Yusei Ikeda

Quantitation of α‐fetoprotein and albumin messenger RNA in human hepatocellular carcinoma

To analyze gene expression of α‐fetoprotein (AFP) and albumin in hepatocellular carcinoma (HCC), messenger RNAs (mRNAs) of these proteins in six human hepatoma cell lines and in 30 cases of HCC were quantitatively analyzed by competitive reverse transcription (RT) followed by polymerase chain reaction (PCR). The transcriptional levels of both AFP and albumin genes in HepG2 and Huh 7 cell lines were 1010 copies/μg RNA, in contrast to approximately 105 copies/μg RNA in HLE and HLF cell lines. AFP and albumin mRNA levels in three normal livers were 105 and 1010 transcripts/μg RNA, respectively. In 30 cases with HCC AFP mRNA level in neoplasm was 10 to 105‐fold enhanced as compared with that of nonneoplastic portion, and correlated with serum AFP level and tumor size (P < .01). In contrast, albumin mRNA level was not reduced in the neoplasms presenting enhanced AFP mRNA levels, indicating that AFP and albumin gene expression in situ is not necessarily mutually exclusive. Prospective analysis revealed that an increased serum AFP was shown at the time of recurrence among patients with enhanced AFP mRNA levels in neoplasm only, indicating that AFP mRNA levels in neoplasm could be a clinically predictable tool.

A multi-center double-blind controlled trial of ursodeoxycholic acid for primary biliary cirrhosis.

SummaryA multi-center double-blind controlled trial of ursodeoxycholic acid (UDCA) for treatment of primary biliary cirrhosis (PBC) was carried out. Twenty two and 23 patients were treated with 600mg/day UDCA and placebo, respectively, for 24 weeks. In UDCA - treated patients, fall of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma glutamyltranspeptidase activities started within 4 weeks after start of the trial and continued throughout the trial period. The serum IgM level fell in 7 UDCA-treated patients examined but not in 10 placebo-treated patients examined. Serum bilirubin concentration showed no significant change at the end of the study in either of UDCA- and placebotreated group of patients. There was no significant difference between these two groups with respect to the frequency of improvement of pruritus. In UDCA-treated patients, serum bile acid composition changed markedly, though its concentation showed no significant change. The percentage of total bile acid which ursodeoxycholic acid took up increased, whereas those which cholic acid, chenodeoxycholic acid and deoxycholic acid took up were decreased.

Uneven distribution of enzymatic alterations on the liver cell surface in experimental extrahepatic cholestasis of rat.

Abstract The effect of bile duct ligation on enzyme activities in the subfractions of the rat liver plasma membrane was investigated. Two subfractions were isolated from the rat liver plasma membrane by homogenization and subsequent centrifugation in a discontinuous sucrose gradient. The light subfraction contained fragments of the bile canalicular surface and the heavy fraction contained fragments of the sinusoidal and lateral surfaces of the hepatocyte. Bile duct ligation decreased NaK ATPase and Mg-ATPase activity in the light subfraction, whereas it had no significant effect on these enzyme activities in the heavy fraction. Leucyl-β-naphthylamidase activity was reduced and alkaline phosphatase activity was increased in both subfractions. These facts suggested that ligation of the bile duct led to loss of the secretory polarity of the liver cell. The in vitro effects of some bile acids on the membranebound enzymes in the light subfraction were investigated, and a possible involvement of chenodeoxycholic acid in the alteration of enzyme activities in the bile canalicular membrane was suggested.

Naturally occurring anti-interferon-α2a antibodies in patients with acute viral hepatitis

The occurrence of antibodies against recombinant human interferon‐α2a (IFN‐α2a) in patients with acute viral hepatitis (AVH) was examined by ELISA. Naturally occurring IgG anti‐IFN‐α2a were found in 50% of patients with type A, 50% of those with type B and in 8.3% of those with non‐A, non‐B AVH. The corresponding frequencies of IgM antibodies were 80%, 30% and 33.3%, respectively. IgM anti‐IFN‐α2a were found more frequently in patients with AVH type A than in normal control subjects (P < 0.01). Anti‐IFN‐α2a were detectable at the highest frequency 3 weeks after acute onset and then became negative. An absorption experiment revealed that IgM anti‐IFN‐α2a did not cross‐react with recombinant human IFN‐α2b. Immunoblotting analysis confirmed the binding of antibodies to IFN‐α2a. Sera positive for IgG and/or IgM anti‐IFN‐α2a were unable to neutralize IFN‐α2a. The appearance of anti‐IFN‐α2a was not correlated with disease severity. There was no evidence to suggest that anti‐IFN‐α2a impaired the elimination of hepatitis virus. This is the first study to demonstrate the occurrence of anti‐IFN‐α2a in patients with AVH. Detection of anti‐IFN‐α2a may be useful for clarifying any underlying immune events in various diseases.

Characterization of Rose Bengal binding to sinusoidal and bile canalicular plasma membrane from rat liver

The binding of Rose bengal, a model organic anion, to sinusoidal and bile canalicular membrane fractions isolated from rat liver was compared. The fluorescence change of Rose bengal after being bound to liver plasma membranes was utilized for measuring the binding. The dissociation constants (Kd = 0.1-0.12 microM) and the binding capacities (n = 11-15 nmol/mg protein) for Rose bengal are comparable between the two membrane fractions, although the n value for sinusoidal membrane is somewhat larger than that for bile canalicular membrane. The Rose bengal binding to both membrane fractions was inhibited by various organic anions at relatively low concentrations, i.e., the half-inhibition concentrations (IC50) for Indocyanine green, sulfobromophthalein, Bromophenol blue and 1-anilino-8-naphthalene sulfonate were 0.1, 100, 1.5-2.5 and 100 microM, respectively, while taurocholate did not inhibit the Rose bengal binding to either membrane fraction at these low concentration ranges. The type of inhibition of sulfobromophthalein and Indocyanine green for Rose bengal binding is different between the two membrane domains. That is, in sinusoidal and bile canalicular membrane fractions, these organic anions exhibit mixed-type and competitive-type inhibition, respectively. It was suggested that the fluorescence method using Rose bengal may provide a simple method for detecting the specific organic anion binding protein(s) in the liver plasma membrane.

Antibodies against sulphatide in sera from patients with autoimmune rheumatic diseases

We tested sera of patients with various autoimmune rheumatic diseases for the presence of antibodies against sulphatide (an acidic glycosphingolipid), identified as a target antigen for antibodies against the liver cell membrane. Thirty‐five percent (7/20) of patients with lupus in the active stage possessed anti‐sulphatide antibodies, whereas 10% (2/20) of those in the inactive stage and 20% (4/20) of those in the stationary stage possessed such antibodies. Moreover, 10%. (3/29) of patients with other autoimmune rheumatic diseases also possessed anti‐sulphatide antibodies. The level of anti‐sulphatide antibodies was significantly correlated with the levels of anti‐double‐stranded (ds) DNA antibodies (r = 0.634, P <0.001) and dextran sulphate‐binding IgG (r = 0.407, P < 0.001). The serum levels of antibodies against sulphatide were correlated with a history of seizures or psychosis in patients with autoimmune rheumatic diseases. Gels coupled with polyanionic dextran sulphate, monoanionic sulphanilic acid and DNA were shown effectively to adsorb anti‐sulphatide antibodies in the sera of patients with active systemie lupus erythematosus (SLE) and autoimmune chronic active hepatitis (AI‐CAH). These results suggest that the observed reactivity with sulphatide is due to the presence of antibodies capable of reacting with various anionic molecules in the sera of patients with autoimmune rheumatic diseases as well as those with AI‐CAH.

Down-regulation of prostaglandin E2 receptors in regenerating rat liver and its physiological significance.

The properties of prostaglandin (PG) E2 receptors in regenerating liver were studied using rat hepatocytes in primary culture. The control cells possessed stereo-specific PGE2 receptors with Bmax and Kd values, at 4 degrees C, of 526 fmol/mg protein and 6.5 nM respectively. In cells from regenerating liver after 70% hepatectomy, Bmax was reduced to 42-43% that of the controls; Kd did not change. Administration of indomethacin before surgery prevented Bmax reduction. These results indicate that PGE2, produced during the regeneration process, evoked cellular events and regulated the density of its receptors.

Potential for differential diagnosis of autoimmune pancreatitis and pancreatic cancer using carbonic anhydrase II antibody.

Objectives: Pancreatic ductal epithelia contain an abundance of carbonic anhydrase (CA), and the presence of antibodies to this enzyme has been described in autoimmune disorders. We previously found a small amount of an immunoglobulin G-like material in purchased CAII reagents, which led to pseudopositive reactions. Methods: We determined the optimum measurement conditions for detecting anti-CAII antibody using an enzyme-linked immunosorbent assay and sera from 140 patients with pancreatic diseases. Results: Compared with the prevalence of anti-CAII antibody in healthy subjects, a significantly higher seroprevalence of the antibody was detected in patients with autoimmune pancreatitis (AIP) (88.9%, P < 0.02), Sjögren syndrome (67.6%, P < 0.01), and alcoholic chronic pancreatitis (45.8%, P < 0.01). No positive results were obtained among patients with pancreatic cancer. Moreover, the antibody value obtained in the pancreatic cancer patients was actually lower than that obtained in healthy subjects. Conclusions: The anti-CAII antibody is probably not a specific marker of AIP because it was present at a higher frequency in the sera of patients with other pancreatic diseases. Nevertheless, the anti-CAII antibody may be a useful tool for the differential diagnosis of AIP and pancreatic cancer.

Genetic Analysis of the Capsid Region of Human Astrovirus Serotype 3 Isolated in Japan

The total capsid region of astrovirus serotype 3 was analyzed using five isolates including four from Japan and one from the United Kingdom. The nucleic acid and deduced amino acid sequences in the region were homologous (over 97%). However, the sequences of serotype 3 were different from those of serotypes 1, 2, 4, 5, 6 and 8, especially in the C terminus.

Detection of human immunodeficiency virus-1 DNA, RNA and antibody, and occult blood in inactivated saliva: availability of the filter paper disk method.

Eighty three samples of saliva were collected from 60 subjects who were asymptomatic carriers of AIDS related complex (ARC) and AIDS. They included hemophiliacs, homosexuals and heterosexuals. Occult blood, human immunodeficiency virus‐1 (HIV‐1) antibody and DNA were assayed after heat treatment at 56°C by strip method, particle agglutination and polymerase chain reaction (PCR), respectively. HIV‐1 RNA was assayed by reverse transcription (RT)‐PCR after heat treatment at 56°C or 90°C, or after application to filter paper disks with drying and heat treatment at 90°C. Positive results were found in 53% (occult blood), 73% (HIV‐1 antibody), 23% (HIV‐1 DNA, 56°C), 34% (HIV‐1 RNA, 56°C), 33% (HIV‐1 RNA, 90°C) and 25% (HIV‐1 RNA, 90°C, filter paper disk), respectively.