Human CC chemokines macrophage inflammatory protein (MIP)-1␣, MIP-1␤, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates. To explore the biological significance of chemokine aggregation, nonaggregating variants were sought. The phenotypes of 105 hMIP-1␣ variants generated by systematic mutagenesis and expression in yeast were determined. hMIP-1␣ residues Asp 26 and Glu 66 were critical to the self-association process. Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml. Substitution of identical or analogous residues in homologous positions in both hMIP-1␤ and RAN-TES demonstrated that they were also critical to aggregation. Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present. Solution of the three-dimensional NMR structure of hMIP-1␣ has enabled comparison of these residues in hMIP-1␤ and RANTES. Aggregated and disaggregated forms of hMIP-1␣, hMIP-1␤, and RANTES generally have equivalent G-protein-coupled receptormediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1␣, hMIP-1␤, and RANTES aggregation in vitro and in vivo. The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M-and T-tropic HIV isolates/strains. This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.

Cell proliferation in the bone marrow and blood of two patients with metastatic breast cancer who were treated with granulocyte colony-stimulating factor was studied by using [3H]thymidine labeling and autoradiography. Additionally, the fate of neutrophils labeled with "Tc-hexamethylpropyleneamineoxime was observed following granulocyte colony-stimulating factor infusion. Proliferation increased in all stages of granulopoiesis, but a significant amount of the increased production stemmed from a greater input to the myeloblast compartment. Changes in the myelogram combined with the increased labeling indicated a faster throughput of cells, which resulted in labeled cells appearing in the circulation within 1 day compared to the normal 4 or 5 days. The ""'Tc studies demonstrated no sequestration of circulating neutrophils by spleen, lungs, or liver. The halflife of the circulating neutrophils was not significantly changed, and calculations from the flow of labeled cells to the peripheral blood indicated an increase of 3.2 extra amplification divisions during neutrophil development. The dramatic neutrophil response to granulocyte colony-stimulating factor can therefore be accommodated by a relatively modest increase in granulopoietic activity.The recent availability of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (1) has stimulated considerable interest in its potential applications for promoting hemopoietic regeneration following bone marrow damage by cytoreductive agents. Stimulation, in vivo, of granulopoiesis by rhG-CSF was demonstrated in mice (2) and in humans (3-7). In patients, 2 or 3 days of continuous infusion or repeated injections of rhG-CSF resulted in a 10-fold increase in peripheral neutrophil levels, which were maintained for the duration of infusion and, following chemotherapy, significantly shortened the duration of the subsequent neutropenia (3,(5)(6)(7). These neutrophils demonstrated normal function and competence (4). Only a small, nonsignificant increase in granulocyte/macrophage colony-forming cell (GM-CFC) cycling and in the GM-CFC/burst-forming unit-erythroid ratio was observed, suggesting that most of the expansion in neutrophil production probably arises after the GM-CFC stage (4). Our intention was to investigate the changes in kinetics induced by G-CSF and required to maintain this elevated neutrophilia. We therefore treated two patients with rhG-CSF and, by means of autoradiography, studied the kinetics of marrow cell proliferation and efflux into the peripheral blood following labeling in vivo with tritiumlabeled thymidine. Since rhG-CSF in vivo initially produces an early fall in peripheral neutrophils followed by rapid influx of mature neutrophils into the circulatory pool (4), we also studied the early effects of this growth factor on circulating neutrophils by labeling them with 99mTc-hexamethylpropyleneamineoxime (9mTc-HMPAO) (8) and observing their fate with a y camera. MATERIALS AND METHODSPatients and Therapy. Two patients with metastatic breast c...

ABSTRACT+ AC133 + population was also enriched (sevenfold) in dendritic cell precursors, and the dendritic cells generated were functionally active in a mixed lymphocyte reaction assay. AC133 + cells should be useful in the study of cellular and molecular mechanisms regulating primitive hemopoietic cells.

Femoral bone marrow was divided longitudinally into two groups of cells of varying size. By assaying CFU and CFU in the two zones of the marrow, their distributions across the diameter of the femur was determined. It is shown that the concentration of CFU increases from the femoral axis (15 CFU/105 bone marrow cells) to the bone surface (44 CFU/105 cells), obeying approximately a square-law relationship. The CFU concentration, on the other hand, increases from the femoral axis (32CFU/105 cells) to a peak value (260 CFU/105 cells) at about 330 um from the axis and thence falls off against to the bone surface (77 CFU/105 cells). Selective kinning cells in DNA synthesis using the tritiated thymidine suicide technique, in vivo, showed that CFU, near the bone surface are proliferating at a faster rate than those more distant from bone, but that CFU have a fast proliferation rate irrespective of their position in the distribution. Thus, bone marrow cell populations are shown to conform to a well-defined spatial organization corresponding to the chronologic relationships between marrow cells.