Transplantation of CD34+ peripheral blood progenitor cells after high- dose chemotherapy for patients with advanced multiple myeloma

A major potential problem of autologous transplantation in the treatment of advanced malignancy is the infusion of tumor cells. A multi-institutional study of purified CD34-selected peripheral blood progenitor cell (PBPC) transplantation was conducted in 37 patients with advanced multiple myeloma receiving myeloablative chemotherapy. Fourteen days after intermediate-dose cyclophosphamide, prednisone, and granulocyte colony-stimulating factor (G-CSF), a median of 3 (range, 2 to 5) 10-L leukaphereses yielded 9.8 x 10(8)/kg (range, 3.7 to 28.3) mononuclear cells. The adsorbed (column-bound) fraction contained 5.9 x 10(6) cells/kg (range, 1.6 to 25.5) with 4.65 x 10(6) CD34 cells/kg (range, 1.2 to 23.3). Using Poisson distribution analysis of positive polymerase chain reactions with patient-specific complementarity- determining region 1 (CDR1) and CDR3 Ig-gene primers, tumor was detected in leukapheresis products from 8 to 14 unselected patients and ranged from 1.13 x 10(4) to 2.14 x 10(6) malignant cells/kg. After CD34 selection, residual tumor was detected in only three patients' products. Overall, a greater than 2.7- to 4.5-log reduction in contaminating multiple myeloma cells was achieved. CD34 PBPCs were infused 1 day after busulfan (14 mg/kg) and cyclophosphamide (120 mg/kg), and granulocyte-macrophage colony-stimulating factor was used until hematologic recovery. The median time to both neutrophil and platelet recovery was 12 days (range, 11 to 16 days and 9 to 52 days, respectively). The median number of erythrocyte and platelet transfusions was 7 (range, 2 to 37) and 3 (range, 0 to 85), respectively. Patients receiving fewer than 2 x 10(6) CD34 cells/kg had significantly prolonged neutropenia, thrombocytopenia, and an increased red blood cell and platelet transfusion requirement. Thus, CD34 selection of PBPCs markedly reduces tumor contamination in multiple myeloma and provides effective hematopoietic support for patients receiving myeloablative therapy.     

Kinetics of factor VIII-von Willebrand factor association

The binding of factor VIII to von Willebrand factor (vWF) is essential for the protection of factor VIII against proteolytic degradation in plasma. We have characterized the binding kinetics of human factor VIII with vWF using a centrifugation binding assay. Purified or plasma vWF was immobilized with a monoclonal antibody (MoAb RU1) covalently linked to Sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden). Factor VIII was incubated with vWF-RU1-Sepharose and unbound factor VIII was separated from bound factor VIII by centrifugation. The amount of bound factor VIII was determined from the decrease of factor VIII activity in the supernatant. Factor VIII binding to vWF-RU1-Sepharose conformed to the Langmuir model for independent binding sites with a Kd of 0.46 +/- 0.12 nmol/L, and a stoichiometry of 1.3 factor VIII molecules per vWF monomer at saturation, suggesting that each vWF subunit contains a binding site for factor VIII. Competition experiments were performed with a recombinant vWF (deltaA2-rvWF), lacking residues 730 to 910 which contain the epitope for MoAB RU1. DeltaA2-rvWF effectively displaced previously bound factor VIII, confirming that factor VIII binding to vWF-RU1-Sepharose was reversible. To determine the association rate constant (k(on)) and the dissociation rate constant (k(off)), factor VIII was incubated with vWF-RU1-Sepharose for various time intervals. The observed association kinetics conformed to a simple bimolecular association reaction with k(on) = 5.9 +/- 1.9 x 10(6) M(-1) s(-1) and k(off) = 1.6 +/- 1.2 x 10(-3) s(-1) (mean +/- SD). Similar values were obtained from the dissociation kinetics measured after dilution of preformed factor VIII-vWF-RU1-Sepharose complexes. Identical rate constants were obtained for factor VIII binding to vWF from normal pooled plasma and to vWF from plasma of patients with hemophilia A. The kinetic parameters in this report allow estimation of the time needed for complex formation in vivo in healthy individuals and in patients with hemophilia A, in which monoclonally purified or recombinant factor VIII associates with endogenous vWF. Using the plasma concentration of vWF (50 nmol/L in monomers) and the obtained values for K(on) and K(off), the time needed to bind 50% of factor VIII is approximately 2 seconds.     

Functional abnormalities of human neutrophils collected by continuous flow filtration leukopheresis.

Continuous flow filtration leukopheresis (FL) is a relatively simple, inexpensive, and efficient technique of harvesting blood neutrophils from normal donors for transfusion into neutropenic recipients. There has been concern, however, that neutrophils may be functionally altered druing this leukopheresis procedure. Human neutrophils obtained by various FL techniques were studied for in vitro chemotaxis by a 51Cr-radiolabel method and for in vitro killing and phagocytosis of Staphylococcus aureus. We compared their function with neutrophils obtained by the NCI-IBM cell separator and by dextran sedimentation from whole blood. FL neutrophils eluted from nylon filters after 3-hr collection periods were functionally abnormal by all parameters tested, while neutrophils obtained by cell separator after similar collection times were not significantly different from control cells. However, neutrophils from 3-hr FL collections were found to include both normal and abnormal populations of cells. Loosely adherent cells, eluted after tapping the filters, were functionally normal; more adherent cells, eluted after tapping the filters and representing the bulk of cells collected, were progressively more abnormal the less readily they were eluted. Shortened FL collection times (1-2 hr) were found to decrease the functional defects. Also, administration of dexamethasone to donors prior to filtration leukopheresis diminished the functional defects of FL neutrophils perhaps by altering adherence characteristics of the cells. These studies show that neutrophils obtained by filtration leukopheresis are functionally abnormal in relation to the time and extent of adherence to nylon filters.     

Efficient surface expression of platelet GPIIb-IIIa requires both subunits

Platelet membrane GPIIb-IIIa is a member of the integrin family of heterodimeric adhesion receptors. Processing and export of certain leukocyte and melanoma integrins is disrupted in cells lacking one subunit. We found that surface expression of GPIIb-IIIa, measured by fluorescent activated cell sorting or by surface labeling, required cotransfection of both subunits. In contrast, surface expression was not detected when the subunits were transfected individually. Immunoprecipitation of metabolically labeled transfected cells confirmed the presence of comparable levels of intracellular protein in all cases. When both subunits were transfected, post-translational cleavage of Pro-GPIIb to yield GPIIb heavy chain was also seen, while transfection with GPIIb alone resulted in coprecipitation of Pro-GPIIb with a second band that may be an endogenous beta subunit. Pro-GPIIb in these transfectants was not processed to yield GPIIb heavy chain. When transfected into COS cells alone, transiently expressed GPIIIa remained intracellular and did not appear to complex with any endogenous proteins. Thus, surface expression of processed GPIIb-IIIa depends on the presence of both subunits; the coordinate reduction of both subunits observed in some cases of Glanzmann's thrombasthenia may result from mutation affecting only one.     

The beta subunit of the interleukin-2 receptor mediates interleukin-2 induction of anti-CD3 redirected cytotoxic capability in large granular lymphocytes

The interleukin 2 (IL 2) receptor was studied in three cases of large granular lymphocyte (LGL) lymphocytosis. All cases were nonreactive with anti-Tac monoclonal antibody (MoAb; recognizing the p55 alpha subunit of the IL 2 receptor). Sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoretic analysis (PAGE) of cells to which radio-labeled rIL 2 had been chemically crosslinked revealed uniform expression of the p70/75 beta subunit of the IL 2 receptor in the absence of the alpha subunit. Stimulation of this receptor with 2 nmol/L rIL 2 for five days led to acquisition of anti-CD3 redirected cytotoxicity. This was accompanied by a fivefold to tenfold elevation in the activity of intracellular N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl esterase, an LGL granule marker enzyme. These effects of IL 2 did not require induction of the Tac peptide.     

Lack of plasminogen activator inhibitor-1 effect in a transgenic mouse model of metastatic melanoma

Tumor cell invasion and metastasis is a complex, multistep process that is postulated to require degradation of extracellular matrix at several steps. Urokinase-type plasminogen activator (uPA) is expressed on the cell surface of B16 murine melanoma cells and is thought to contribute to the pericellular proteolysis necessary for tumor cell migration. In vitro modification of B16 melanoma cell surface uPA activity has been shown to alter the invasive and metastatic potential of these murine melanoma cells in vivo. Plasminogen activator inhibitor-1 (PAI-1), a rapid inhibitor of both uPA and tissue-type plasminogen activator (tPA) is the major physiologic regulator of plasminogen activator activity. To test the role of host PAI-1 in the invasive and metastatic capacity of B16 melanoma cells we analyzed local tumor growth and pulmonary metastasis in transgenic mice engineered to overexpress murine PAI-1 in multiple tissues including lung, and in mice completely deficient in PAI-1. No significant difference in the number of pulmonary metastases was observed after intravenous inoculation of tumor cells into PAI-1- overexpressing and PAI-1-deficient mice when compared with wild-type controls. Similarly, in a spontaneous metastasis model, PAI-1- overexpressing and PAI-1-deficient mice demonstrated no difference in primary tumor size or overall survival. These data demonstrate that wide variations of host PAI-1 expression, from complete absence to marked overexpression, does not significantly influence the metastatic potential of B16 melanoma cells in a murine model.     

Is activation of the granulocyte by concanavalin-A a reversible process?

The stimulation of granulocyte O2- production by concanavalin-A can be reversed with alpha-methylmannoside. Such cells can be reactivated to generate O2- by adding phorbol myristate acetate or N-formyl-methionyl- leucyl-phenylalanine. Opsonized zymosan, however, is not an effective stimulant to these cells. alpha-Methylmannoside prevents, but does not reverse, depolarization of granulocytes by concanavalin-A. Previously activated cells have a shorter lag time for reactivation by phorbol myristate acetate. Incubation in 2-deoxyglucose of cells previously treated with concanavalin-A and alpha-methylmannoside prevents reactivation. EGTA prevents concanavalin-A-stimulated O2- production only when added prior to the stimulant. EGTA has only a slight effect on reactivation. alpha-Methylmannoside prevents concanavalin-A- stimulated release of lysozyme only when added prior to the stimulant. Prior treatment of cells with concanavalin-A and alpha-methylmannoside inhibits subsequent ingestion of complement-coated particles. We conclude that although the O2--generating system can be reversibly activated with concanavalin-A followed by alpha-methylmannoside, these cells are different from untreated cells. Cells treated in such a way do not respond to all stimuli, remain depolarized, have shortened lag times, no longer require calcium for activation, continue to degranulate, and do not ingest well. Thus, although some changes that accompany the interaction of stimuli with granulocytes are reversible, some are not, and the previously activated cell does not return to a true resting state.     

Generation of natural killer cells from Thy 1.1+ bone marrow precursor cells in the rat

We have recently described a long-term bone marrow culture (LTBMC) system in the rat for the generation of natural killer (NK) cells from bone marrow (BM) precursors in the presence of interleukin-2 (IL-2). We found that the LTBMC-conditioned medium was essential to render the NK precursor cells responsive to IL-2. In this report, we isolated by flow cytometric cell sorting Thy 1.1+ BM cells, which have been shown to contain pluripotent stem cells and early precursor cells of various hematopoietic lineages. These Thy 1.1+ immature BM precursors did not generate any detectable NK activity when cultured for 7 days with IL-2 alone. However, when the cells were cultured with IL-2 in the presence of LTBMC-conditioned medium, NK cells were generated as demonstrated by cytolytic activity against NK-sensitive tumor targets, large granular lymphocyte (LGL) morphology, and the acquisition of NK cell-associated phenotype (72% of the cells were 3.2.3+/OX41-/CD5-). This study demonstrates the existence of an IL-2 unresponsive Thy 1.1+ NK precursor in the BM of the rat, that can differentiate to a mature NK cell in the presence of LTBMC-conditioned medium and IL-2.     

Synthesis of cobalamin coenzymes by human lymphocytes in vitro and the effect of folates and metabolic inhibitors

The uptake of 57Co-cyanocobalamin (CN-Cbl) and its conversion to 5- deoxyadenosylcobalamin (Ado-Cbl), methylcobalamin (Me-Cbl), and hydroxocobalamin (OH-Cbl) has been studied in phytohemagglutinin (PHA)- transformed lymphocytes from normal subjects and patients with patients with pernicious anemia. Uptake and conversion were much greater by PHA- stimulated lymphocytes than by mature non-transformed lymphocytes. In normal cells, uptake of 57Co-CN-Cbl and synthesis of the cobalamin coenzymes were approximately linear between 3 and 48 hr incubation. Ado- Cbl was the major cobalamin formed, and after 72 hr the cells contained about twice as much Ado-Cbl as Me-Cbl. Uptake by lymphocytes from patients with untreated pernicious anemia (PA) was greater than that by normal lymphocytes, but the proportions of Ado-Cbl and Me-Cbl synthesized by each were similar. Folic acid and methyltetrahydrofolate enhanced synthesis of Me-Cbl both in normal and in PA cells, while methotrexate and 5-fluorouracil depressed it. This depression was overcome by 5-formyltetrahydrofolate, suggesting that an uninterrupted folate cycle may play an important role in Me-Cbl synthesis.     

The acute chest syndrome in sickle cell disease: incidence and risk factors. The Cooperative Study of Sickle Cell Disease

The acute chest syndrome (ACS), a pneumonia-like illness in sickle cell patients, is one of the most frequent causes of their morbidity and hospitalizations. Repeated ACS events may predict the development of chronic lung disease. ACS is reported as a frequent cause of death in these patients. We examine here the incidence and risk factors of ACS in 3,751 patients with sickle cell disease who were observed prospectively for at least 2 years (19,867 patient-years [pt-yrs]) as part of a multicenter national study group. The ACS, defined by a new pulmonary infiltrate on x-ray, occurred at least once in 1,085 patients (2,100 events). ACS incidence was higher in patients with homozygous sickle cell disease (SS; 12.8/100 pt-yrs) and in patients with sickle cell-beta(0) -thalassemic (9.4/100 pt-yrs), and lower in patients with hemoglobin (Hb) SC disease (5.2/100 pt-yrs) and patients with sickle cell-beta(+) thalassemia (3.9/100 pt-yrs). alpha-Thalassemia did not affect the rate of ACS incidence in SS patients. Within each Hb type the incidence was strongly but inversely related to age, being highest in children 2 to 4 years of age (25.3/100 pt-yrs in SS) and decreasing gradually to its lowest value in adults (8.8/100 pt-yrs in SS). In SS children (< 10 years of age), we documented an age-related within- person reduction in ACS attack rates. Adults with a higher ACS rate had a higher rate of mortality (from all causes) than those with low ACS rates. This increased rate of mortality might also have contributed to the decline in ACS rate with age. In multivariate analysis, other factors affecting incidence in SS patients were degree of anemia (lower ACS rates in patients with lower steady-state Hb levels) and fetal Hb (lower rates in patients with high fetal Hb). There was also a positive association between ACS rate and steady-state leukocyte count. The relationship of ACS rate to higher steady-state Hb levels in SS patients is unexplained but might be caused by increased blood viscosity.