Rheumatoid arthritis-related antigen 47 kDa (RA-A47) is a product of colligin-2 and acts as a human HSP47

We previously isolated RA-A47, which is recognized as an antigen of rheumatoid arthritis (RA), from a human chondrosarcoma-derived cell line (HCS-2/8). The N-terminal 21-amino-acid sequence of RA-A47 had 81% homology to the deduced amino acid sequence of the human heat-shock protein (HSP) 47 gene, the colligin gene, and 100% homology to that of the colligin-2 gene. Moreover, as is HSP47, RA-A47 was a heat-inducible collagen-binding protein. To further characterize RA-A47, we isolated ra-a47 cDNA from HCS-2/8 cells and human periodontal ligament fibroblast (HPLF) cells. The isolated ra-a47 cDNAs from both cells were almost the same as that of colligin-2. C₅₀₄ and G₅₀₅ in the cDNA sequences of both cells and C₅₉₈ in the cDNA of HCS-2/8 were different from the corresponding bases of colligin-2 cDNA. These differences were also observed in genomic DNA. colligin cDNA was not isolated. To show that the isolated cDNA encodes RA-A47 protein, it was expressed in Cos-7 cells. The produced protein was 47 kDa and was recognized both with RA sera and antirat HSP47 antibody, indicating that it is RA-A47 and has structural similarity to HSP47. These results taken together with our previous findings show that RA-A47 is the putative colligin-2 gene product and behaves as a human HSP47. Although colligin has been considered the human HSP47 gene, failure to detect the colligin gene and its mRNA suggests that colligin does not exist in human cells and that the HSP47 gene is identical with colligin-2, which encodes RA-A47. Received: January 19, 2000 / Accepted: May 1, 2000     

Elevation of serum MAGE-4 protein levels and prediction of hepatocellular carcinogenesis in patients with liver cirrhosis.

Early detection of hepatocellular carcinoma (HCC) is clinically important because advanced HCC limits treatment modalities for the cancer. We have previously reported that serum levels of MAGE-4 protein are strongly associated with the development of HCC. The present study was designed to determine whether elevated serum MAGE-4 protein levels can predict hepatocellular carcinogenesis in patients with liver cirrhosis before clinical diagnosis. Among 62 cirrhotic patients, 28 patients were diagnosed with HCC during the follow-up period. The levels of MAGE-4 protein and alpha-fetoprotein (AFP) were significantly elevated in cirrhotic patients with HCC. Univariate and multivariate analyses suggest that elevated serum MAGE-4 protein is more significant than AFP. Importantly, retrospective analysis of prefrozen sera of cirrhotic patients revealed a transient or continuous elevation of serum MAGE-4 protein levels in 14 of 28 cirrhotic patients with HCC (50%) before clinical diagnosis. In contrast, elevated serum MAGE-4 protein levels were observed in 3 of 33 cirrhotic patients without HCC (9%), and in 2 of 34 hepatitic patients (6%). These results indicate that elevated serum MAGE-4 protein levels can be a predictive marker of hepatocellular carcinogenesis in cirrhotic patients, thereby enabling us to treat patients at an earlier stage.     

Effects of mechanical stress on expression of differentiated phenotypes of chondrocytes

In the patients with dislocated hip arthropathy, cartilage gene expression was confirmed in weight-bearing inner layer tissues of the joint capsule. Because these inner layer tissues of the joint capsule formed joint-like structures with the femoral head for a long period, cartilaginous genes may have been expressed in the weight-bearing inner surface of the joint capsule. There was a difference in expression of the genes between weight-bearing and non-weight-bearing parts. From a quantitative comparison between GAPDH and aggrecan gene expression, aggrecan gene expression was 30-fold higher in the weight-bearing part than in the non-weight-bearing part. Aggrecan gene expression was not detected in outer layer tissues of the joint capsule. Type II collagen and TGF-beta genes were also detected, and both genes showed differences in expression between the weight-bearing and non-weight-bearing parts like the aggrecan gene. This may have been because mechanical stress caused cartilaginous differentiation in undifferentiated mesenchymal tissues in the inner layer of the joint capsule. Cell differentiation and proliferation caused by mechanical stress are indicate key role to osteoarticular tissues, and it is considered important for orthopedic treatment to evaluate the process in detail.     

Expression of differentiated phenotype in the pseudo-tendon sheath formed by a silicon rod

We examined the cartilaginous gene expression in the inner layer of the pseudo-tendon sheath formed by an silicon rod. While cartilaginous gene expression was not detected in the outer layer of the tissue, gene expressions of aggrecan and type II collagen were detected in the inner layer of the the newly formed pseudo-tendon sheath around the silicon rod. Relative expression of aggrecan and Type II collagen were 0.15 and 0.28, respectively, compared to that of GAPDH. The expression of type II collagen was 0.57-folds of that of type I collagen. In these tissues, a sliding surface was formed by a silicon rod and the surrounding tissues, and its mechanical stress may induce cartilaginous gene expression. Mechanical stress together with various growth factors and cytokines may be critically important for the formation of more physiological tendon sheath structures. Therefore, we will further examine the changes detected in the tissues and evaluate mechanical stress for formation of the tendon sheath.     

Expression of the SART1 tumor-rejection antigen in human osteosarcomas

We recently reported a tumor-rejection antigen, SART1259, possessing tumor epitopes capable of inducing cytotoxic T lymphocytes (CTLs). This study investigated the expression of SART1259 antigen in osteosarcoma and other skeletal malignant tumors to explore for a potential molecule for use in specific immunotherapy. The SART1259 antigen was detected in the cytosol fraction of 13 of 21 (62%) osteosarcoma cell lines and 3 of 8 (38%) osteosarcoma tissues, and 3 of 10 (30%) malignant fibrous histiocytoma (MFH) tissues. The HLA-A24+ and SART1259+ osteosarcoma cells were recognized by the HLA-A24 restricted and SART1 specific CTLs. These results raise a possibility that the SART1259 would be an appropriate molecule for use in specific immunotherapy of approximately one-third of HLA-A24+ patients with osteosarcoma and MFH.     

Altered expression of natural killer cell inhibitory receptors (KIRs) on T cells in bronchoalveolar lavage fluid and peripheral blood of sarcoidosis patients

BACKGROUND AND AIM OF THE WORK: The role for natural killer cell inhibitory receptors (KIRs) on T cells is not fully understood, but signalling through KIRs on T cells may inhibit T cell receptor mediated activation, and KIR expression has been suggested to be one mechanism of controlling T cell mediated immune responses. An aberrant KIR expression on T cells could thus be of importance in autoimmune as well as infectious disorders. Sarcoidosis patients have several immunological impairments that have not been clarified, and we here examined the KIR expression on CD4+ and CD8+ peripheral blood (PBL) and bronchoalveolar lavage (BAL) T cells of sarcoidosis patients and controls. METHODS: We used three KIR specific monoclonal antibodies, namely DX9 (specific for p70), DX27 (p58) and DX22 (specific for CD94, that belongs to another major group of KIRs) and flow cytometry. RESULTS: p70 was expressed lower in patient CD8+ PBL (median 2.3%) compared to controls (6.3%) (p < 0.01). In patients, p58 was expressed by less CD8+ BAL lymphocytes (median 1.2%) compared to PBL (6.8%) (p < 0.01) while CD94 was expressed by more CD8+ BAL lymphocytes (median 14.5%) compared to PBL (9.6%) (p < 0.01). Moreover, in CD8+ PBL, CD94 and p58 were expressed significantly lower in patients with an active vs. inactive disease, and in patients with chest radiographic stage I vs. stage II, respectively. CONCLUSIONS: The significantly altered expression of distinct KIRs on CD8+ T cells in sarcoidosis, especially in patients with signs of an active disease, indicate these cells to be dysregulated and implicate them in the pathogenesis of the disease.     

Influence of cigarette smoke on the L-ascorbic acid metabolism and the activities of drug-metabolizing enzyme in rats

This study clarified the influence of cigarette smoke on the L-ascorbic acid (AsA) metabolism and the activities of drug-metabolizing enzyme in rats. The test rats (group T) were exposed to weak sidestream smoke from cigarettes for 2 h, everyday for 57 days. AsA concentration in the tissues and excreted amount of AsA in urine of group T tended to be higher than those of control group (group C). The plasma AsA concentration and the activities of aniline hydroxylase and 7-ethoxycoumarin O-deethylase of group T were significantly higher than those of group C. There was no significant difference in the activity of UDP glucuronosyltransferase or in the liver cytochrome P-450 content between these two groups.