Flow cytometric analysis of intercellular adhesion between B-cell precursor acute lymphoblastic leukemic cells and bone marrow stromal cells.

The growth of B-cell precursor acute lymphoblastic leukemic (BCP ALL) cells in vitro is dependent on interactions with bone marrow (BM) stromal cells. We have recently demonstrated that the rate of cell division of BCP ALL cells increases when cultured in direct contact with BM stromal cells. In this study we describe a new method for examining the direct binding of BM stromal cells and BCP ALL cells at a cellular level. For this binding assay, BCP ALL cells from six patient samples were first stained with the lipophilic fluorescent probe PKH 26 GL and mixed with BM stromal cells in suspension. In all cases, aggregates between BCP ALL and BM stromal cells were identified by flow cytometry and isolated. Using this assay we have examined some of the mechanisms involved in this binding process. The pattern of aggregate formation at various leukemic/stromal cell ratios showed that the aggregate formation increased by increasing the number of either cell type and that the binding could not be saturated. This suggests that the interaction between these cells is an equilibrium reaction. Functional studies showed that the majority of BCP ALL-BM stromal cell binding is dependent on the presence of divalent cations and requires active cellular metabolism. Finally, by use of inhibitory monoclonal antibodies (moAbs) directed against cell adhesion molecules including anti-CD29, VCAM and CD18, we have demonstrated that the involvement of these molecules in the direct cellular interactions could be detected by this method. However, the maximum inhibition observed was 36% which suggests either that the avidity is low or that other adhesion molecules are involved. The data show that the use of flow cytometric analysis of aggregate formation (rather than cell binding to intact cell layers) allows the study of cell interactions at the individual cell level which can reveal additional cellular adhesion mechanisms.

Use of granulocyte-macrophage colony-stimulating factor in two children treated with cord blood transplantation.

Cord blood contains stem cells in amounts similar to or slightly less than those present in a bone marrow collection to be used for bone marrow transplantation (BMT). Too few cord blood transplants (CBT) have yet been performed to define the ability to achieve engraftment and the rate of engraftment. Two cord blood transplants have been performed using granulocyte-macrophage colony stimulating factor (GM-CSF) to hasten engraftment. Two children, aged 5 and 6 years received a CBT using HLA-identical stem cells collected at the birth of a sibling. One child had X-linked lymphoproliferative disease (XLP), and the other, acute lymphoblastic leukemia in second complete remission. One had an ABO and one an Rh blood group mismatch. Conditioning therapy consisted of cyclophosphamide, melphalan, and antithymocyte globulin or busulphan and cyclophosphamide. Graft-versus-host disease prophylaxis was methotrexate and cyclosporine or cyclosporine. Both children were given GM-CSF at 5 micrograms/kg/day from day 1 until the absolute neutrophil count (ANC) reached 1.0 x 10(9)/L for 3 consecutive days. If this level was not reached by day 14, the dose of GM-CSF was doubled. Both children engrafted rapidly, with ANCs reaching 0.5 x 10(9)/L in 12 and 16 days. Engraftment was confirmed by blood group in both and sex chromosome typing in one. Both children developed mild GVHD localized to skin, which resolved with steroid therapy. The child with XLP was cured and has survived for 34 months; the second child has survived 27 months with normal marrow function but has had a relapse of leukemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Selection of two human bone marrow stromal cell subpopulations with different effects on the maintenance and differentiation of myeloid progenitor cells.

The simple selection of two human bone marrow stromal cell populations is described. Adherent stromal cell layers were formed in primary cultures of low-density marrow cells. At time of confluence, persistent nonadherent cells were collected and transferred to new culture flasks, where they formed a secondary stromal layer. These primary and secondary stromal layers differed in their ability to support myelopoiesis, as tested by progenitor cell production after inoculation with fresh bone marrow cells. In the presence of primary stromal layers the number of granulocyte-macrophage colony-forming cells (GM-CFC) decreased gradually, but in the presence of secondary layers production of GM-CFC was evident during the first 3 weeks. The regulation of the two stromal types on hemopoietic cell proliferation and differentiation was investigated by determining the kinetics of the transitions within the differentiation sequence of three myeloid progenitor cells. Pre-CFC, day-14 CFC, and day-7 CFC were fractionated by cell sorting on the basis of forward light scatter and cocultured with the two stromal layer types. It was found that the decrease in CFC numbers in the presence of primary stromal layers could be explained by the stimulation of hemopoietic cells into rapid differentiation with loss of proliferative capacity at an early stage of culture. Secondary layers appeared to promote survival and self-renewal of later types of progenitor cells and to trigger more immature cells to proliferate and differentiate at a later time of culture.

Bromacil and diuron residue levels in Florida citrus soils.

The widespread use of herbicides in Florida citrus groves raises the possibility of residue accumulation following repeated applications. To determine residue levels of commonly used herbicides, soil samples were taken from large experimental plots in commercial groves in Polk and Hardee Counties. Bromacil and diuron had been applied in combination at both locations for 7-8 years. Analyses of samples showed low levels of both herbicides at various soil depths to 60 cm. Only a small amount of bromacil was detectable one year after applications, but diuron levels were higher. Continuous applications at recommended rates and frequencies have resulted in maximum bromacil and diuron levels of 3.9 percent and 13.1 percent, respectively, of their total application.