Yamamoto, Kazuhide

We have previously developed an enzyme-linked immunosorbent assay (ELISA) to measure stool decay-accelerating factor (DAF) and found that stool DAF concentrations were significantly elevated in patients with colorectal cancer, suggesting that the measurement of stool DAF may be valuable for the detection of colorectal cancer. In order to refine the assay for the measurement of stool DAF, we investigated 1) effects of centrifugation of stool samples, 2) effects of detergents, and 3) adequate combination of various anti-DAF monoclonal antibodies for the ELISA system using only monoclonal antibodies. We found that high-speed centrifugation could be omitted and that only the removal of large undigested food residues by centrifugation of short duration in a low-speed benchtop microcentrifuge sufficed to adequately prepare the stool samples. Addition of 2 detergents, octyl beta-glucoside and sodium deoxycholate, known to solubilize glycosyl-phosphatidylinositol-anchored proteins such as DAF, did not influence stool DAF values. By using 2 mouse anti-DAF monoclonal antibodies (clone 4F11 and 1C6), we were able to achieve a stable ELISA for the measurement of stool DAF using a uniform source of antibodies. The results should allow us to consistently apply the DAF assay for routine use in the detection of colorectal cancer.

The preS2 region of the hepatitis B virus (HBV) has been reported to have human polymerized albumin receptor (PAR) activity, which correlates with viral replication. Here, we studied the genomic sequence of the preS region from rare patients lacking PAR activity, despite active viral replication. PAR and DNA polymerase activity was identified in 178 HBe antigen-positive HBV carriers, and a significant correlation between 2 markers was shown, except in 2 hepatitis patients lacking PAR activity. Nucleotide sequences of the preS region of HBV from both patients were examined by direct sequencing of PCR products. In one patient, a 45-base deletion was found to overlap half of the putative polymerized human albumin binding site in the preS2 region. In the other patient, a point mutation at the first nucleotide of the start codon of the preS2 region of HBV was found. There was no such genomic change in the 3 control HBV sequences. These results indicate that the preS2 region is necessary for binding of polymerized human albumin, and this is the first report of naturally existing mutant virus with no or low PAR activity.

Phalloidin, a toxin from the plant Amanita phalloides, irreversibly polymerizes actin filaments and causes cholestasis. Three-dimensional structural changes induced by phalloidin in the bile canaliculi and the intra-acinar localization of these changes were studied in the rat liver by scanning and transmission electron microscopy. After 3 days of treatment, canalicular changes appeared mainly in zones 2 and 3 of Rappaport's acinus, but after 7 days of treatment changes occurred in bile canaliculi of the whole acinus. The changes in the bile canaliculi included tortuosity, saccular dilatation, loss of microvilli, bleb formation and elongation of canalicular side branches. Some side branches extended near to Disse's space, leaving only a thin cytoplasmic rim between the canalicular lumen and Disse's space. Kupffer cells were occasionally situated near such extended bile canaliculi and protruded their processes into the hepatic cord. These results suggest that bile canaliculi in zone 3 are more susceptible to phalloidin toxicity than those in zone 1 and that biliary constituents may leak from such altered bile canaliculi.

To characterize primary sclerosing cholangitis (PSC) in Japanese patients and its association with inflammatory bowel disease (IBD), 155 reported cases of PSC, including 6 cases of our own, were reviewed. The prevalence of IBD was less in Japanese PSC patients than in Western patients (23% versus 62-100%). Japanese PSC patients with IBD were younger (mean age, 33.1 versus 51.8 years) and were more often women (51% versus 36%) than those without IBD. Seventy-four percent of PSC patients with IBD had extensive colonic lesions, and 89% of those developed IBD simultaneously, with or prior to PSC. There were 3 cases of neutrophilic cholangitis among the PSC patients with IBD but none in those without IBD. Based on these observations, we speculate that there may be subtypes of PSC which differ pathophysiologically.