Tetsuya Tachikawa

Altered mRNA expression of specific molecular species of fucosyl- and sialyl-transferases in human colorectal cancer tissues

Human colorectal cancers express various cancer‐associated carbohydrate determinants such as Lewis Y or sialyl Lewis A, suggesting a considerable alteration in glycosyltransferase activities occurring upon malignant transformation. We investigated the mRNA amounts of fucosyltransferase (Fuc‐T) and sialyltransferase (ST) isoenzymes, including Fuc‐T III, IV, V, VI and VII and ST‐3N, ST‐30 and ST‐4, in human colorectal cancer tissues by Northern blotting and RT‐PCR. Regarding fucosyltransferases, mRNA of Fuc‐T III and VI was not significantly altered, and only Fuc‐T IV mRNA showed a moderate increase in cancer tissues when compared with adjacent non‐malignant colonic epithelia taken from the same patient (273 ± 96%; p < 0.001). The moderate increase of Fuc‐T IV message may be related to an enhanced expression of Lewis Y in colon cancer tissues. In the ST isoenzymes, mRNA for ST‐3N remained unchanged, whereas that for ST‐4 decreased significantly in cancer tissues, to 32 ± 29%, (p < 0.005). The most remarkable finding was that the message of ST‐30 was prominently increased in cancer tissues compared with non‐malignant colorectal mucosa. When further investigated by quantitative RT‐PCR assays on a larger series of patients with colorectal cancers, the average increase in mRNA for ST‐30 was 459 ± 200% compared with that in adjacent non‐malignant epithelium (significant at p < 0.0001). The increase of ST‐30 message was more prominent in the cancer tissues strongly expressing sialyl Lewis A than in the cancer tissues expressing sialyl Lewis A only weakly or moderately (significant at p < 0.05). The marked increase in the message of ST‐30 is suggested to be related to an enhanced expression of sialylated carbohydrate determinants in colon cancer tissues including sialyl Lewis A, since the enzyme exhibited a significant activity against the type I chain carbohydrate substrate and produced the precursors for sialyl Lewis A synthesis, when its cDNA was expressed in Cos‐7 cells. Int. J. Cancer 71:556‐564, 1997

Highly sensitive urine-based enzyme-linked immunosorbent assay for detection of antibody to Helicobacter pylori.

Enzyme‐linked immunosorbent assay (ELISA) has been widely used for detection of Helicobacter pylori (H. pylori), but sample collection is often invasive, complicated, and expensive. Urine samples can be obtained noninvasively and are easier and safer to handle than serum samples. A urine‐based ELISA, if found to be accurate, would therefore be a useful alternative to serum‐based tests for H. pylori.

Clinical Usefulness of Urine‐based Enzyme‐linked Immunosorbent Assay for Detection of Antibody to Helicobacter pylori: A Collaborative Study in Nine Medical Institutions in Japan

Background. A urine‐based enzyme‐linked immunosorbent assay (ELISA) kit for detection of antibody to Helicobacter pylori has been developed in Japan. Urine samples can be obtained noninvasively and are easier and safer to handle than are serum samples. The aim of this study was to examine the clinical usefulness of this urine‐based ELISA kit.

A New Rapid Test for Detecting Anti–Helicobacter pylori Antibody Excreted into Urine

Background. Helicobacter pylori infection has been one of the most common infectious diseases in the world, whereas a gold standard for identifying its infection has not yet been established. The specific test will depend on the particular clinical, epidemiological, and scientific requirements. We recently developed a new type of rapid test to detect H. pylori antibody excreted into urine; the test requires only 20 minutes. The purpose of this study was to examine the accuracy of this rapid test.

ELEVATION OF AN ALPHA (1,3)FUCOSYLTRANSFERASE ACTIVITY CORRELATED WITH APOPTOSIS IN THE HUMAN COLON ADENOCARCINOMA CELL LINE, HT-29

We studied changes in the carbohydrate expression following apoptotic cell death induced by treatment with interferon (IFN)-γ and anti-Fas antibody using human colon adenocarcinoma HT-29 cells. An apoptotic cell death of HT-29 accompanied with typical DNA fragmentation was observed when the cells were cultured sequentially with IFN-γ and anti-Fas antibody. In flow cytometric analyses, the expression of Lex and Ley antigen was strongly and slightly enhanced, respectively, on the cell surface in accordance with the apoptosis. When the fucosyltransferase (Fuc-T) activities of the lysates from the treated cells were examined relative to those from untreated cells, a 2.5-fold increase of α(1,3)-Fuc-T activities and a slight increase of α(1,2)-Fuc-T activities were observed, but little or no increase of α(1,4)-Fuc-T activity was detected. In Northern blot analyses using probes for Fuc-T III, IV, V, VI and VII genes, strong RNA messages for Fuc-T III, V and/or VI and a weak RNA message for Fuc-T IV were detected in the untreated HT-29 cells. On the other hand, in the treated cells, the messages for Fuc-T III, V and/or VI were found to almost disappear and the 2.3 kb message for Fuc-T IV was observed to elevate 2.8-fold. Therefore, we suggest that the strongly increased expression of Lex antigen found on the HT-29 cell surface might be involved in the process of apoptosis, and that the enhancement of the antigen expression seems to result from the increased activity of α(1,3)-Fuc-T encoded mainly by the Fuc-T IV gene.

α2→3Sialyltransferase associated with the synthesis of CA 19‐9 in colorectal tumors

A novel assay method specific for α2→3sialyltransferase that seems to be responsible for the synthesis of CA 19‐9 antigen was developed and the levels of the enzyme in colorectal tumor tissues were measured and compared with the levels of α1→4fucosyltransferase and the CA 19‐9 antigen.

Development and Characterization of a Novel Anti-fucosylated Antigen Monoclonal Antibody YB-2 and Its Usefulness in the Immunohistochemical Diagnosis of Colorectal Cancer

A novel monoclonal antibody, YB‐2 was obtained after immunization of mice with fucosylated antigens isolated from human saliva. The antibody was demonstrated to react with Y (Fucα1→2Gal‐ β1→4[Fucα1→3]GlcNAcβ),Leb(Fucα1→2Galβ1→3[Fucα→4]GlcNAcβ)andHtype2 (Fucα→2Gal‐βl→4GlcNAcβ) antigens, but not with H type 1 (Fucα→2Galβ1→3GlcNAcβ), Le→ (Galβ1→3[Fucα1→4]GlcNAcβ), X (Galβ1→4[Fucα1→3]GlcNAcβ) or with non‐fucosylated antigens. Inhibition assays of YB‐2 antibody with such reactive antigens showed that YB‐2 antibody preferentially reacted with Y antigen. Formalin‐fixed and paraffin‐embedded sections prepared from normal and malignant colorectal tissues were examined immunohistochemically with YB‐2. The positive rates of staining with YB‐2 antibody were 88.6% in malignant and 12.0% in normal tissues. The expression of fucosylated antigens detected by YB‐2 antibody seemed to be correlated with survival among patients with primary colorectal cancer. Therefore, YB‐2 antibody could be useful as an immunochemical tool for diagnosis and evaluation of the prognosis of colorectal cancer.

On-site diagnosis of H. pylori infection by urine

We have recently developed an on-site diagnostic kit for H. pylori infection using urine (utilizing immunochromatographic method employing a nitrocellulose membrane coated by extracted H. pylori antigen). Accordingly, we investigated its usefulness in 155 consecutive dyspeptic patients using the 13C urea breath test as a gold standard and further compared its performance with two commercially available rapid diagnostic kits that use whole blood (Helisal Rapid Blood, and ImmunoCard H. pylori). As the results, the urine based on-site diagnostic kit provided 95.9% sensitivity and 87.9% specificity with 92.9% accuracy, which were comparable or even better than that of both rapid whole blood tests, suggesting its usefulness in screening of H. pylori infection.

The usefulness of anti-fucosylated antigen antibody YB-2 for diagnosis of hepatocellular carcinoma.

Levels of fucosylated antigens in sera from patients with liver diseases were examined by a newly developed sandwich-type enzyme immuno assay with the aid of anti-fucosylated antigen antibody, YB-2 which reacts simultaneously with Y, Leb and H type 2 antigens. When the cut-off value was set arbitrarily at mean [3 SD values of normal, 30 (69.8%) of the 43 patients with HCC, 14 (53.8%) of the 26 patients with liver cirrhosis (LC) and 24 (45.3%) of the 53 patients with chronic hepatitis (CH) were found to be positive, whereas all of the 30 samples from healthy controls were negative. The levels of α-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-II (PIVKA-II) in HCC were not correlated with those of YB-2 antigens. The positive rates of the combination YB-2 and AFP assay and YB-2 and PIVKA-II assay in HCC were significantly higher (83.7 and 86.0%, respectively) than that of the AFP and PIVKA-II combination (65.1%) which had been reported to be the best combination up to this time.

A Highly Sensitive Enzyme-Linked Immunosorbent Assay Method for Blood-Group A and B Transferase Activities using H-Disaccharide.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) method using H-disaccharide (Fucα1, 2 Galβ-BSA) was developed to measure α (1, 3)-N-acetyl-D-galactosaminyltransferase (A-transferase) and α (1, 3)-D-galactosyltransferase (B-transferase) activities in human plasma. Fifty-eight and 43 plasma samples from various subgroups of A and B, respectively, were assayed with UDP-GalNAc or UDP-Gal in microtiter plate wells coated with H-disaccharide covalently attached to BSA, and the products formed by transferases were detected by horse-radish peroxidase-conjugated anti-A or anti-B monoclonal antibody. Transferase activities measured by this method were compared with those by the conventional method based on the conversion of O-type erythrocytes into A- or B-type erythrocytes.The present method allowed detection of weak enzyme activities from A- and B-type subgroups and variants such as A2, A3, AXB, cisAB (for A-transferase) and AB3, BX, cisAB (for B-transferase). In contrast, these activities could not be detected at all by the conventional method. Further, enzyme activities of all samples (n=101) except one from AX were quantified by this ELISA method, whereas only 21% (12/58) of the A-transferase and 58% (25/43) of B-transferase from the same individuals were determined to be present by conversion of blood types on erythrocytes.These results demonstrate that the present method was sufficiently sensitive for the assay of weak A- and B-transferase activities and the typing of A and B blood groups.