Alterations in circulating lymphoid cell populations in systemic small vessel vasculitis are non-specific manifestations of renal injury.

Innate lymphocyte populations, such as innate lymphoid cells (ILCs), γδ T cells, invariant natural killer T (iNK T) cells and mucosal-associated invariant T (MAIT) cells are emerging as important effectors of innate immunity and are involved in various inflammatory and autoimmune diseases. The aim of this study was to assess the frequencies and absolute numbers of innate lymphocytes as well as conventional lymphocytes and monocytes in peripheral blood from a cohort of anti-neutrophil cytoplasm autoantibody (ANCA)-associated vasculitis (AAV) patients. Thirty-eight AAV patients and 24 healthy and disease controls were included in the study. Patients with AAV were sampled both with and without immunosuppressive treatment, and in the setting of both active disease and remission. The frequencies of MAIT and ILC2 cells were significantly lower in patients with AAV and in the disease control group compared to healthy controls. These reductions in the AAV patients remained during remission. B cell count and frequencies were significantly lower in AAV in remission compared to patients with active disease and disease controls. Despite the strong T helper type 2 (Th) preponderance of eosinophilic granulomatosis with polyangiitis, we did not observe increased ILC2 frequency in this cohort of patients. The frequencies of other cell types were similar in all groups studied. Reductions in circulating ILC2 and MAIT cells reported previously in patients with AAV are not specific for AAV, but are more likely to be due to non-specific manifestations of renal impairment and chronic illness. Reduction in B cell numbers in AAV patients experiencing remission is probably therapy-related.

A crosslinked cofactor in lysyl oxidase: redox function for amino acid side chains.

A previously unknown redox cofactor has been identified in the active site of lysyl oxidase from the bovine aorta. Edman sequencing, mass spectrometry, ultraviolet-visible spectra, and resonance Raman studies showed that this cofactor is a quinone. Its structure is derived from the crosslinking of the epsilon-amino group of a peptidyl lysine with the modified side chain of a tyrosyl residue, and it has been designated lysine tyrosylquinone. This quinone appears to be the only example of a mammalian cofactor formed from the crosslinking of two amino acid side chains. This discovery expands the range of known quino-cofactor structures and has implications for the mechanism of their biogenesis.

Differential modulation of K(+)-evoked (3)H-neurotransmitter release from human neocortex by gabapentin and pregabalin.

Anticonvulsant, analgesic, and anxiolytic effects have been observed both in preclinical and clinical studies with gabapentin (GBP) and pregabalin (PGB). These drugs appear to act by binding to the alpha(2)delta subunit of voltage-sensitive Ca(2+) channels (VSCC), resulting in the inhibition of neurotransmitter release. In this study, we examined the effects of GBP and PGB (mostly 100 microM, corresponding to relatively high preclinical/clinical plasma levels) on the release of neurotransmitters in human neocortical slices. These slices were prelabeled with (3)H-dopamine ((3)H-DA), (3)H-choline (to release (3)H-acetylcholine ((3)H-ACh)), (3)H-noradrenaline ((3)H-NA), and (3)H-serotonin ((3)H-5-HT), and stimulated twice in superfusion experiments by elevation of extracellular K(+) in the presence and absence of GBP and PGB. The alpha(2)delta ligands produced significant inhibitions of K(+)-evoked (3)H-ACh, (3)H-NA, and (3)H-5-HT release between 22% and 56% without affecting (3)H-DA release. Neither drug reduced (3)H-NA release in the presence of L: -isoleucine, a putative alpha(2)delta antagonist. Interestingly, this antagonism did not occur using the enantiomer, D: -isoleucine. These results suggest that GBP and PGB are not general inhibitors of VSCC and neurotransmitter release. Such alpha(2)delta ligands appear to be selective modulators of the release of certain, but not all, neurotransmitters. This differential modulation of neurotransmission presumably contributes to their clinical profile.

Alpha-plutonium's polycrystalline elastic moduli over its full temperature range.

alpha-plutonium's volume-corrected polycrystal elastic moduli were measured between 18 K and the upper limit of its occurrence, near 400 K. The two independent moduli for a polycrystal-bulk and shear-behave smoothly, indicating no phase transition. Both moduli show the same 50% increase on cooling, an order of magnitude larger than in other metals. The Debye temperature obtained from low-temperature elastic moduli, 207 K, significantly exceeds most previous estimates. The Gruneisen parameter gamma=5.3, obtained from the temperature dependence of the bulk modulus, is intermediate among previous estimates using other approaches, alpha-plutonium's Poisson ratio nu is low: 0.18, nearly temperature independent, and its small decrease on warming opposes usual behavior. The high gamma, large but equal bulk modulus and shear modulus fractional stiffening on cooling, and near-temperature-invariant nu are attributed to a single mechanism: 5-f electron localization-delocalization.

Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 A resolution.

BACKGROUND: Copper-containing amine oxidases catalyze the oxidative deamination of primary amines to aldehydes, in a reaction that requires free radicals. These enzymes are important in many biological processes, including cell differentiation and growth, would healing, detoxification and signalling. The catalytic reaction requires a redox cofactor, topa quinone (TPQ), which is derived by post-translational modification of an invariant tyrosine residue. Both the biogenesis of the TPQ cofactor and the reaction catalyzed by the enzyme require the presence of a copper atom at the active site. The crystal structure of a prokaryotic copper amine oxidase from E. coli (ECAO) has recently been reported. RESULTS: The first structure of a eukaryotic (pea seedling) amine oxidase (PSAO) has been solved and refined at 2.2 A resolution. The crystallographic phases were derived from a single phosphotungstic acid derivative. The positions of the tungsten atoms in the W12 clusters were obtained by molecular replacement using E. coli amine oxidase as a search model. The methodology avoided bias from the search model, and provides an essentially independent view of a eukaryotic amine oxidase. The PSAO molecule is a homodimer; each subunit has three domains. The active site of each subunit lies near an edge of the beta-sandwich of the largest domain, but is not accessible from the solvent. The essential active-site copper atom is coordinated by three histidine side chains and two water molecules in an approximately square-pyramidal arrangement. All the atoms of the TPQ cofactor are unambiguously defined, the shortest distance to the copper atom being approximately 6 A. CONCLUSIONS: There is considerable structural homology between PSAO and ECAO. A combination of evidence from both structures indicates that the TPQ side chain is sufficiently flexible to permit the aromatic grouf to rotate about the Cbeta-Cgamma bond, and to move between bonding and non-bonding positions with respect to the Cu atom. Conformational flexibility is also required at the surface of the molecule to allow the substrates access to the active site, which is inaccessible to solvent, as expected for an enzyme that uses radical chemistry.

Copper-containing oxidases.

Major advances have been made during 1997 and 1998 toward understanding the structure/function relationships of the active sites in copper-containing oxidases. Central to this progress has been the elucidation of crystal structures for many of these enzymes. For example, studies of the mechanisms of biogenesis and/or catalysis of amine oxidase and galactose oxidase have been both stimulated and directed by the availability of structures for these proteins. Similarly, it is anticipated that the recently published crystal structures of peptidylglycine alpha-hydroxylating monooxygenase and laccase will contribute greatly toward understanding the roles of copper in these two proteins.

Crystallization and preliminary crystallographic characterization of the copper-containing amine oxidase from pea seedlings.

The copper-containing amine oxidase from pea seedlings has been crystallized using lithium sulfate as precipitant at pH 5.2. The unit cell is orthorhombic, space group P2(1)2(1)2(1), with dimensions a = 89.3 A, b = 113.4 A, c = 199.0 A. The mass of the asymmetric unit is 131(+/- 13) kDa, consistent with independent evidence that the molecule has two approximately 66 kDa subunits. The crystals diffract to 2.5 A in a synchrotron X-ray beam.

Candidal sinusitis and diabetic ketoacidosis. A brief report.

A 55-year-old man presented with diabetic ketoacidosis and pansinusitis due to infection with Candida albicans. The infection responded to local drainage procedures, the administration of amphotericin B (2 g), and aggressive medical therapy of the ketoacidosis. Sinusitis due to C albicans is rare but may be more frequently seen in the immunocompromised host. Unlike those infections caused by Mucor or Aspergillus species, sinusitis due to C albicans may respond to local drainage and amphotericin B therapy.

Spook house sporotrichosis. A point-source outbreak of sporotrichosis associated with hay bale props in a Halloween haunted-house.

While isolated cases of sporotrichosis typically occur following contact with contaminated plant materials, outbreaks are distinctly unusual. A temporal increase in the incidence of sporotrichosis in a dermatology practice at a military installation in southwestern Oklahoma prompted an investigation. Patients with sporotrichosis presenting to a single dermatologist in the winter of 1992-1993 were interviewed, epidemiological data were collected, and fungal cultures were obtained from incriminated hay fields. Five patients presented with cutaneous sporotrichosis during a 5-week period beginning in December 1992. Four patients had maintained hay bales in a Halloween haunted house and the fifth patient had visited the house once. As in 3 previous reports, this outbreak was associated with stored hay or hay bales harvested in the US plains states. Contact with hay should be recognized as a risk factor for infection with Sporothrix schenckii. Outbreaks are possible given adequate intensity of exposure and may be difficult to recognize because of the delayed presentation of clinical illness.

The effects of estrogen replacement therapy on airway function in postmenopausal, asthmatic women.

BACKGROUND: Data from multiple clinical, epidemiologic, and in vitro studies are conflicting regarding the effect of estrogen replacement therapy (ERT) on airway function in postmenopausal women with asthma. OBJECTIVE: To determine the impact of withdrawal of estrogen administration in postmenopausal, asthmatic women. METHODS: Twenty asthmatic women who were postmenopausal for at least 2 years and undergoing ERT were recruited for this prospective crossover study. Subjects continued taking baseline estrogen for 28 days, stopped taking estrogen for 28 days, and then resumed taking the medication for 14 days. Objective measurements were obtained by recording daily peak flows in the morning and evening and formal spirometry at days 14, 28, 42, 56, and 70. Compliance was measured by evaluating serum estradiol levels at days 28 and 56. Daily use of short-acting beta-agonist bronchodilators was also recorded. RESULTS: Differences in estradiol levels indicated compliance with the medication regimen. The combined day 14 and 28 (taking estrogen) mean percent predicted forced expiratory volume in 1 second (FEV(1)) was 77% compared with the combined day 42 and 56 (not taking estrogen) mean FEV(1) of 78% and the day 70 (taking estrogen again) FEV(1) of 76% (P>.05). Average peak flow measurements were 295.5 L/min for the duration of ERT, 293.9 L/min while not undergoing ERT, and 291.8 L/min when ERT was restarted for the final 2 weeks of the study (P>.05). Use of short-acting beta-agonist bronchodilators did not differ between study periods. CONCLUSION: These data indicate that neither the discontinuation nor reinitiation of ERT in postmenopausal, asthmatic women has any effect on objective measures of airway obstruction.

Glycerol permeation of the human granulocyte.

This communication presents a study of the glycerol permeability of the human granulocyte. The study was undertaken to facilitate the use of glycerol as a cryoprotective agent. Granulocytes were purified by centrifugation-elutriation and permeability measured both in monolayer and suspension. Cell permeability was determined by following the passage of 3H-glycerol across the cell membrane. Glycerol efflux was measured at concentrations ranging from 1 microM to 1750 mM (2100 mOsm) under isosmotic and hypotonic conditions. Influx was determined at concentrations between 1 and 100 mM. The monolayer and suspension assays yielded similar kinetics for efflux. Both influx and efflux kinetics were biphasic. The permeability coefficient for efflux was independent of concentration. Since the kinetics were curvilinear, they could not be completely described by a single permeability coefficient. However, "average" values were computed and found to be 1.7 x 10(-5) cm/min for efflux, and 2.8 x 10(-5) cm/min for influx.

The effect of osmotic stress on the function of the human granulocyte.

Clinical investigations have shown that the transfusion of human granulocytes can protect the neutropenic patient against acute bacterial infection. The increasing utilization of granulocyte therapy plus an inability to store these cells in liquid media have created a requirement for a cryopreservation technique. However, the cryopreservation of human granulocytes has proven quite difficult to achieve. It is likely that one of the contributing factors is the lability of these cells under osmotic stress. The purpose of this paper was to precisely define the osmotic tolerance limits of the human granulocyte and thereby provide guidelines for the laboratory manipulation of this cell. To accomplish this, cells were exposed to hypertonic or hypotonic media and then tested for viability under isotonic conditions. Function tests included phagocytosis of particles, bacterial killing, migration in the Boyden chamber, and migration under agarose. The experiments revealed that granulocytes will tolerate a narrow range of osmolalities extending from 200 mOsm to 600-700 mOsm. The osmotic damage occurs in less than 30 seconds and appears to be irreversible. The hypertonic limit of the cell cannot be extended by reducing the temperature to 0 degrees C during the osmotic challenge.

Transforming growth factor-beta 1 and T-lymphocytes modulate the growth of human peripheral blood progenitor cells.

This study examined the effects of transforming growth factor-beta 1 (TGF-beta 1) and autologous T-lymphocytes on the growth of day-14 granulocyte-monocyte progenitor cells (CFU-GM). Mononuclear cells from human peripheral blood were depleted of accessory cells, yielding "B/null" or "null" cell populations enriched 17-fold and 94-fold, respectively, for CFU-GM. Addition of TGF-beta 1 to B/null or null cells caused a dose-dependent decline in CFU-GM cloning efficiency. The addition of unstimulated autologous T cells to B/null or null cells enhanced CFU-GM growth both in the absence and presence of TGF-beta 1. T cells enhanced growth of all CFU-GM colony types to equal extents, whereas TGF-beta 1 inhibition preferentially reduced the proportion of macrophage colonies. TGF-beta 1 concentrations that blocked T-cell proliferation did not interfere with the capacity of T cells to enhance CFU-GM growth. The data suggest that noncycling T cells can reduce the inhibitory effects of TGF-beta 1, most likely through production of one or more stimulatory cytokines.

Detection and quantitation of long-term culture-initiating cells in normal human peripheral blood.

The objective of this study was to utilize the Dexter long-term culture system to characterize and quantitate early hematopoietic cells in normal human blood. Peripheral blood mononuclear cells were depleted of T cells. B cells, and monocytes, yielding a population (null cells) highly enriched for hematopoietic precursors. One to 2 x 10(7) null cells containing an average of 5.4 x 10(4) granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM) were cultured on irradiated allogeneic marrow stromal layers in T-25 flasks. Nonadherent progenitors were detected for an average of 6.8 +/- 0.7 (standard error of the mean) weeks and exceeded 1 x 10(4) per flask for 4 weeks. Null cells were treated with 5-fluorouracil (5-FU) to determine whether long-term growth was due to survival of CFU-GM or to maturation of more primitive precursors. When 5-FU treatment killed greater than 99% of CFU-GM, an increase in progenitors was observed after 12 days of cultivation with stroma. CFU-GM cloning efficiency of 5-FU-treated cells peaked after 24 days of incubation, whereas controls peaked after 7 days. The total number of nonadherent CFU-CM in 5-FU-treated and control cultures converged during the 5-8 weeks of incubation. The frequency of long-term culture-initiating cells (LTCIC) in peripheral blood was determined by limiting dilution analysis in micro-stromal cultures. In two donors, the frequencies of LTCIC were 6.3 and 19.6 per 1 x 10(5) null cells. The CFU-GM:LTCIC ratios were 12.6:1 and 12.8:1. We conclude that normal peripheral blood contains primitive hematopoietic precursors demonstrable as 5-FU-resistant precursors of CFU-GM or as LTCIC. The frequency of the latter is 13-fold lower than day-14 CFU-GM.

Transforming growth factor-beta(1) induces apoptosis in CD34(+)CD38(-/low) cells that express Bcl-2 at a low level.

OBJECTIVE: Transforming growth factor-beta(1) (TGF-beta(1)) strongly inhibits the proliferation and differentiation of primitive CD34(+)CD38(-) hematopoietic cells. In contrast, Flt3 ligand (FL) is a positive effector of CD34(+)CD38(-/low) cell proliferation. Because apoptosis plays a critical role in hematopoietic development, TGF-beta(1) and FL were analyzed as possible modulators of apoptosis. Specifically, this report examined expression of apoptotic promoters Bax and Bad and apoptotic inhibitors Bcl-2 and Bcl-x (all members of the Bcl-2 protein family). Protein levels were determined in fresh and cultured CD34(+)CD38(+) cells and CD34(+)CD38(-/low) cells with and without treatment with TGF-beta(1) and FL. MATERIALS AND METHODS: Cells fractions were purified by sorting CD34(+)-enriched mononuclear cells from mobilized peripheral blood. Expression of Bcl-2, Bcl-x, Bax, and Bad and the extent of apoptosis were determined by flow cytometric analysis of freshly isolated cells and cells cultured with TGF-beta(1) and FL effectors. RESULTS: TGF-beta(1) reduced CD34(+)CD38(+) cell expansion and arrested cell division. Inhibition of growth was not accompanied by an increase in apoptosis. In CD34(+)CD38(-)(/low) cells, serum TGF-beta(1) and added TGF-beta(1) inhibited cell growth and significantly increased apoptotic cell death. Freshly isolated CD34(+)CD38(+) and CD34(+)CD38(-/low) cells expressed Bcl-2 at similar low levels. However, after 3 days, Bcl-2 expression was markedly higher in cultured CD34(+)CD38(+) cells. TGF-beta(1) significantly increased Bax expression in both fractions after 3 days cultivation (p = 0.0034). Thus, addition of TGF beta-1 further reduced the already low Bcl-2:Bax ratio in CD34(+)CD38(-/low) cells. CONCLUSIONS: Compared to CD34(+)CD38(+) cells, CD34(+)CD38(-/low) cells were slow to up-regulate expression of Bcl-2 during ex vivo culture. TGF-beta(1) up-regulated Bax expression by both CD34(+)CD38(+) and CD34(+)CD38(-)(/low) cells and promoted apoptosis in the latter fraction. This suggests that the preferential induction of apoptosis in primitive cells by TGF-beta(1) may be due to its further reduction of the Bcl-2:Bax ratio.

A new density gradient for the separation of large quantities of rosette-positive and rosette-negative cells.

Allogeneic transplantation of peripheral blood stem cells will not be feasible until new techniques are developed for large-scale depletion of T-lymphocytes. Small quantities of cells can be depleted of T-lymphocytes by sheep erythrocyte rosetting and Ficoll-diatrizoate discontinuous gradient fractionation. However, when processing 10(9)-10(10) cells, the fractionation step is inefficient because of the limited capacity of Ficoll-diatrizoate. We therefore developed a new discontinuous gradient for the separation of large numbers of sheep red blood cell (RBC) rosette-positive and negative cells. The gradient was designed so that sheep cells and rosettes would not interfere with the banding of rosette-negative cells. In that way, nonspecific entrapment was reduced, and high cell capacity and yield were achieved. The system fractionated rosetted cell suspensions into four populations: free sheep RBC, rosetted T cells, nonrosetted T cells, and low-density non-T cells. The 15-ml gradient routinely separated 3-24 X 10(8) cells. Progenitor yield ranged from 83% to 99%, with 95% depletion of T-lymphocytes. The method is rapid, reproducible, and inexpensive. This preparative technique could prove useful clinically when large-scale separation of E-rosette-positive and negative cells is required.

Purification of peripheral blood hematopoietic cells using Campath-1M monoclonal antibody.

The purpose of this study was to develop a method for exhaustive depletion of T cells in preparation for allogeneic peripheral blood (PB) hematopoietic stem cell (HSC) transplantation. Our goal was to achieve 4 logs of T-cell reduction while minimizing the loss of colony-forming progenitor cells. Campath-1M, a rat IgM cytolytic to T cells, B cells, and monocytes in the presence of human complement, was chosen for the investigation. To determine the best target population for Campath treatment, three different PB mononuclear cell (MNC) populations were examined: 1) total MNC isolated with Ficoll-sodium diatrizoate; 2) MNC after T-cell depletion by sheep red blood cell rosetting and high capacity Percoll density gradient fractionation (E-MNC); and 3) B/Null cells, a population of E-MNC obtained by lysing monocytes with L-phenylalanine methyl ester (PME). Of the three target MNC populations, Campath-treated B/Null cells had the highest cloning efficiencies (CE) for granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFUe) (56.7 and 38.5 colonies per 5 x 10(3) cells, respectively) and the highest enrichments for CFU-GM (108.2-fold, range 18- to 440-fold) and BFUe (178.5-fold, range 78- to 314-fold). Recoveries of progenitor cells were comparable for Campath-treated MNC (49.7% and 84.5%, respectively, for CFU-GM and BFUe) and Campath-treated B/Null (44.8% and 76.1%), and significantly higher than those from Campath-treated E-MNC (19.5% and 34.3%). The T-cell content in the Campath-treated B/Null cells were reduced by 5.0 logs from the starting MNC, as determined by limiting dilution analysis (LDA). We conclude that Campath-1M can be used for T-cell depletion of PB HSC. The best results were obtained when T cells and monocytes were removed prior to Campath treatment. This cell population is highly enriched for hematopoietic cells and may prove useful for in vitro studies as well allogeneic transplantation.

Glycerolization of the human neutrophil for cryopreservation: osmotic response of the cell.

The human neutrophil probably requires a very high intracellular concentration of cryoprotectant to avoid cellular injury during cryopreservation. Accordingly, new techniques were developed so that high concentrations of protectant could be safely used. To accomplish that, it was necessary to overcome the osmotic limitations of the neutrophil as well as problems with cytotoxicity and membrane permeability. To reduce complications from toxicity, glycerol was selected as the protective agent. To minimize osmotic stress, the protectant concentration was changed slowly and gently by a system combining cross-flow filtration and exponential gradient glycerolization. With that system, it was possible to introduce and remove high concentrations of glycerol with little loss of neutrophil viability. For both ascending and descending gradients, calculations were used to follow the changes in intracellular glycerol concentration. Similar calculations were used to determine the relationship between cell volume, gradient slope, and medium osmolality. From those data, guidelines were formulated for the glycerolization and deglycerolization of the human neutrophil.

Expression of Flt3 and c-kit during growth and maturation of human CD34+CD38- cells.

Studies of murine stem cells suggest that the cytokine receptors Flt3 and c-kit are expressed differentially on the earliest reconstitutional cells, such that Flt3 is not expressed until after stem cell activation. Much less is known about the expression of Flt3 and c-kit on primitive human cells, especially those mobilized into circulation for transplantation. In this study, early circulating precursors were analyzed for expression of Flt3 at the gene and protein levels. Flow cytometric studies showed that >90% of CD34+CD38- cells expressed Flt3 antigen (CD135). The proportion of fresh CD34+ cells expressing Flt3 decreased as CD38 staining increased. These results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) analyses, which showed that Flt3 gene expression generally was limited to the CD34+CD38- population. Because Flt3 ligand (FL) enhances the growth and/or maintenance of primitive cells, it was important to know how long early cells retain Flt3 receptor expression in expansion culture. Both RT-PCR analyses and functional tests demonstrated that primitive cells are capable of expressing Flt3 for as long as 2 weeks in liquid medium. During the first week of culture, FL enhanced the generation of cells and progenitors without causing a loss of primitive CD34+CD38-Flt3+ cells. Flt3 expression in cell cultures was limited to precursors retaining a CD34+CD38(-/lo) phenotype. Because the most primitive human precursors are believed to express c-kit at a low level, we examined the FL responsiveness of CD34+CD38-c-kit(-/lo) cells and CD34+CD38-c-kit+ cells. CD34+CD38-c-kit(-/lo), cells constituted a small fraction (12%) of the CD34+CD38- population. Whereas both c-kit(-/lo) and c-kit+ subsets were stimulated by FL, cell expansion (p < 0.01) and colony formation (p < 0.01) were greater and maintained longer with CD34+CD38-c-kit(-/lo) cells. Furthermore, the rapid response to FL suggests that primitive CD34+CD38-c-kit(-/lo) cells express Flt3 at the time of isolation or shortly thereafter. These results demonstrate the presence of Flt3 on CD34+CD38 blood cells and suggests that Flt3 also may be present on a c-kit(-/lo) subset, among the most primitive in circulation. Flt3 is lost during maturation to committed (CD34+CD38+) lineages. Addition of FL to primitive cell cultures stimulates cell expansion while maintaining early CD34+CD38-Flt3+ precursors for at least 7 days. The possible existence of a more primitive CD34+CD38-c-kit(-/lo) Flt3(-/lo) precursor remains to be determined.

Isolation of large numbers of fully viable human neutrophils: a preparative technique using percoll density gradient centrifugation.

Techniques were developed for the isolation of human neutrophils from large volumes of human blood. This preparative procedure avoids exposure to Ficoll/Hypaque and offers a low cost alternative to centrifugal elutriation. First, leukocyte-enriched suspensions were obtained by treating buffy coats with 2% dextran. The leukocyte suspensions were then fractionated on three steps Percoll density gradients. The gradients were designed to carry large numbers of cells while maintaining resolution of the cell peaks. As a result, at least 320 X 10(6) leukocytes could be processed on a single 12 ml gradient. Extensive testing of the procedure showed that the purity of the neutrophil band was 96.5 +/- 3.16% (n = 50). The band contained 82.4 +/- 14.6% (n = 39) of the neutrophils applied to the gradient. Viability assays (bacterial killing, chemotaxis) demonstrated that neutrophils isolated on Percoll gradients were just as active as those harvested by centrifugal elutriation. Recovery data are presented and compared to the results obtained by others.