Macrophage-stimulating protein (MSP) and its receptor, RON, stimulate human osteoclast activity but not proliferation: effect of MSP distinct from that of hepatocyte growth factor.

Stem cell-derived tyrosine kinase (STK) is a member of the hepatocyte growth factor (HGF) receptor family. The ligand for STK, macrophage-stimulating protein (MSP), is a serum protein activated by members of the coagulation cascade. The RON gene is a human homolog of the murine STK. In this study we examined the role of MSP-RON in the signal pathway of human osteoclasts. Using anti-RON antibody, we detected RON expressed in multinucleated osteoclast-like cells (OCLs) formed in cultures of human bone marrow cells. To determine bone resorption, we placed OCLs on thin films of ceramic calcium phosphate formed on quartz plate-coated slides (Millenium Biologix) and measured pit formation. MSP stimulated pit formation by OCLs in a dose-dependent manner. MSP (50 ng/mL) caused a fourfold increase in pit area compared with the control. Furthermore, we examined the effects of MSP and HGF on OCL formation by purified populations of hematopoietic progenitors. OCLs were phenotypically identified by their cross-reactivity with 23c6, a monoclonal antibody that preferentially binds to osteoclasts. HGF (50 ng/mL) stimulated the differentiation of progenitors to 23c6-positive OCLs but did not enhance bone absorption. In contrast, MSP did not affect proliferation of osteoclast precursors but stimulated bone resorption by OCLs. We conclude that the MSP signal transduction pathway plays a role in bone resorption that is distinct from that of HGF.

Counting and differential of bone marrow cells by an electronic method.

The authors studied the cell count and size distribution curves of nucleated cells in bone marrow aspirate, using a fully automatic blood cell counter, and compared the results with those of the traditional manual method. The cell count by the electronic method correlated well with that by the manual method when the cell concentration was 100 X 10(9)/L or less. The size distribution curve had two peaks. The small cell fraction found electronically correlated well with the lymphocyte and erythroblast fractions found manually on Giemsa-stained smears. The method was highly reproducible, and technical problems did not arise, so the authors think it can be used in routine clinical testing.

[Combination chemotherapy with cyclophosphamide, adriamycin, vincristine and prednisolone (VEPA) for non-Hodgkin's lymphoma].

Combination Chemotherapy with Adriamycin (VEPA) was applied to 16 patients with non-Hodgkin's lymphoma. The effects of VEPA therapy were compared with those of combination chemotherapy without Adriamycin (non-VEPA). Complete remission rate achieved with VEPA therapy was 37.5% while that with non-VEPA therapy was 27.6%. Histologically, the complete remission rate in cases of large-cell type treated with VEPA therapy was 40%, while that with non-VEPA therapy was 14.3%. No cases of stage IV showed complete remission, whereas the complete remission rate for cases of stage III was 37.5% for VEPA therapy, but 27.6% for non-VEPA therapy. From these results we concluded that VEPA therapy is more effective for non-Hodgkin's lymphoma, especially large-cell type, than non-VEPA therapy.

Macrophage-stimulating protein activates STK receptor tyrosine kinase on osteoclasts and facilitates bone resorption by osteoclast-like cells.

Recently we cloned a novel receptor tyrosine kinase, STK. STK belongs to the hepatocyte growth factor receptor family and was identified as the receptor for macrophage-stimulating protein (MSP). STK is expressed on a restricted, macrophage population such as peritoneal macrophages, but not on mononuclear phagocytes of peripheral blood, bone marrow, or alveoli. Using an anti-STK monoclonal antibody, we observed STK expression on multinuclear osteoclast-like cells (OCLs) formed by murine bone marrow cultures in the presence of 1,25-dihydroxyvitamin D3, and interleukin-3. The OCLs expressed both the calcitonin receptor and STK. We also detected STK expression in bone-derived mouse osteoclasts. The addition of MSP to OCLs induced rapid morphologic changes such as cytoplasmic contraction and formation of ruffled border. In addition, MSP caused rapid redistribution of src to the borders of cytoplasm. These phenomena were associated with enhanced bone resorption. MSP caused a threefold increase in pit formation compared with control OCLs. These findings suggest that by involving src kinase, the MSP/STK signal transduction pathway induces rapid cytoskeletal reorganization in osteoclasts and facilitates bone resorption by osteoclasts.

[Clinical application of sintered bone (II). Effects of collagen coating on implant materials in vitro].

In vitro studies with osteoblast-like cells, revealed that in addition to preventing the immediate outflow and promoting stabilization of implant materials, these cells have the additional effect of promoting the mending and early calcification by coating the materials with collagen. The materials used for the experiment were minute hydroxyapatite, beta-tricalcium phosphate and bovine sintered bone. To these, atelocollagen which is solble in pepsin extracted from calf corium and crosslinked by using ultraviolet rays was added. We observed the cells very closely after these materials were added to alpha-modified eagle's medium containing 10 mM beta-glycero phosphate, 10% fetal bovine serum and osteoblast-like cells and the cultures incubated at 37 degrees C and 5% CO2. After 14 and 21 days, cells were fixed and stained with Alizarin Red S and Von Kossa stains to measure calcification. As a result of the collagen coating, positive areas appeared and, compared to the control improvement of the early mending was apparent. The results suggest that by coating the materials with collagen, the osteoblast-like cells show better mending and early calcification.

[Fixation effect of phospholipids using tannic acid. Part 1: Artificial lung surfactant].

In order to study the preservation of phospholipids in specimens for electron microscopic study, two procedures were compared; routine double fixation with glutaraldehyde followed-by osmium tetroxide and fixation with a mixture of tannic acid-glutaraldehyde followed by osmium tetroxide, utilizing both glass slide smear and ultrathin section methods, using artificial lung surfactant (mainly composing of phospholipids). The following results were obtained. (1) In routine double fixation with glutaraldehyde-osmium tetroxide, although the lamellar structure, mainly composed of phospholipids, was often visualized, prefixation with mixed tannic acid-glutaraldehyde always resulted in a lamellar structure with a regular periodicity and good contrast. (2) The saturated phospholipid was better preserved with acetone dehydration than with alcohol dehydration. (3) Depending on the outcome of the first fixation, there was extensive loss of phospholipids during the process due to alcohol dehydration and propylene infiltration. (4) When the specimens were fixed with osmium tetroxide prior to tannic acid treatment, the multilamellar structure of the bilayer was usually irregular. Moreover, if the specimens were fixed with osmium tetroxide without tannic acid, phospholipid preservation was not good. From the above results, it became, apparent that prefixation by a mixture of tannic acid-glutaraldehyde followed by osmium tetroxide postfixation and dehydration by acetone was the most appropriate method for preserving saturated phospholipids and thus a stable ultrastructure of phospholipids an lamellar will obtained.