Summary Monoclonal antibodies were raised against human placental soluble E-cadherins and used in an immunoenzymometric assay to detect soluble E-cadherins in biological fluids. The E-cadherin assay was accurate enough to quantitate the concentration of soluble E-cadherin in the cell culture supernatants. Immunoreactive E-cadherins, identified as existing in the soluble form in normal serum, were shown to have apparent lower molecular mass (approximately 80 kDa) than intact molecules of E-cadherin. We found that the immunoreactive E-cadherin levels in the serum of the studied cancer patients were significantly elevated (mean ± s.d. 3.80 ± 2.36 tig ml', P<0.0001) when compared with the normal levels (1.99 ± 0.50 1tg ml-'). We also found that serum E-cadherin levels in the 22 patients with gastric cancer (3.51 ± 1.78 tLg ml-, P <0.02) or the 11 patients with hepatocellular cancer (5.55 ± 3.11 Lg ml-', P<0.001) were significantly higher than those in the 26 diabetic patients (2.33 ± 1.58 iLg ml-'). Of the 54 cancer patients, 53.7% exhibited an elevated amount of soluble E-cadherin in serum. Thus, it is evident that soluble E-cadherin in circulation can be used as a prospective tumour marker that accurately reflects the progressive regeneration of E-cadherin at tumour sites, potentially induced by tumour-associated proteolytic degradation.

Basic fibroblast growth factor (bFGF) may be involved in the development and repair of dentine and pulp because bFGF, its related peptides, and FGF receptors are expressed in dental mesenchymal cells. In this study, we examined the effects of bFGF on DNA synthesis, osteonectin/SPARC levels, alkaline phosphatase (ALPase) activity, their mRNA levels, and calcium levels in cultures of human pulp cells. Pulp cells were isolated from three healthy upper wisdom teeth of three patients and maintained separately. These cells produced SPARC, ALPase, and calcified nodules and there was a close correlation between the SPARC-synthetic activity of the cell lines and their levels of ALPase and calcification. The levels of SPARC, ALPase and calcium deposits in the three pulp cell cultures were 10-250 times those of human foreskin fibroblasts. Western blots showed that the pulp cells produced 38-kDa SPARC. Northern blots showed that the pulp cells expressed flg (FGF receptor type 1) transcripts throughout all culture stages, irrespective of the presence or absence of bFGF. The addition of bFGF to the pulp cultures suppressed the increases in ALPase activity, SPARC synthesis, and their mRNA levels, although it increased the incorporation of [3H]thymidine into DNA> 10-fold. The effects of bFGF on ALPase activity and SPARC synthesis were reversible. Furthermore, bFGF abolished the calcification of the extracellular matrix; the calcium content of bFGF-free cultures. These findings suggest that bFGF is a potent mitogen for human pulp cells and that it inhibits the expression of the odontoblast phenotype by the cells at least partly at pretranslational levels.

Objective. To investigate the roles of SPARC (secreted protein, acidic and rich in cysteine) (osteonectin) in arthritis, using cartilage and synovium specimens and synovial fluids (SF) from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), and to examine the effects of cytokines, growth factors, and hormones on SPARC synthesis by chondrocytes in culture.Methods. SPARC in cartilage and synovium was immunostained with monoclonal antibodies. SPARC synthesis by cultured chondrocytes was measured by Northern blot analysis, immunoblotting, and sandwich enzyme-linked immunosorbent assay.Results. SPARC was identified in numerous chondrocytes in the superficial and middle zones and in Shigeo Nakamura, DDS, Hisashi Satakeda, DDS, Hiroshi Okamoto, DDS, PhD, Mitsuhide Noshiro, PhD, Yukio Kato, DDS, PhD: Hiroshima University, Hiroshima, Japan; Kyouko Kamihagi, PhD, Masahiko Katayama, PhD: Biotechnology Research Laboratones, Takara Shyuzo, Ohtu, Japan; Haiou Pan, DDS: Drug Discovery Research Laboratories, Hoechst Japan Limited, Kawagoe, Japan; Koichiro Takahashi, PhD: Clinical Pharmacology Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., Itabashi-ku, Tokyo, Japan; Yasuo Yoshihara, MD, Masayuki Shimmei, MD: National Defense Medical College, Tokorozawa, Japan; Yasunori Okada, MD, PhD: Cancer Research Institute, Kanazawa University, Kanazawa, Japan.Address reprint requests to Yukio Kato, DDS, PhD, Department of Biochemistry, School of Dentistry, Hiroshima University, 1-2-3, Kasumi, Minamiku, Hiroshima City, 734, Japan.Submitted for publication May 11, 1995; accepted in revised form November 6, 1995.regenerating chondrocytes of RA and OA joints, whereas such staining was absent in these zones of normal cartilage, except For weak signals from a few chondrocytes in the deep zone. In addition, SPARC synthesis was enhanced in synovial cells of RA and OA joints. The average SPARC level in SF was 10-fold higher in the RA than in the OA population. In rabbit articular chondrocyte cultures, administration of transforming growth factor pl (TGFp1) and bone morphogenetic protein 2 increased SPARC levels at 24-48 hours, whereas interleukin-lp (IL-lp), IL-la, tumor necrosis factor a, lipopolysaccharide, phorbol myristate acetate, basic fibroblast growth factor, and dexamethasone decreased SPARC levels at 2&72 hours. TGFP increased SPARC messenger RNA (mRNA) levels at 24 hours, whereas IL-IP caused a marked decrease in SPARC mRNA levels at 24 hours. Furthermore, IL-1 decreased the glycosylation of SPARC. Conclusion. These findings suggest that various growth factors and cytokines, including TGFm and IL-lp, regulate the production of SPARC by chondrocytes at pre-and posttranslational levels, and that SPARC synthesis is markedly enhanced in arthritic joints.SPARC (secreted protein, acidic and rich in cysteine) (osteonectin) (BM-40) is a 3543-kd Ca++-binding glycoprotein, which was initially isolated from 539

Increased levels of circulating undercarboxylated osteocalcin (ucOC), measured indirectly with the hydroxyapatite (HAP) binding assay, have been shown to predict hip fracture risk in a small group of elderly institutionalized women. The aim of this study was to confirm these findings in a prospective cohort study (EPIDOS prospective study) of 7598 healthy, independently living women over 75 yr of age. One hundred and four women who sustained a hip fracture during a 22-month follow-up period were age matched with 255 controls who did not fracture. Baseline samples were collected before hip fracture for measurement of total OC and ucOC, assessed either with the HAP binding assay or directly with a new enzyme-linked immunosorbent assay (ELISA). This direct ELISA uses human recombinant noncarboxylated OC as a standard and two monoclonal antibodies, one of which was raised against the 14-30 Glu synthetic peptide. We found that the intra- and interassay variations are less than 11%, and this assay exhibits a 5% cross-reactivity with purified human bone OC, used as a source of carboxylated OC. ucOC levels measured with this ELISA correlated well with the HAP binding assay in the population of 359 elderly women (r = 0.82; P < 0.0001). We estimated the risk of hip fracture for women with levels of ucOC in the highest quartile of values for the 255 controls. We found that increased levels of ucOC measured by ELISA were associated with increased hip fracture risk with an odds ratio (OR) of 1.9 (95% confidence interval, 1.2-3.0), and the ELISA had a greater sensitivity than the HAP assay. In contrast, total OC was not associated with hip fracture risk. After adjustment for femoral neck bone mineral density (BMD) and mobility status assessed by gait speed, ucOC still predicted hip fracture with an OR of 1.8 (1.0-3.0). Women with both femoral neck BMD in the lowest quartile and ucOC in the highest quartile were at higher risk of hip fracture, with an OR of 5.5 (2.7-11.2), than those with only low BMD or high ucOC levels. In conclusion, we have developed a new specific ELISA for serum ucOC, with low cross-reactivity with carboxylated OC and increased specificity and sensitivity over the HAP assay. Using this new ELISA, we found that ucOC, but not total OC, predicts hip fracture risk independently of femoral neck BMD in elderly women drawn from the general population. Thus, ucOC measurement could be combined with bone mass determination to improve the assessment of hip fracture risk in elderly women.

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