therapy, in accordance with histologic improvement. Multivariate analysis showed that s-collagen IV and hepatitis C virus genotype were the most important factors affecting the response to interferon therapy of all variates. Thus, s-collagen IV is one of the most useful aids for the evaluation of liver fibrotic grade in CH-C and a potent predicting indicator for the responsiveness to interferon therapy.

A case of primary low grade mucosa associated lymphoid tissue (MALT) lymphoma of the oesophagus

We report a very rare case of primary low grade mucosa associated lymphoid tissue (MALT) lymphoma of the oesophagus. An 83 year old woman was referred to our hospital in June 1999 for further examination and treatment of oesophageal tumour. Although a physical examination and laboratory data showed no significant abnormalities, endoscopic observation revealed two slightly elevated submucosal tumour-like lesions of the oesophagus. Tissue specimens were obtained by endoscopic mucosal resection of the oesophagus using a cap fitted panendoscope. The lesions were composed of diffuse small atypical lymphoid cells—that is, centrocyte-like cells—which were stained with CD20, L26, BCL-2, and κ, but not with CD3, CD5, CD10, or cyclin D1. Monoclonality was detected by polymerase chain reaction analysis using the primer for CDR-3 of immunoglobulin H and diagnosed as low grade MALT lymphoma of the oesophagus. The tumours were considered to be completely resected and therefore additional treatment was not administered. The patient is alive and well 22 months after treatment and diagnosis.

Comparative Study of CA-50 (Time-Resolved Fluoroimmunoassay), Span-1, and CA19-9 in the Diagnosis of Pancreatic Cancer

The clinical diagnostic utility of CA-50 (time-resolved fluoroimmunoassay) and Span-1 was compared with that of CA19-9 by measuring their levels in sera from patients with pancreatic cancer and other diseases. In pancreatic cancer CA-50, Span-1, and CA19-9 showed similar positive rates (84%, 82%, and 81%, respectively). With regard to the ability to distinguish pancreatic cancer from chronic pancreatitis, however, the specificity of CA-50 and Span-1 was higher than that of CA19-9 (85%, 85%, and 79%, respectively). Despite the similar positive rates of CA-50 and Span-1 in pancreatic cancer, the correlation between these two markers was low. Thus, used in combination, they compensated for each other in the diagnosis of pancreatic cancer. In chronic liver diseases, serum levels of both CA-50 and Span-1 were correlated with that of biliary tract enzymes, alkaline phosphatase, and r-glutamyl transpeptidase. And these two markers were more affected by the biliary system than CA19-9, resulting in the significantly higher positive rates. In these diseases immunohistochemical study showed that all three markers were localized in the epithelial cells of the bile duct, with CA-50 and Span-1 showing a similar tissue distribution.

Hepatocarcinogenesis inhibition by caffeine in ACI rats treated with 2-acetylaminofluorene

The inhibitory effects of caffeine have been demonstrated on the development of various organs in animals. The purpose of the present study was to examine the inhibitory effect of caffeine on hepatocarcinogenesis and to determine the responsive dose of caffeine on hepatocarcinogenesis in young male ACI rats. Animals given a diet containing 2-acetylaminofluorene (2-AAF) for 12 weeks and then a basal diet and tap water containing caffeine for 18 weeks showed statistically significant decreases in the incidence, multiplicity (the number of hepatic tumors per rat) and histological grade compared with rats fed a diet containing carcinogen for 12 weeks followed by tap water alone. Dose-dependent inhibition of hepatocarcinogenesis by caffeine was also seen. The inhibitory effect of caffeine on hepatocarcinogenesis in rats was found when caffeine was administered during the initiation phase.

Analysis of T cell repertoire in the liver of patients with chronic hepatitis C

Many T cells infiltrate into the liver of patients with chronic hepatitis C (CH‐C). They are believed to play a crucial role in the immunopathogenesis of hepatic inflammation, but their clonality and specificity are unknown. The aim of this study was to clarify the characteristics of these T cells. We analysed the complementarity‐determining region (CDR)3 size lengths of T cell receptor (TCR) β‐chains by size spectratyping, and determined the sequences of Vβ CDR3 after subcloning Vβ‐specific polymerase chain reaction products. Spectratyping showed clonal expansions in all liver specimens, most of which showed more than two T cell clones. Moreover, many non‐clonal T cells also accumulated in the liver. Clonality of the T cells suspected by spectratyping was confirmed by CDR3 sequencing. Although the sequences revealed no whole CDR3‐shared clones among different patients, some common motif sequences were observed. Our data suggest that T cells are stimulated by several hepatitis C virus (HCV) epitopes, then accumulate in the liver of CH‐C patients. Shared motifs of expanded T cell clones suggest that they might recognize the same regions of HCV peptides, but have differences due to HCV peptide mutational changes. These clones might also interact with non‐clonal T cells and play a crucial role in the immunopathogenesis of CH‐C.

Case report: Pneumatosis cystoides intestinalis associated with post-surgical bowel anastomosis: a report of three cases and review of the Japanese literature.

We report three cases of pneumatosis cystoides intestinalis (PCI) occurring in association with post‐surgical bowel anastomosis. A 74‐year‐old man, a 58‐year‐old woman, and a 62‐year‐old woman were found to have PCI at the colonic side of a bowel anastomosis at 4 years, 3 years and 1 year after operation, respectively, for right colon carcinoma, although all were asymptomatic. They all had a positive anti‐nuclear antibody test and had received postoperative cancer chemotherapy. The clinical features of 123 cases of PCI reported in Japan between 1981 and 1995 were also reviewed. On the basis of the present and previous cases, we propose that post‐surgical anastomosis, cancer chemotherapy, and predisposition to collagen vascular disease might be responsible for the damage to intestinal mucosa that leads to the development of PCI.