Akira Yachi

Detection of circulating intercellular adhesion molecule-1 antigen in malignant diseases.

A new monoclonal antibody (MoAb) H A58 (IgG 1) was prepared, which recognizes the binding site on the intercellular adhesion molecule‐1 (ICAM‐1) antigen to the lymphocyte function‐associated antigen‐1 (LFA‐1). The double‐determinant immunoassay (DDIA) was established with use of MoAb HAS8 and another anti‐ICAM‐1, MoAb CL207, to detect the soluble, shedding ICAM‐1 antigen. Human recombinanl interferon‐gamma (IFN‐γ) induced not only the expression of cell surface ICAM‐1, but also the shedding ICAM‐I antigen in an IFN‐y concentration‐dependent and incubation‐time‐dependent manner. DDIA was applied to detect the shedding ICAM‐1 antigen in the sera of patients with malignant or benign diseases. The incidence of posilivity for ICAM‐I antigen in malignant diseases was higher than that in benign diseases or in healthy controls. Furthermore, the sera of cancer patients with liver metastasis showed higher levels of the shedding ICAM‐1 antigen. These findings suggest that serum ICAM‐1 antigen may be a useful marker to monitor tumor burden in cancer patients.

EXPRESSION OF CD10/NEUTRAL ENDOPEPTIDASE IN NORMAL AND MALIGNANT TISSUES OF THE HUMAN STOMACH AND COLON

The expression of common acute lymphoblastic leukemia antigen (CALLA; CD10), which is identical to neutral endopeptidase (NEP, EC3.424.11), was examined in the malignant and adjacent noninvaded tissues of the human stomach and colon (n = 27). All of 27 normal and 18 well or moderately differentiated adenocarcioma tissue specimens were positive for monoclonal antibody (mAb) NL-1 against CD10/NEP, whereas the expression level was clearly decreased in all of 9 specimens of poorly differentiated adenocarcinoma. In addition, all of 7 gastric or colorectal carcinoma cell lines tested showed decreased expression of CD10/NEP. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the crude antigen J5 from the normal colon tissue lysate by mAb J5 detected a single band of approximately 100 kDa that was consistent with that of NALM-6 cells used as a positive control. These findings suggest that CD10/NEP is expressed in normal epithelial cells of the human stomach and colon, whereas the expression level is decreased in the poorly differentiated type of adenocarcinoma.

Circulating intercellular adhesion molecule-1 (ICAM-1) antigen in sera of patients with idiopathic pulmonary fibrosis

Intercellular adhesion molecule‐1 (ICAM‐1), a member of immunoglobulin supergene family with a five‐domain structure, is known to play an important role in inflammatory diseases. An ELISA was developed using two MoAbs against human ICAM‐1 in order to detect the soluble shedding ICAM‐1 antigen in sera. We measured levels of circulating ICAM‐1 antigen in sera of patients with idiopathic pulmonary fibrosis (IPF), pulmonary sarcoidosis, hypersensitive pneumonitis, bacterial and mycoplasmal pneumonia, and inflammatory diseases of other organs. The results clearly demonstrated that IPF had significantly high levels of circulating ICAM‐1 in sera as compared with other disorders or normal controls. Moreover, immunohistochemical analysis with MoAb against human ICAM‐1 disclosed that in IPF, the expression of ICAM‐1 was intensively enhanced on alveolar epithelial cells. These results suggest that ICAM‐1 may contribute to the pathogenesis of IPF.

Expression of c-kit and kit Ligand in Human Colon Carcinoma Cells

To determine whether c-kit and kit ligand (KL) mRNAs could be expressed in human epithelial tumors, reverse transcriptase-polymerase chain reaction and Northern blot analysis were performed. KL mRNA was shown to be expressed in a variety of epithelial tissues and cell lines. The expression of c-kit mRNA was then examined in hepatocellular and colon carcinoma cell lines. While hepatocellular carcinoma cell lines did not express c-kit mRNA as far as we could ascertain, 2 of 5 colon carcinoma cell lines showed the expression of both c-kit and KL mRNAs. Furthermore, the expression of c-kit in these cells was demonstrated at the protein level by flow cytometry. These data suggest that c-kit and KL may play an important role as an autocrine loop in the proliferation of some colon carcinoma cells.

Colonization of Helicobacter pylori in the Gastric Mucosa of Mongolian Gerbils

Helicobacter pylori was orally inoculated into Mongolian gerbils. The organisms were able to colonize in the gastro‐mucosal layer of the gerbils, especially in those gerbils which had mucosal lesions caused by indomethacin treatment. The pathological changes developed by H. pylori infection were restricted to the stomachs, and only slightly inflammatory cells were observed.

Soluble Intercellular Adhesion Molecule-1 (ICAM-1) Antigen in Patients with Rheumatoid Arthritis

Intercellular adhesion molecule‐1 (ICAM‐1), a member of the immunoglobulin supergene family, is known to play an important role in inflammatory diseases. Using a previously developed enzyme‐linked immunosorbent assay (ELISA) with two monoclonal antibodies (MoAbs) against human ICAM‐1, levels of soluble ICAM‐1 (sICAM‐1) were measured in sera from patients with collagen diseases and in synovial fluids (SF) from patients with rheumatoid arthritis (RA). Although the results did not demonstrate that RA and other collagen diseases, as a group, had significantly higher levels of sICAM‐1 in sera as compared with healthy controls, 21 of 138 cases (15%) with collagen diseases and 11 out of 57 patients (19%) with RA clearly showed higher levels of sICAM‐1 in the sera. Comparisons between RA patients of radiological stages I and II and between stage I and other stages showed significantly higher levels of sICAM‐1 in the sera of patients in the latter stages. RA patients with vasculitis and/or pneumonitis showed significantly higher levels of sICAM‐1 than those without vasculitis or pneumonitis. Significant correlations were demonstrated between sICAM‐1 and the factors IgG‐RF, IgM‐RF, erythrocyte sedimentation rate (ESR) and TNF‐α in sera of RA patients. In addition, it was noted that the levels of sICAM‐1 in SF were as high as those in the sera of patients with RA.

Molecular cloning and chromosomal mapping of a human protein-tyrosine phosphatase LC-PTP†

We isolated cDNA clones encoding a protein-tyrosine phosphatase (PTP) from a human T cell PEER cDNA library. The predicted open reading frame encodes a approximately 40-kDa protein composed of 360 amino acids and has no apparent hydrophobic segments, suggesting that it is a nontransmembrane PTP, which was designated as LC-PTP (leukocyte PTP). Northern blot analysis revealed that the LC-PTP mRNA was preferentially expressed in a variety of hematopoietic cells and the transcriptional sizes were approximately 4.0 kilobases and approximately 2.9 kilobases. Fluorescent in situ hybridization revealed that the human LC-PTP gene is located on the chromosome region 1q32.1, which is known to be a site associated with chromosomal deletion in malignant lymphomas, where candidate tumor suppressor genes might be present.

Effect of radiation on the expression of carcinoembryonic antigen of human gastric adenocarcinoma cells

The changes of antigenic expression of cultured human gastric adenocarcinoma MKN45 cells caused by irradiation were investigated to elucidate the immune responses to localized irradiation. The expression of carcinoembryonic antigen (CEA) showed remarkable increases in the culture supernatant and on the surface of the membrane of irradiated cells. The expression of major histocompatibility complex Class I antigen on the membrane also was enhanced by irradiation. In addition, the irradiated cell groups, when analyzed using a CEA‐specific probe, showed remarkable increases in the CEA mRNA. These enhancements increased in the 10‐Gy and 15‐Gy irradiated populations compared with the 5‐Gy irradiated population. These results suggest that the enhancement of expression of CEA by radiation takes place at the CEA gene expression (mRNA) level but not at the protein level.

Soluble intercellular adhesion molecule‐1 (ICAM‐1) in sera and bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis

ICAM‐1 plays an important role in inflammatory diseases. To assess level of soluble ICAM‐1 in the circulation and inflamed lesions, we measured levels of soluble ICAM‐1 in the circulation and bronchial veolar lavage fluid (BALF) of patients with idiopathic pulmonary fibrosis (IPF) and with pulmonary sarcoidosis (PS) and of healthy volunteers (HV), and we also analysed ICAM‐I expression of BALF cells in some patients and HV. IPF patients had significantly higher levels of circulating ICAM‐1 than HV, while PS patients did not. By contrast, significantly increased levels of BALF soluble ICAM‐1 were found in PS patients compared with those of HV, but not in IPF patients. There were no significant differences in the proportions of ICAM‐1+ BALF lymphocytes in IPF patients, PS patients and HV. whereas significantly increased proportions of ICAM‐1 pulmonary alveolar macrophages were found in PS patients compared with those of HV, but not in IPF patients. There was a significant positive correlation of BALF soluble ICAM‐1 levels to BALF lymphocyte proportions in PS patients. Although the source of BALF soluble ICAM‐1 is unclear. BALF soluble ICAM‐1 appears to reflect the grade of local activity of sarcoidosis. An interesting discrepancy between soluble ICAM‐1 levels in the circulation and BALF was found in IPF patients, and this might be an important clue to an understanding of this disorder.

Cloning and characterization of a human cDNA encoding a novel putative cytoplasmic protein-tyrosine-phosphatase

We have cloned and characterized a human cDNA encoding a new member of the family of cytosolic type protein-tyrosine-phosphatase (PTP), designated as PTPG1, from an adult colon tissue cDNA library by using the PCR product as probe. We obtained 5 cDNA clones, which cover the predicted open reading frame encoding a 88-kDa protein composed of 780 amino acids, and it had no apparent signal or transmembrane sequences, suggesting that it is a cytosolic protein. The N-terminal region had a PTP catalytic domain that is 30-40% identical to previously reported human PTPs. This revealed that the enzyme composes an additional family of human PTPs. PTPG1 was characterized by a long non-enzymatic domain located at the C-terminus, including PEST sequences which are characteristic for short half-life proteins in eukaryotes. Northern blot analysis of PTPG1 mRNA showed a 4.6-kb transcript that was detected in a wide variety of cell lines to suggest its extensive expression.