Initial effects of partially purified bone morphogenetic protein on the expression of glycosaminoglycan, collagen, and alkaline phosphatase in nonunion cell cultures.

Bone morphogenetic protein (BMP) stimulates mesenchymal cells to differentiate, resulting in de novo endochondral ossification in vivo. The response of fibrocartilage and periosteal cells from human and canine nonunion tissues to partially purified BMP was examined in culture. Cells derived from neonatal rat muscle explants were used for comparison. Alkaline phosphatase activity and expression of alkaline phosphatase and Types I and II collagen mRNAs were compared to that of rat chondrocytes. Synthesis of Type II collagen by the muscle cells was verified by enzyme-linked immunosorbent assay (ELISA). Addition of BMP to the muscle cell and nonunion cell cultures resulted in a dose-dependent decrease in cell number. There was a decrease in matrix vesicle and plasma membrane alkaline phosphatase activity concomitant with an increase in mRNA levels for alkaline phosphatase and collagen genes. Synthesis of immunoreactive Type II collagen increased. These data indicate that neonatal rat muscle cells and nonunion cells may respond in a similar fashion to BMP. Bone morphogenetic protein stimulated hyaluronic acid synthesis at three days, but chondroitin sulfate synthesis did not increase until ten days exposure to BMP. These data, together with those summarized above, suggest that more than three days may be required for complete expression of the chondrocyte phenotype typical of endochondral ossification.

Phenotypic changes of rabbit mandibular condylar cartilage cells in culture.

The present study describes the behavior of mandibular condylar cartilage (MCC) cells as a function of time in primary culture, since it is not yet clear whether these cells maintain their phenotype in culture. MCC cells from New Zealand white rabbits were seeded at high density and cultured in DMEM containing 50 micrograms/mL ascorbic acid and 10% fetal bovine serum. These cells appeared as a heterogeneous population and changed their shape, size, and refractivity as cultures aged. Cartilage-like cells, which always dominated the culture, were infiltrated with a minority of fibroblast-like cells. Cell number increased progressively, and cultures reached confluence at nine days. Antibody activity for cartilage-specific glycosaminoglycan was determined by ELISA assay. This reaction reached a maximum at six days and decreased thereafter. Cultures stained with Alcian blue (pH 1.0) supported these results. Cytoplasmic mRNA analysis indicated that the transcription of type II collagen gene was present at all time points. Type I collagen and alkaline phosphatase mRNA levels showed progressive increases from 12 h to nine days, with significantly higher values in cells cultured for six, nine, and 12 days than in cells collected from earlier time points. These results suggest that in our present culture system, MCC cells undergo phenotypic changes that resemble their maturation processes in vivo.

In vitro generation of lymphocytotoxicity to autochthonous leukaemic cells in chronic myeloid leukaemia.

Lymphocytes from 13 chronic myeloid leukaemia (CML) patients in remission were tested for their ability to differentiate in vitro into a cell population cytotoxic to autochthonous target leukaemic cells. CML remission lymphocytes were cultured in vitro with autochthonous leukaemic cells and allogeneic normal lymphocytes from an unrelated donor, singly or in combination. The cytotoxic lymphocytes obtained after 7 days of culture were tested for their ability to kill autochthonous leukaemic cells in a 3h 51Cr-release assay. It was found that with the allogeneic stimulus alone, cytotoxicity was generated in 5/13 cases, whilst stimulation of lymphocytes with autochthonous leukaemic cells alone induced cytotoxicity in 7/13 cases. In contrast, anti-leukaemic cytotoxicity was shown in 12/13 cases when responder lymphocytes were stimulated with both autochthonous leukaemic and unrelated allogeneic normal lymphocytes. The specificity of cytotoxicity was confirmed using other targets such as autochthonous PHA-transformed lymphoblasts and mouse L1210 cells. In 1/5 cases, CML remission lymphocytes stimulated with autochthonous leukaemic cells showed cytotoxicity to PHA-transformed autochthonous normal lymphoblasts, whilst 1/4 patients showed nonspecific cytotoxicity to L1210 cells when lymphocytes were cultured in MLC or MLTC, as well as in a 3-cell assay.

Cellular sensitization in chronic myeloid leukaemia patients to leukaemic blast antigens.

Sixteen chronic myeloid leukaemia (CML) patients in remission were tested with solubilized membrane antigens from CML leukaemic cells, CML blasts, AML blasts and ALL blasts for cellular immunity in vitro by lymphocyte transformation (LT) and leucocyte migration inhibition (LMI) assays. Twelve CML patients in remission were tested with allogeneic PHA-transformed normal lymphoblasts. As controls, peripheral-blood leucocytes from 9 healthy persons were tested with the same antigen preparations. It was seen that 8/16 (50%) CML patients responded to CML antigens by both LT and LMI assays, while 5/16 (31%) patients reacted to CML blasts and 44% (7/16) patients reacted to AML blast antigens. It was interesting to note that 5/11 (45%) CML patients reacted to ALL blast antigens by both assays. One out of 12 patients reacted to PHA-transformed lymphoblasts. None of the healthy controls reacted to leukaemia-associated antigens. The results suggest the sharing of antigens between myeloid leukaemic cells, myeloid blasts and lymphoid blasts.

A case of Sézary syndrome with non-rosetting cells and unusual cytogenetic findings.

A rare case of Sézary syndrome with typical, clinical and haematological picture is described. The absence of any lymphoid surface markers on Sézary cells and the consistent presence of distinct clone 45 XY-F cytogenetic abnormality are very unusual features observed in the present case. In spite of these peculiar findings, response to chlorambucil and prednisolone was excellent.