Characterization of polyvalent allogeneic vaccines.

Allogeneic polyvalent vaccines have significant therapeutic and manufacturing advantages including: (i) the presence of multiple tumour-associated antigens; (ii) the potential benefit of viable but non-replicating cells which can provide persistent antigen presentation to the patient, and (iii) the ability to be consistently manufactured in large lots that can be used to treat multiple patients and be fully tested before release. Canvaxin is an example of a multi-cell allogeneic, polyvalent active immunotherapy and has been extensively tested in phase I and II clinical trials. Results of these clinical studies show a statistically significant increase in median and five-year survival of stage III and stage IV surgically resected patients with melanoma as compared to matched historical controls. Phase III randomized double-blind trials in both stage III and stage IV patients with melanoma are in progress. Manufacture of allogenic whole cell vaccines requires identity testing and assurance that the vaccine is replication-incompetent. Since allogeneic whole-cell vaccines contain multiple antigens and patient immune responses may occur to several different antigens, it may be essential to test-for multiple antigens on the cells in the vaccine. This can be accomplished using quantitative flow cytometry to assess cell-surface antigens on viable cells and intracellular antigens on fixed and permeablilized cells. In addition, multi-cell allogeneic vaccines may also be assessed for content using PCR assays to identify unique cell line-associated DNA microsatellites to verify that each of the cell lines is present in the final product. Finally, a critical issue with allogeneic tumour cell vaccines is the assurance that irradiation of the final product is effective and that cells have been rendered replication-incompetent. This can be achieved using both high sensitivity visual assessment of the vaccine following extended culture and comparing cell cultures from irradiated vaccine and irradiated vaccine spiked with replication competent vaccine cells. This approach has been shown to have a limit of detection of one replication-competent cell in one million non-replicating, irradiated vaccine cells. DNA-based assays such as BrdU incorporation can also be used to assess cell replication, but in our hands these assays are generally less sensitive. Phase I/II data with Canvaxin are encouraging and support the further testing and extension of this approach to other tumour vaccines. In addition, the manufacture, testing, and release of whole cell active immunotherapies can be achieved using quantitative antigen testing and proliferation assays to ensure the consistent manufacturing necessary to produce a commercial product.

A competitive fluorescence assay to measure the reactivity of compounds.

A sensitive competitive method was developed for assessing the reactivity of compounds toward glutathione and toward thiols in general. The method employs the reaction of the fluorogenic reagent fluorescein-5-maleimide (FM) with glutathione (GSH) to generate a large increase in fluorescence emission. When the reaction is measured in the presence of a compound that competes with FM toward GSH, the rate constant for fluorescent product formation increases while the total amount of product formed at the end of the reaction decreases. These changes in the presence of a series of competitor concentrations allow one to calculate the rate constant of the reaction of the competitor with GSH. At 23 degrees C, pH 7.40 in PBS buffer the second-order rate constant of the FM-GSH reaction is k2 = (1.67 +/- 0.32) x 10(4) M(-1) x s(-1). Two GSH-reactive compounds were evaluated: the second-order rate constant for the reaction of PNU-27707 with GSH under our experimental conditions is k(i) = 5660 +/- 266 M(-1) x s(-1), while that of PNU-37802 is k(i) = 21,200 +/- 1600 M(-1) x s(-1). The method is easily adaptable to a high-throughput screening format.

Use of anti-CD3 x anti-HER2/neu bispecific antibody for redirecting cytotoxicity of activated T cells toward HER2/neu+ tumors.

Relapse after adjuvant chemotherapy or high-dose chemotherapy with stem cell transplant for high-risk breast cancer remains high and new strategies that provide additional antitumor effects are needed. This report describes methods to generate highly effective HER2/neu-specific cytotoxic T cells by arming activated T cells with anti-CD3 x anti-HER2/neu bispecific antibody (BsAb). OKT3 and 9184 (anti-HER2) monoclonal antibodies (mAb) were conjugated and used to arm T cells that were subsequently tested in binding, cytotoxicity, and cytokine secretion assays. Armed T cells aggregated and specifically killed HER2/neu(+) breast cancer cells. Cytotoxicity emerged after 6 days of culture, was higher in armed T cells than unarmed T cells at all effector to target ratios (E/T) tested, and increased as the arming dose was increased. At an E/T of 20:1, the mean cytotoxicity of armed activated T cells (ATC) from 10 normal subjects increased by 59 +/- 11% (+/-SD) over that seen in unarmed ATC (p < 0.001) and the mean cytotoxicity of armed ATC from 6 cancer patients increased by 32 +/- 9% above that seen for unarmed ATC (p < 0.0004). After arming, the BsAb persisted on ATC up to 72 h and armed ATC continued to be cytotoxic up to 54 h. The amount of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted was 1699, 922, and 3092 pg/ml/10(6) cells per 24 h, respectively, when armed T cells were exposed to a HER2/neu(+) breast carcinoma cell line. These studies show the feasibility and clinical adaptability of this approach for generating large numbers of anti-HER2-specific, cytotoxic T cells for clinical trials.

The ligand affinity of proteins measured by isothermal denaturation kinetics.

An isothermal denaturation kinetic method was developed for identifying potential ligands of proteins and measuring their affinity. The method is suitable for finding ligands specific toward proteins of unknown function and for large-scale drug screening. It consists of analyzing the kinetics of isothermal denaturation of the protein-with and without the presence of potential specific ligands-as measured by long-wavelength fluorescent dyes whose quantum yield increases when bound to hydrophobic regions exposed upon unfolding of the proteins. The experimental procedure was developed using thymidylate kinase and stromelysin as target proteins. The kinetics of thermal unfolding of both of these enzymes were consistent with a pathway of two consecutive first-order rate-limiting steps. Reflecting the stabilizing effect of protein/ligand complexes, the presence of specific ligands decreased the value of the rate constants of both steps in a dose-dependent manner. The dependence of the rate constants on ligand concentration obeyed a simple binding isotherm, the analysis of which yielded an accurate equilibrium constant for ligand binding. The method was validated by comparing its results with those obtained under the same conditions by steady-state fluorescence spectroscopy, circular dichroism, and uv spectrophotometry: The corresponding rate constants were comparable for each of the analytical detection methods.

Clinical impact of ex vivo differentiated myeloid precursors after high-dose chemotherapy and peripheral blood progenitor cell rescue.

The infusion of ex vivo differentiated myeloid precursors may be able to shorten the period of obligatory neutropenia after high-dose chemotherapy and peripheral blood progenitor cell rescue by providing cells capable of differentiating to mature neutrophils within days of infusion. To test this hypothesis, 21 female patients with metastatic breast cancer underwent progenitor cell mobilization with cyclophosphamide, etoposide and G-CSF. CD34+ cells from one to two leukapheresis products were isolated and placed in suspension culture with a serum-free growth medium supplemented with PIXY321. The cultures were maintained for 12 days with subcultures initiated on day 7. The remaining leukapheresis products were cryopreserved in an unmanipulated state. Forty-eight hours after completing high-dose cyclophosphamide, thiotepa and carboplatin, the cryopreserved progenitors were infused, followed 1 to 24 h later by infusion of the differentiated myeloid precursors. In one patient, the cultured cells were labeled with Indium-111 with nuclear imaging performed up to 48 h post infusion. The differentiated myeloid precursors were suitable for infusion in 17 of the patients with a median 13-fold expansion of total nucleated cells. A range of 5.6 to 1066 x 10(7) nucleated cells were infused. Morphologically the cells were predominantly of myeloid lineage (63%) with a median 41% of the cells expressing CD15. No untoward effects were noted with the infusion of the cultured cells. The median days to neutrophil and platelet recovery were 8 and 10 days, respectively. There was a significant relationship (r = 0.67, P = 0.007) between the dose of differentiated myeloid precursors (CD15+ cells) and the depth and duration of neutropenia; a similar relationship, however, was also observed with the dose of cryopreserved CD34+ cells. After infusion of the radiolabeled myeloid precursors, a pattern of distribution similar to radio-labeled granulocytes was noted with uptake detected initially in the lungs and subsequently the reticulo-endothelial system. The impact of differentiated myeloid precursors on neutropenia as an adjunct to high-dose chemotherapy and peripheral blood progenitor cell rescue remains unclear from this study. Further study with controlled doses of cryopreserved progenitors and escalating doses of differentiated myeloid precursors is required.

A fluorescence resonance energy transfer method for measuring the binding of inhibitors to stromelysin.

A sensitive fluorescence resonance energy transfer method was developed for the direct measurement of the dissociation constants of stromelysin inhibitors. The method is applied to the thiadiazole class of stromelysin inhibitors and it takes advantage of the fact that, upon binding to the active site of enzyme, the thiadiazole ring, with its absorbance centered at 320 nm, is able to quench the fluorescence of the tryptophan residues surrounding the catalytic site. The changes in fluorescence are proportional to the occupancy of the active site: Analysis of the fluorescence versus inhibitor concentration data yields dissociation constants that are in agreement with the corresponding competitive inhibitory constants measured by a catalytic rate assay. The affinity of nonthiadiazole inhibitors of stromelysin-such as hydroxamic acids and others-can be determined from the concentration-dependent displacement of a thiadiazole of known affinity. Using this displacement method, we determined the affinities of a number of structurally diverse inhibitors toward stromelysin. Since the three tryptophan residues located in the vicinity of the active site of stromelysin are conserved in gelatinase and collagenase, the method should also be applicable to inhibitors of other matrix metalloproteinases.

Identification of a peptide directed against the anti-CD34 antibody, 9C5, by phage display and its use in hematopoietic stem cell selection.

A peptide sequence was identified by phage display technology that could be used as an alternative to chymopapain for the release of hematopoietic progenitor cells captured by anti-CD34 monoclonal antibodies. This was achieved by affinity selection screening (biopanning) of a random hexapeptide sequence phage display library. Four rounds of biopanning were performed to enrich for phage clones with specific affinity for anti-CD34 monoclonal antibody, 9C5. DNA sequence analyses of these phage clones revealed an enrichment of two predominant sequences, QQGWFP and TQGSFW. These two clones also shared a consensus sequence motif, QGxF, that exhibited 50% and 67% homology with a region spanning amino acids 14-19 of the mature CD34 antigen. Based on these data, synthetic peptides were generated and assessed for their ability to release 9C5 from CD34+ cells. Using a flow cytometric assay, it was found that the synthetic peptide, 9069N, effectively released 9C5 from the CD34-expressing cell line, KG1a, in a concentration-dependent manner (77% and 99% release of 9C5 at 0.14 and 0.70 mM peptide concentrations, respectively). In the Isolex 300i immunomagnetic selection system, this peptide was shown to be effective at releasing 9C5 sensitized CD34+ hematopoietic progenitors from sheep anti-mouse IgG Dynabeads. Thus, a synthetic peptide, which specifically and efficiently released immunomagnetically selected hematopoietic progenitor cells from paramagnetic beads, was identified. This reagent is a significant advance in the selection of hematopoietic progenitors in that it does not alter cell surface antigens. As such, further phenotypic characterization or immunoselection can be performed.

Determination of the affinity of drugs toward serum albumin by measurement of the quenching of the intrinsic tryptophan fluorescence of the protein.

Binding of new chemical entities to serum proteins is an issue confronting pharmaceutical companies during development of potential therapeutic agents. Most drugs bind to the most abundant plasma protein, human serum albumin (HSA), at two major binding sites. Excepting fluorescence spectroscopy, existing methods for assaying drug binding to serum albumin are insensitive to higher-affinity compounds and can be labour-intensive, time-consuming, and usually require compound-specific assays. This led us to examine alternative ways to measure drug-albumin interaction. One method described here uses fluorescence quenching of the single tryptophan (Trp) residue in HSA excited at 295 nm to measure drug-binding affinity. Unfortunately, many compounds absorb, fluoresce, or both, in this UV wavelength region of the spectrum. Several types of binding phenomenon and spectral interference were identified by use of six structurally unrelated compounds and the equations necessary to make corrections mathematically were derived and applied to calculate binding constants accurately. The general cases were: direct quenching of Trp fluorescence by optically transparent ligands with low or high affinities; binding of optically transparent, non-fluorescent ligands to two specific sites where both sites or only one site result in Trp fluorescence quenching; and chromophores whose absorption either overlaps the Trp emission and quenches by energy transfer or absorbs light at the Trp fluorescence excitation wavelength producing absorptive screening as well as fluorescence quenching. Unless identification of the site specificity of drug binding to serum albumin is desired, quenching of the Trp fluorescence of albumin by titration with ligand is a rapid and facile method for determining the binding affinities of drugs for serum albumin.

The constituent tryptophans and bisANS as fluorescent probes of the active site and of a secondary binding site of stromelysin-1 (MMP-3).

The active site of the catalytic domain of stromelysin-1 (matrix metalloproteinase-3, MMP-3) was probed by fluorescence quenching, lifetime, and polarization of its three intrinsic tryptophans and by the environmentally sensitive fluorescent reporter molecule bisANS. Wavelength-dependent acrylamide quenching identified three distinct emitting tryptophan species, only one of which changes its emission and fluorescence lifetime upon binding of the competitive inhibitor Batimastat. Significant changes in the tryptophan fluorescence polarization occur upon binding by any of the three hydroxamate inhibitors Batimastat, CAS108383-58-0, and Celltech CT1418, all of which bind in the P2'-P3' region of the active site. In contrast, the inhibitor CGS27023A, which is thought to bind in the P1-P1' region, does not induce any change in tryptophan fluorescence polarization. The use of the fluorescent probe bisANS revealed the existence of an auxiliary binding site extrinsic to the catalytic cleft. BisANS acts as a competitive inhibitor of stromelysin with a dissociation constant of Ki=22 microM. In addition to this binding to the active site, it also binds to the auxiliary site with a dissociation constant of 3.40+/-0.17 microM. The auxiliary site is open, hydrophobic, and near the fluorescing tryptophans. The binding of bisANS to the auxiliary site is greatly enhanced by Batimastat, but not by the other competitive inhibitors tested.

Ex vivo generation of dendritic cells from CD34+ cells in gas-permeable containers under serum-free conditions.

Dendritic cells (DC) are efficient and potent APCs that can be generated ex vivo. For them to be used clinically, however, a closed culture system using serum-free medium should be used. Our goal was to differentiate DC from human blood CD34+ cells in serum-free media in a new gas-permeable culture container, PL2417. Apheresis products were collected from healthy G-CSF-mobilized donors, and CD34+ cells were selected using the Isolex immunomagnetic cell selection system. Cells were cultured in the presence of GM-CSF and tumor necrosis factor-alpha (TNF-alpha) in various serum-free media and compared with serum-containing medium in 4-well plates. One of the serum-free media was then selected and used in PL2417 containers and compared with serum-containing medium in standard flasks. The cells were evaluated at days 0, 7, and 14 for the presence of DC, which were identified morphologically after Wright-Giemsa staining by cytoplasmic processes extending from the surface of the cell. The cultures were evaluated phenotypically by flow cytometry and immunohistochemistry. The stimulatory capacity was examined in MLR. Overall, results from serum-free media and PL2417 containers were comparable results obtained under the other conditions. These data indicate that culture-deriving DC from CD34+ cells in PL2417 closed system containers using serum-free media is as effective as using standard flasks and serum-supplemented media.

Large-scale production of CD4+ T cells from HIV-1-infected donors after CD3/CD28 costimulation.

We describe a procedure for large-scale enrichment, growth, and harvesting CD4+ T cells. This method may be effective for HIV-1 immunotherapy, as the mode of stimulation, with anti-CD3 plus anti-CD28 coated beads (CD3/CD28 beads) induces a potent antiviral effect. PBMC were obtained by density gradient centrifugation of an apheresis product. Monocytes/macrophages were removed by incubating PBMC with beads coated with IgG. The cells were then magnetically depleted of B cells and CD8+ cells with mouse anti-CD20 and anti-CD8 MAbs and sheep antimouse coated beads. The remaining cells were >80% CD4+ and were transferred to gas-permeable bags containing CD3/CD28 beads and cultured in a closed system. After 14 days, the cell number increased an average of 37-fold, and cells were nearly 100% CD4+. Viral load, assessed by DNA PCR for HIV-1 gag, decreased >10-fold during culture in the absence of antiretroviral agents. Removal of CD3/CD28 beads from the cell suspension was accomplished by passing cells plus beads (3-30 x 10(9) cells in 2-12 L) over a MaxSep magnetic separator using gravity-driven flow. The cells were then concentrated to 300 ml in an automated centrifuge. This process allows safe and efficient growth of large numbers of CD4+ T cells from HIV-1+ donors.

Neutrophil precursor generation: effects of culture conditions.

The influence of feeding schedules on the expansion and differentiation of enriched PB CD34+ cells (84.9+/-14.7% purity) was studied after 12-13 days of serum-free liquid culture. CD34+ cell cultures were initiated (n=6) on day 0 (2 x 10(5) cells) in X-VIVO 10 medium containing 1% human albumin (HA) and 100 ng/ml each of rIL-3, rIL-6, rSCF, and rG-CSF. The cultures were supplemented on days 3, 6, and 9 as follows: condition 1, unfed (static culture); condition 2, 100 ng/ml rG-CSF; condition 3, split 1:2 medium + 100 ng/ml each rIL-3, rIL-6, rSCF, and rG-CSF; condition 4, split 1:2 medium + 100 ng/ml rG-CSF. The proliferative capacities (fold increase) of condition 2 (49.1+/-21.3), condition 3 (75.6+/-33.4), and condition 4 (63.1+/-23.8) cultures were significantly higher (p < 0.05) than that of the condition 1 unfed (35.5+/-14.0) cultures. Flow cytometric analysis (CD15-FITC/CD11b-PE) showed that the highest CD15+ cell purity (neutrophil precursors) was found in the condition 3 (1.18 x 10(7)+/-4.29 x 10(6)) cultures, followed by condition 4 (9.84 x 10(6)+/-3.57 x 10(6)), condition 2 (7.54 x 10(6)+/-2.06 x 10(6)), and condition 1 (4.78 x 10(6)+/-9.80 x 10(5)), respectively. The average cloning efficiency of the day 0 enriched CD34+ cells, 15.1%+/-10.3%, decreased to less than 0.2% in all of the day 12-13 cultures. These data suggest that feeding CD34+ cell cultures with rG-CSF alone, medium + rG-CSF, or medium + rIL3, rIL-6, rSCF, and rG-CSF enhances CD15+ neutrophil precursor (promyelocytes, myelocytes, metamyelocytes) production in vitro.

Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease.

Little is known about the potential for engraftment of autologous hematopoietic stem cells in human adults not subjected to myeloablative conditioning regimens. Five adult patients with the p47(phox) deficiency form of chronic granulomatous disease received intravenous infusions of autologous CD34(+) peripheral blood stem cells (PBSCs) that had been transduced ex vivo with a recombinant retrovirus encoding normal p47(phox). Although marrow conditioning was not given, functionally corrected granulocytes were detectable in peripheral blood of all five patients. Peak correction occurred 3-6 weeks after infusion and ranged from 0.004 to 0.05% of total peripheral blood granulocytes. Corrected cells were detectable for as long as 6 months after infusion in some individuals. Thus, prolonged engraftment of autologous PBSCs and continued expression of the transduced gene can occur in adults without conditioning. This trial also piloted the use of animal protein-free medium and a blood-bank-compatible closed system of gas-permeable plastic containers for culture and transduction of the PBSCs. These features enhance the safety of PBSCs directed gene therapy.

Neutrophil maturation of CD34+ cells from peripheral blood and bone marrow in serum-free culture medium with PIXY321 and granulocyte-colony stimulating factor (G-CSF).

Bone marrow (BM) or peripheral blood (PB) CD34+ cells were cultured for 12 days in serum-free culture medium containing PIXY321 (IL-3/ GM-CSF fusion protein) with or without periodic supplements of granulocyte-colony stimulating factor (G-CSF). The cultures were evaluated at day 12 for total cell proliferation (fold increase from day 0), neutrophil differentiation by flow cytometry, using dual staining with CD15-FITC and CD11b-PE, and morphology using Wright-Giemsa and granule staining. In cultures containing PIXY321 where 6000 U/ml of G-CSF was added days 0 and 6, there was no significant difference (p > or = 0.05) in cell proliferation or the percent of CD15+/CD11b+ cells when compared with cultures with PIXY321 alone. ELISA analysis showed G-CSF levels had declined by 90% after 3 days of culture. Further studies were performed to assess the benefit of supplementing lower concentrations of G-CSF (600 U/ml) at more frequent intervals. A significant increase (p < or = 0.05) in cell proliferation and percent CD15+/CD11b+ was observed when G-CSF was added on days 0, 3, 6, and 9 (every 3 days) as compared with those cultures with PIXY321 alone. CD34+ cell proliferation without G-CSF was 19.6 +/- 4.8-fold, with G-CSF added on days 0 and 6 was 28.7 +/- 6.4-fold, and with G-CSF added on days 0, 3, 6, and 9 was 45.9 +/- 10.6-fold. Percent of CD15+/CD11b+ cells was 19.0 +/- 4.6%, 38.2 +/- 7.2%, and 58.5 +/- 6.5%, respectively, in these cultures. We observed more CD15+/CD11b+ cells, myelocytes/metamyelocytes, and secondary granule staining in cultures with G-CSF added on day, 0, 3, 6, and 9 as compared with cultures with G-CSF added on days 0 and 6 or no G-CSF added. We conclude that PIXY321 and G-CSF act synergistically on the in vitro proliferation and neutrophil differentiation of BM and PB CD34+ cells and that frequent supplements of G-CSF facilitate neutrophil differentiation.

The pretransition of dipalmitoyllecithin bilayers as probed by the fluorescent pyrrolopyrimidine, U-104067.

The amphiphilic pyrrolopyrimidine, U-104067, is a fluorophore ideally suited to report on the relative hydrophobicities of different microenvironments. It forms stable monomolecular layers at the air/water interface with a limiting molecular area of 51.9 +/- 0.3 A2/molecule and a collapse pressure of about 18 dyn/cm. Differential scanning calorimetry of its mixed liposomes with dipalmitoyllecithin shows full solubility of the compound in the liquid disordered phase and insolubility in the solid ordered phase. In aqueous solutions, the compound binds to phospholipid bilayers with a stoichiometry of 13.2 +/- 1.2 moles of lipid per mole of U-104067, with Kd = 0.33 +/- 0.05 microM toward egg lecithin/phosphatidylserine bilayers and Kd = 1.5 +/- 0.3 microM toward pure egg lecithin bilayers. In liquid crystalline phospholipid bilayers the compound behaves as two independently emitting species, one accessible to acrylamide and the other one not. Doxyl fatty acid methyl esters quench both species and show that the average position of the fluorophore is at a depth corresponding to that of the 7th carbon of a fatty acyl chain. Dissolved in the liquid disordered (L alpha) phase of dipalmitoyllecithin at 45 degrees C, U-104067 shows a single ionizable group, pKa = 3.19 +/- 0.03 while in the solid ordered (L beta) phase it displays two ionizable groups, pKa1 = 4.99 +/- 0.10 and pKa2 = 6.96 +/- 0.13. The most unusual property of this molecule is that it is miscible with the tilted (L beta) and liquid (L alpha) phases of dipalmitoyllecithin but totally immiscible with the rippled (P beta) phase. Because of this, U-104067 is a sensitive reporter for the tilted/rippled phase transition as monitored by its fluorescence anisotropy and its quantum yield changes.

Fluorescence and site-directed mutagenesis studies of interleukin 1 beta.

The authors mutated two key residues in the sequence of the cytokine interleukin 1 beta, namely the double mutant Phe46 to Trp46 and Trp120 to Phe120 and the single point mutation Lys103 to Leu103 and measured the resulting receptor binding and biological activities. The biological and receptor binding activities of the Trp46 mutein was reduced by a factor of 12 and 25, respectively, and surprisingly, those of the Leu103 mutein, 2600 and 600-fold relative to the wild-type protein. The authors had previously showed that Lys103 was unusually reactive to a variety of derivatizing agents. Furthermore, the Trp to Phe mutation allowed us to monitor the local environment of that residue by studying its intrinsic fluorescence properties, as well as any change in the fluorescence properties of Trp120 of the Leu103 mutein. The results of these studies show that mutation of Lys103 to Leu103 produces subtle long-range changes in the micro-environment of Trp120, indicative of a key role for this residue in the folding of the entire protein.

Ex vivo expansion of frozen/thawed CD34+ cells isolated from frozen human apheresis products.

Human CD34+ cells purified from frozen mobilized peripheral blood apheresis products (n = 7) were studied immediately (freshly isolated) or refrozen and studied after > 30 days storage in liquid nitrogen (refrozen/thawed). The proliferation and differentiation of freshly isolated or refrozen/thawed CD34+ cells were examined after 10 days of serum-supplemented suspension culture with recombinant human hematopoietic growth factors. The proliferative capacity (fold increase) of the refrozen/thawed CD34+ cells (mean +/- SD, 54.3 +/- 34.3) was comparable to the freshly isolated CD34+ cell cultures (49.0 +/- 42.4). Two-color flow cytometry of the CD34+ cultured cell populations, fresh and refrozen/thawed, displayed typical patterns of neutrophil differentiation into CD15/CD11b neutrophil precursors. The colony-forming ability of freshly isolated and refrozen/thawed CD34+ cells showed no significant differences (p > 0.05) in the total number or type of colony-forming units (CFU-GM, CFU-M, BFU-E, CFU-GEMM) obtained. In addition, the cloning efficiencies of freshly isolated (19.5 +/- 7.6%) and refrozen/thawed CD34+ cells (21.9 +/- 12.7%) were comparable (p = 0.366). These data suggest that CD34+ cells enriched from frozen apheresis blood products can be either used immediately or stored in liquid nitrogen and thawed with minimal effect on their ability to proliferate and differentiate in liquid culture.

Large-scale selection of CD34+ peripheral blood progenitors and expansion of neutrophil precursors for clinical applications.

Hematopoietic recovery after high-dose chemotherapy is characterized by an obligate period of neutropenia of approximately 8-10 days. It is postulated that if a pool of neutrophil precursors and progenitors were expanded in vitro and reinfused, the duration of neutropenia may be substantially shortened by these cells capable of providing mature neutrophils within days of reinfusion. In this study, peripheral blood progenitor cell products were obtained from six normal donors mobilized with rhG-CSF and two patients mobilized with cyclophosphamide and rhG-CSF. CD34+ cells were isolated using the Isolex immunomagnetic bead method. A mean of 8.26 x 10(7) CD34+ cells with a mean purity of 74.5% were seeded at a concentration of 1 x 10(5)/ml into a 12 day stroma-free liquid culture using gas-permeable bags. A serum-free growth medium supplemented with PIXY321 was used. On day 7, there was a mean cellular expansion of fourfold, at which time the cells were resuspended at the initial concentration, yielding a mean culture volume of 3L (1-6 L). On day 12, there was an additional mean fold cellular expansion of 10 x, achieving an overall mean fold expansion of 41 +/- 16. Cellular characterization of the expanded cells revealed predominantly neutrophil precursors by morphology (mean 70.1%) and flow cytometric analysis. A mean of 52.3% of the expanded cells expressed CD15. Immunohistochemical staining revealed a mean of 7.1% CD41a+ megakaryocytic progenitors in the final cultured cell product. Detectable CD34+ cells were maintained only in those cultures initiated with greater than 90% CD34+ cells. Colony-forming units-granulocyte-macrophage (CFU-GM) were maintained in the 12 day culture at a level similar to the preculture number, whereas CFU mixed were depleted in all samples. On day 0, there were few CFU clusters (colonies containing fewer than 50 cells) identified, but by day 12, a mean total of 8.3 x 10(6) CFU clusters were identified. On day 12, the expanded cells were harvested and pooled using the Fenwal CS3000 Plus blood cell separator and resuspended in Plasma-Lyte-A with 1% human serum albumin. The mean harvest recovery of expanded progenitors was 91%, with a mean viability of 86%.

Infusion of ovine C5a into sheep mimics the inflammatory response of hemodialysis.

Previous studies in our group have explored the inflammatory response in sheep to dialysis with a variety of different hemodialysis membranes. In the present study we investigated the potential role of C5a in mediating inflammatory responses that have been attributed to complement activation in the extracorporeal setting. Sheep C5a was infused into sheep in a manner that simulated exposure to this anaphylatoxin during dialysis. C5a infusion into sheep was shown to produce a dose-dependent neutropenia that was quantitatively and temporally identical to the response of sheep undergoing dialysis with complement-activating membranes. The two lowest doses used (0.25 and 0.50 micrograms/kg), which resulted in concentrations below the detectable limits of current assays (10 ng/ml), produced significant neutropenia (21.8% and 78.1%, respectively). The ability of the neutrophils (PMNs) to bind fluorescein isothiocyanate-C5a or initiate a respiratory burst in response to phorbol myristate acetate were also affected in a dose-dependent manner. In contrast, C5a alone was not able to produce significant release of lactoferrin, a specific granule constituent, suggesting that degranulation of PMN-specific and primary granules requires secondary stimuli. The production of thromboxane A2 and thromboxane's consequent cardiopulmonary effect of increasing mean pulmonary artery pressure were both observed in a dose-dependent fashion. However, larger amounts of C5a were required to elicit these latter responses as compared with the PMN activities. These results suggest that C5a may be a primary mediator of complement-dependent events that occur during extracorporeal therapies such as hemodialysis, and they also suggest that very little complement activation is necessary to activate leukocytes, whereas higher thresholds are required to produce cardiopulmonary responses.

Immunocytochemistry and flow cytometry evaluation of human megakaryocytes in fresh samples and cultures of CD34+ cells.

Adhering platelets on the cell surface can give misleading results when doing flow cytometry analysis of platelet/megakaryocyte-specific glycoprotein (GP) antigens to enumerate megakaryocytes (MK) in mobilized peripheral blood (PB), apheresis products, or normal bone marrow (BM). For adequate quantification and characterization of human MK, we examined samples with parallel flow cytometry and immunocytochemistry. MK expression of GP IIb/IIIa (CD41a), GP Ib (CD42b), GP IIIa (CD61), CD45, CD33, and CD11b, and their light scatter properties were evaluated. Fresh samples of low density mononuclear cells (MNC) or purified CD34+ cells contained 10-45% of platelet-coated cells. Platelet-coated cells decreased dramatically after several days of incubation in a serum-free medium supplemented with stem cell factor, IL-3, IL-6, and/or GM-CSF. Between d 9-12, flow cytometry detected a distinct CD41a+ MK population, 8.3 +/- 1.3% in BM CD34 cell cultures (n = 7) and 13.1 +/- 2.1% in PB CD34 cell cultures (n = 14), comparable to immunocytochemistry data (7.8 +/- 1.9% and 16.4 +/- 2.6%, respectively). CD41a stained a higher proportion of MK than CD42b or CD61, while CD42b+ or CD61+ cells contained more morphologically mature MK than CD41a+ cells in cultures containing aplastic serum. When fluorescence emission of CD41a was plotted against forward-light scatter (FSC), subpopulations of small and large MK were observed. Such subpopulations overlapped in CD41a intensity and side-light scatter (SSC) property. Most MK co-expressed CD45 (98.8% positive) but not CD33 (80.7% negative) or CD11b (88.9% negative). Our data indicate that flow cytometry can be used effectively to identify MK. However, caution should be taken with samples containing adherent platelets.