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Interaction between the receptor for advanced glycation end products (RAGE) and its ligands has been implicated in the pathogenesis of various inflammatory disorders. In this study, we establish an in vitro binding assay in which recombinant human high-mobility group box 1 (rhHMGB1) or recombinant human S100A12 (rhS100A12) immobilized on the microplate binds to recombinant soluble RAGE (rsRAGE). The rsRAGE binding to both rhHMGB1 and rhS100A12 was saturable and dependent on the immobilized ligands. The binding of rsRAGE to rhS100A12 depended on Ca2 and Zn2, whereas that to rhHMGB1 was not. Scatchard plot analysis showed that rsRAGE had higher affinity for rhHMGB1 than for rhS100A12. rsRAGE was demonstrated to bind to heparin, and rhS100A12, in the presence of Ca2, was also found to bind to heparin. We examined the effects of heparin preparations with different molecular sizesunfractionated native heparin (UFH), low molecular weight heparin (LMWH) 5000Da, and LMWH 3000Da on the binding of rsRAGE to rhHMGB1 and rhS100A12. All 3 preparations concentration-dependently inhibited the binding of rsRAGE to rhHMGB1 to a greater extent than did rhS100A12. These results suggested that heparin's anti-inflammatory effects can be partly explained by its blocking of the interaction between HMGB1 or S100A12 and RAGE. On the other hand, heparin would be a promising effective remedy against RAGE-related inflammatory disorders.

Metaplastic bony tissue along with hyperplastic mucosal epithelium showing no atypism was detected in biopsy materials from a Yamada type I gastric polyp. The tissue was metaplastic woven bone associated with calcification. Histogenesis of the bone formation is as yet unknown. This is the first reported case of the presence of metaplastic bone accompanied by hyperplastic gastric mucosa so far.

The difference in the physiological condition of drivers of manual transmission buses (MTB) and automatic transmission buses (ATB) was examined from the viewpoint of occupational health. This study was based on a self-administered questionnaire which involved items concerning subjective fatigue complaints. No differences in the mental fatigue and stress between MTB drivers and ATB drivers were observed. Although ATB drivers tended to feel less physical fatigue than MTB drivers, the difference was not statistically significant. From these results, it was suggested that there was little difference in the subjective fatigue between ATB drivers and MTB drivers.

The anti-ulcer action of clotiazepam (a thienodiazepine derivative) was studied in mice subjected to non-physical and physical stimuli in a communication box. There were two groups of mice: the "sender" mice that received electric shocks on the feet and responded by squealing and jumping, and the "responder" mice that were affected by the senders' responses without receiving shocks on the feet. Gastric ulcers resulted in both groups. The effect of clotiazepam was compared with that of diazepam. The incidence of gastric ulcers was suppressed by clotiazepam at a dose of 3 mg/kg, per os, in "responder" and "sender" mice, and by diazepam at a dose of 1 mg/kg, per os, in "responder" mice. These results suggest that clotiazepam has a suppressive action against gastric ulcers produced by non-physical or physical stimuli, although its potency is slightly weaker than that of diazepam.

We herein determined by fluorescence in situ hybridization the chromosomal localization of 2 human genes, BRAL1 and BCAN, both of which belong to the link-module superfamily, i.e. to the same band of chromosome 1q21-23. Further analysis of the genomic organization of BRAL1 and BCAN revealed that the BRAL1 gene was located 20-kb upstream of the BCAN start site. We isolated a polymorphic dinucleotide (CA) repeat sequence from a genomic clone containing the BCAN gene. High heterozygosity (0.79) makes this polymorphism a useful marker in the study of genetic disorders. Knowledge of the structure of the genes and the marker provides essential information for further analysis of the gene locus at chromosome 1q21-23.

Using 2-dimensional gel electrophoresis, we previously demonstrated that the S100C protein remarkably decreased after immortalization of normal human fibroblasts, and that this protein caused growth inhibition of human tumor cells when forcibly expressed in these cells, suggesting that S100C plays a significant role in tumor suppression. The present study was carried out to determine what type of human tissues express S100C protein, and, subsequently, whether the S100C content in these tissues changes after normal cells have been transformed into cancer cells. We found that ductal cells in various tissues were positively stained with the S100C protein. In comparison, epithelial cells in digestive organs such as the stomach, small intestine, and colon were not stained as strongly. When 14 pairs of human normal and cancerous tissues were stained with the antibody, decreases in the staining levels of S100C were observed in 6 kinds of cancerous tissues--from the bronchus, mammary duct, renal tubule, prostate, uterus, and testis--in comparison with staining in their normal counterparts. These results suggest that S100C is a new tumor marker protein, the expression of which significantly decreases after malignant transformation of human tissues.

Anti-idiotype antibodies (Ab2) play an important role in the homeostasis of immune responses and are related to the development and the disease activity of certain autoimmune diseases. The asialoglycoprotein receptor (ASGPR) is considered one of the target antigens in the pathogenesis of autoimmune chronic active hepatitis (AIH). We previously developed a mouse monoclonal antibody (clone 8D7) which recognizes rat and human ASGPR. In this study, to help investigate the anti-ASGPR antibody-anti-idiotype antibody network in patients with AIH, we developed a syngeneic mouse monoclonal Ab2 to the 8D7 anti-ASGPR antibody (Ab1). One clone, designated as 3C8, tested positive for specific reactivity to 8D7-Ab1 and did not bind to other irrelevant immunoglobulins. By competitive inhibition assays, the binding of 8D7-Ab1 to liver membrane extracts, i.e., the crude antigen preparation, was inhibited by 3C8-Ab2 in a dose-dependent manner, and the binding of 8D7-Ab1 to 3C8-Ab2 was inhibited by the liver membrane extracts. In the immunohistochemical analysis, 3C8-Ab2 blocked the specific staining of sinusoidal margins of rat hepatocytes by 8D7-Ab1. These results suggest that 3C8 anti-idiotype antibody recognizes the specific idiotypic determinants within the antigen-binding site of 8D7-Ab1.

The celiac and mesenteric arterial system including the left gastric, splenic, common hepatic, and superior mesenteric arteries shows various types of origins, courses, ramifications and anastomoses. In order to explain the various expressions of this system, we have proposed a typological model, in which celiacomesenteric arteries develop as paired or bilaterally symmetrical primordial vessels originated from the anterior aspect of the aorta, and these vessels anastomose each other with longitudinal and horizontal pathways. Here, we report 3 unusual cases characterized by arterial rings, formed by the left gastric, left accessory hepatic, proper hepatic, anterior pancreaticoduodenal, and dorsal pancreatic arteries. The dorsal pancreatic and anterior pancreaticoduodenal arteries are located to the right and left of the embryonic pancreas developing in the dorsal mesentery, respectively. Such hepatopancreatic arterial rings simultaneously containing right and left elements can only be explained using our typological model, in which the concept of paired arteries or bilateral symmetry is introduced.

In autoimmune chronic active hepatitis (AIH) and primary biliary cirrhosis (PBC), various autoantibodies including anti-asialoglycoprotein receptor (ASGPR) antibodies have been found in patients' sera. We have previously developed a mouse monoclonal antibody against rat and human ASGPR. In this study, we developed a capture enzyme-linked immunosorbent assay (ELISA) for detection of anti-ASGPR antibodies using this monoclonal antibody and investigated the occurrence of anti-ASGPR antibodies in the sera of patients with various liver diseases. Serum samples were obtained from 123 patients with various liver diseases, including 21 patients with AIH and 40 patients with PBC. In this capture ELISA, the target antigen in the crude rat liver membrane extracts was captured on the ELISA wells by the ASGPR-specific mouse monoclonal antibody. Thus, the cumbersome process of antigen purification was rendered unnecessary. Using this capture ELISA, we detected the anti-ASGPR antibody in 67% of the patients with AIH, in 100% of the patients with PBC, and in 57% of the patients with acute hepatitis type A. However, the anti-ASGPR antibody was rarely detected in patients with other liver diseases such as primary sclerosing cholangitis and obstructive jaundice. Our findings suggest that this capture ELISA would be useful for the detection of anti-ASGPR antibodies in autoimmune liver diseases.

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.

We developed a monoclonal antibody (MoAb) (clone 5E8) against an antigen on the bile canalicular membrane of rat hepatocyte. By immunoblotting, MoAb 5E8 detected a band of 110 kD. In this study, we used the phage display technique to identify the target antigen recognized by MoAb 5E8. We screened a random phage display library expressing 12-mer peptide sequences and identified a peptide sequence, FHFNPYTGHPLT, as an epitope. We compared this peptide sequence with those of dipeptidyl peptidase IV (DPP IV, E.C.3.4.14.5) and Cell-CAM105, which proteins were located by a database search based on the information of tissue localization and approximate molecular weight of the MoAb 5E8 antigen, and sequence similarity with a region in DPP IV (amino acids 225-233) but not with Cell-CAM105 was found. In addition, we immunohistochemically stained various tissues (liver, small intestine, and kidney) of Japanese Fischer 344 rats, known to be deficient for DPP IV, with MoAb 5E8 and showed that the expression of MoAb 5E8 antigen was negligible or weak. In contrast, tissues sampled from the same organs of Sprague-Dawley rats, known to express DPP IV, were positively stained. These findings suggest that the antigen recognized by MoAb 5E8 is DDPIV and its major epitope is located in amino acids at positions 225-233.

Dynamin is a protein essential to endocytosis. Dynamin 2, a dynamin isoform, is expressed most intensely in testicular tissue; however, precise localization has never been studied. Therefore, we investigated the expression of dynamin 2 in rat testicular tissue using immunohistochemical methods, and discuss here the physiological function of this protein. Testicular tissues were obtained from Wistar rats at 10, 21 and 63 days of age. Immunohistochemistrical examination and Western blot analysis were conducted using dynamin 2 specific antibody. Western blot analysis showed that expression in 21- and 63-day-old rats was more intense than that in 10-day-old rats. Dynamin 2 expression was observed using immunohistochemical method in the seminiferous tubules of all rats. In the 63-day-old rats, the expression was intense, especially in spermatids in the earlier maturation stages and in spermatocytes, and was observed in Sertoli cells. However, in spermatids, the expression gradually declined as spermatids matured to spermatozoa. In the 21-day-old rats, the expression was evident in spermatocytes and Sertoli cells, but that in the 10-day-old rats was weak. Intense expression of dynamin 2 during spermatogenesis suggests that this protein plays an important role in this process.

Corticoids are well known for their immunosuppressive properties. Interleukin-10 (IL-10) is an intrinsic antiinflammatory peptide in immune diseases, originally identified as cytokine synthesis inhibitory factor. We examined the effect of hydrocortisone sodium succinate (HSS) on the production of IL-10 by human peripheral blood mononuclear cells (PBMCs). PBMCs from healthy volunteers and cancer-burden patients were preincubated separately with or without HSS for 1 h, then stimulated with 5 microg/ml lipopolysaccharide (LPS). Production of IL-10 by human PBMCs was detected with LPS stimulation and its production was higher in cancer-burden patients than in normal volunteers, although this was not statistically significant. HSS suppressed production of IL-10 by LPS-stimulated PBMCs in a dose-dependent manner both in normal volunteers and in cancer-burden patients. These results indicate that, in addition to their antiinflammatory properties, corticoids act to restore the immunosuppressive states even in cancer-burden states

We developed a reliable system for the irradiation of xenografted tumors in mice which allows for accurate local irradiation under specific pathogen-free conditions. The system presented here consists of acrylic supports for mice and an acrylic box connected to a pump through 0.22 microns pore-sized filters. Mice with xenotransplanted tumors growing on their right hind legs were set on the supports and put into the box in a laminar flow hood. The tumors of 7 mice were irradiated simultaneously with X-rays of 6 and 10 MV generated by a linear accelerator at a dose rate of 3.1-4.7 Gy/min. The air was ventilated through filters during irradiation in the closed box. Microorganism tests confirmed that no bacteria entered or left the box. One of the significant characteristics of this setup is that it allows for irradiation under conditions of acute hypoxia, which is obtained using an integrated tourniquet. The dose variation among 7 tumors was less than 1%. The rest of the mouse's body was shielded effectively by a half-field technique and a lead block. As a result, the whole body dose for the mice was 0-4% of the total dose absorbed by the tumor. Due to the high dose rate and the ability to irradiate 7 mice simultaneously under specific pathogen-free conditions, this new system can be considered a time-saving and valuable tool for radiation oncology research.

To investigate the correlation between nuclear medicine parameters determined by technetium-99m-DTPA-galactosyl-human serum albumin (Tc-99m-GSA) and liver function tests, canonical correlation analysis was performed. Tc-99m-GSA studies were performed on 47 patients with hepatocellular carcinoma (HCC) who had undergone transcatheter arterial embolization (TAE). The nuclear medicine parameters LU15, HH15 and LHL15, which are results of nuclear imaging tests, were chosen in combination with the following liver function tests: the serum bilirubin level (T.Bil), the serum albumin level (Alb), serum cholinesterase activity (Ch-E), the clearance rate of indocyanine green (KICG), the hepaplastin test (HPT) and the prothrombin time (PT). The canonical correlation coefficient was 0.7345 and the upper tail probability was 0.00167. A significant correlation was observed between the two sets of variables. The high structural coefficients of Ch-E, KICG and HPT indicated a close relationship with the nuclear medicine parameters, supporting the notion that these nuclear medicine parameters are useful for the estimation of liver damage. The structural coefficients of the nuclear medicine parameters were also high, with LU15 being a parameter as useful as both HH15 and LHL15. T.Bil may evaluate a liver function that is not measured by nuclear imaging techniques, so we should take T.Bil results into account before considering TAE.

Seventy-nine shoulders suspected of rotator cuff tears were examined by ultrasonography (US) and forty-three received surgery. Long and short axis scans were performed and findings of each were separately classified according to a five-grade system, and the results were correlated with the actual extent of tear observed during surgery. Internal echogenicity and subacromial impingement were analyzed before and after surgery. A accuracy of US in detecting rotator cuff tears was analyzed. In addition, the correlation between cuff shape observed by US before surgery and actual shape observed during surgery was assessed. It was noted that cuff thinning and abnormalities in shape did not recover to normal after surgery. However, in the cases of discontinuities observed by US before surgery, US findings indicated that the torn cuff was anchored to the greater tuberosity and functional during active motion. Although post-operative US findings were not normal, clinical results were good in most cases. Sensitivity of US for detecting rotator cuff tear was 100% and specificity 94%. US is non-invasive, cost effective and allows the physician to examine the joint while it is in motion. Therefore, at this time, we use US as a screening method for detecting rotator cuff tears. Furthermore, US allows us to check for re-tears while the joint is in motion, which is essential for accurate diagnosis.

Genomic sequencing and chromosomal assignment of the gene encoding rat APEX nuclease, a multifunctional DNA repair enzyme, were performed. An active Apex gene and a processed pseudogene were isolated from a rat genomic library. The active Apex gene consists of 5 exons and 4 introns spanning 2.1 kb. The putative promoter region of the Apex gene lacks the typical TATA box, but contains CAAT boxes and a CpG island having putative binding sites for several transcription factors, such as Sp1, AP-2, GATA-1 and ATF. A putative O-sialoglycoprotease (a homologue of Pasteurella haemolytica glycoprotease, gcp; abbreviated as Prsmg1/Gcpl1) gene consisting of 11 exons and 10 introns spanning 7.3 kb lies immediately adjacent to the Apex gene in a 5'-to-5' orientation. The Apex gene locus was mapped to rat chromosome 15p12 using in situ hybridization. The processed pseudogene (designated as rat Apexp1) has a nucleotide sequence 87.1% identical to that of the rat Apex cDNA, although several stop codons interrupting the coding sequences and multiple nucleotide deletions were observed. The Apexp1 is located in an inactive LINE sequence. Calculation of nucleotide substitution rates suggests that the immediate, active progenitor of Apexp1 arose 23 million years ago and that the non-functionalization occurred 15 million years ago.

The distribution of lectin receptors in the human tonsil was studied using 16 biotinylated lectins. The avidin-biotin-peroxidase complex (ABC) method was used on frozen and paraffin-embedded tissue sections. Cell suspensions were also analysed by dual flow cytometry using respective fluorescein isothiocyanate-conjugated lectins and phycoerythrin-labeled anti-CD3 and anti-human immunoglobulin. Frozen sections fixed with acetone and paraffin-embedded materials fixed in three solutions were compared for lectin affinity; ethanol-fixed sections gave best results followed by frozen and buffered formalin-fixed ones, then nonbuffered formalin. Con-A, RCA-1, LcH, WGA, MPA, PHA, PSA, PNA, SJA and GSA-1 reacted with all tissue components of the tonsil in immunohistochemical studies, but binding intensity was fixative dependent. Binding of Lotus and BPA to lymphocytes was limited to germinal center lymphocytes. Other tissue components were also reactive but staining intensity was weaker in Lotus compared with BPA. SBA and DBA did not react with lymphocytes, but reacted with macrophages/histiocytes, vascular endothelia, and epithelial cells. LBA and LPA were constantly negative with all tissue components irrespective of fixatives. Flow cytometric analyses showed that all but three (DBA, LBA and LPA) partially or totally stained lymphocyte surfaces. Lotus receptors were expressed exclusively on B-lymphocytes.

We performed a long-term follow-up of 4 patients with penile cancer who underwent hyperthermotherapy from August 1985 until August 1992. Hyperthermia was applied using a frequency of 350 MHz with a waveguide applicator twice a week for 60 min each for an average of 9.5 times (varying from 6 to 13 times). The total heating time that the temperature of urethra could be kept above 42 degrees C, was 166 min on the average (ranging from 0 to 463 min). Two patients classified as stage I according to the Jackson classification and 1 patient classified as stage IV underwent combined radiotherapy and received an average radiation dose of 53 Gy (range, 40-70 Gy). Among these patients 2 underwent combined chemotherapy with bleomycin or peplomycin. Malignant cells disappeared posttherapeutically and in August 1992, after an average of 5 years and 9 months (varying from 4 years 6 months to 6 years 10 months), the patients were free of recurrences. The one patient on stage IV had extensive invasion of the abdominal wall, but still recovered completely. One patient on stage III underwent combined chemotherapy and hyperthermotherapy, but heating had obviously been insufficient. There was a residue of malignant cells after the treatment and we performed a penectomy. Regarding functional preservation of the penis a multidisciplinary therapy incorporating hyperthermotherapy can be expected to increase the curativity. This indicates that it could induce in an advanced case, where an operation would be difficult, complete remission.