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The quantitative nuclease protection assay (qNPA) is a very simple and highly sensitive method for measuring mRNA transcripts, can be used on a variety of sample types, and is amenable to high-throughput sample processing. We have combined the power of the qNPA assay with the density of a DNA microarray to create a qNPA Microarray platform. This platform is compatible with common laboratory equipment: it uses fluorescence-based detection, can be analyzed with common microarray scanners, and is in an SBS footprint with 96-well layout for high-throughput applications. Here, we demonstrate the characteristics of a qNPA Microarray slide that contains up to 1700 gene elements per well. We show that the new platform can reliably detect transcripts at levels as low as 10fM with median CVs below 12%. On a standardized set of samples, the qNPA Microarray detected the same trends in gene expression as the original qNPA technology, real time qPCR, and Affymetrix GeneChip DNA Microarrays. Given its ease of use, compatibility with multiple sample types, high-throughput capabilities, and its integration with standard laboratory equipment, the qNPA Microarray is a powerful new platform for gene expression research.

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Nonhuman primates have been used as models for testing the role of interleukin-1 (IL-1) in inflammatory diseases, including endotoxemia. The objective of this investigation was to develop a reproducible and rapid method for in vivo evaluation of IL-1 antagonists using cynomolgus monkeys. IL-1 alone can induce many of the symptoms of endotoxemia in monkeys including fever, loss of appetite, and lethargy, however, test animals are slow to recover and may become desensitized to IL-1. We have developed an ex vivo method using whole blood for analysis of IL-1 antagonists administered in vivo to the monkeys and report here results for the naturally occurring IL-1 receptor antagonist, IL-1ra. In this procedure, animals are given an i.v. infusion of IL-1ra, and blood samples are taken preinfusion and during the infusion. The samples are incubated with or without IL-1 beta and the subsequent ex vivo induction of IL-6 determined. This allows analysis of the effects of in vivo pharmacodynamics on the efficacy of antagonists without exposing the test animals to IL-1. In this ex vivo protocol, each animal serves as its own control, eliminating from the assessment the large animal to animal variation observed with in vivo responses. By testing various doses, we estimate that 50% inhibition of IL-1 induced IL-6 can be achieved with an infusion of IL-1ra at 5 micrograms/kg/15 min. This method allows simple and efficient analysis of inhibitors and antagonists of IL-1 and, potentially, other effectors.

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The rat is commonly used as an animal model to test the efficacy of new anticoagulant and antithrombotic drugs. Many of the protocols require the use of anesthetics for surgical procedures. Three common coagulation assays used to monitor the effects of new anticoagulant and antithrombotic drugs are thrombin time, activated partial thromboplastin time, and prothrombin time. We examined the effects of either urethane- or ketamine/xylazine-induced anesthesia on variables in the three coagulation assays with blood samples obtained from the rats before the induction of anesthesia, compared with samples obtained at 15 or 60 min after anesthesia induction. Statistically significant differences were observed in the ketamine/xylazine group for thrombin time at 60 min (compared with control values) and in the urethane group for activated partial thromboplastin time at 60 min (compared with control values). In both instances a prolongation of times was the effect seen. These results indicate that the choice of anesthetic affects the data in certain coagulation assays in the rat and must be taken into account when one is planning studies and analyzing results.

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Combinatorial libraries employing the one-bead-one-compound technique are reviewed. Two distinguishing features characterize this technique. First, each compound is identified with a unique solid support, enabling facile segregation of active compounds. Second, the identity of a compound on a positively reacting bead is elucidated only after its biological relevance is established. Direct methods of structure identification (Edman degradation and mass spectroscopy) as well as indirect "coding" methods facilitating the synthesis and screening of nonpeptide libraries are discussed. Nonpeptide and "scaffold" libraries, together with a new approach for the discovery of a peptide binding motif using a "library of libraries," are also discussed. In addition, the ability to use combinatorial libraries to optimize initially discovered leads is illustrated with examples using peptide libraries.

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In order to identify regions of C5a that contribute to receptor binding and functional activity of the anaphylatoxin, a series of peptides was synthesized in which core segments have been attached to C-terminal segments via native peptidic or disulfide bonds. It has been found that residues Arg40 and Arg46 in the loop-3 region of the core induce a 1000-fold increase in the affinity of the disordered C-terminal segment of C5a. The results obtained from this work lead to the conclusion that the loop-3 region is most likely the core binding site of C5a.

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We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.

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A method of indirectly determining the structure of nonpeptidic or nonsequenceable compounds that have been synthesized on individual particles of solid support is described. The technique permits the parallel synthesis of a compound that is not susceptible to Edman degradation (e.g., N-terminal-blocked peptide), or one containing components that cannot be identified by amino acid sequencing, together with a corresponding "coding" peptide. Each coupling step in the assembly of the nonsequenceable compound is followed by the coupling of an amino acid to a different attachment site of the same carrier particle, whereby the amino acid unambiguously codes for the previously coupled building block of the nonsequenceable compound. The rationale is to enable the sequence determination of a biologically active compound that has been identified through the screening of a library of nonequenceable compounds, by translating the sequence of its "coding" peptide, determined by Edman degradation, into the structure of the active compound. The technique facilitates the construction and screening of nonpeptidic libraries for the discovery of important pharmaceutical compounds.

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The fifth component of the complement cascade, C5a, was iodinated using the Bolton-Hunter reagent. Results from the present study, using the high affinity ligand, [125I]Bolton-Hunter-labeled C5a ([125I]BH-C5a), revealed a single binding site on membranes prepared from human neutrophils, U-937 cells and human monocytes. Saturation studies demonstrated Bmax values in these cells of 11.5, 47.3 and 16.6 fmol/10(6) cells, respectively. The C5a receptor demonstrated a very high affinity for [125I]BH-C5a of approximately 4 pM in each cell type. Competition studies using analogs of C5a generated a similar order of potency in each of the cell types of C5a > or = C5a(1-74), Ser66Ala > C5a(1-73) > C5a(1-69). These studies indicate that [125I]BH-C5a labels a similar receptor in neutrophil, U-937 cell and monocyte membranes. Furthermore, C5a(1-73) produced shallow inhibition curves in competition experiments in each cell type. Computer analysis of the binding data revealed two components of binding. When 10 nM unlabeled C5a was used to initiate dissociation of [125I]BH-C5a binding in neutrophil membranes, two binding components were observed. In addition, dissociation of [125I]BH-C5a binding by 10 nM unlabeled C5a in the presence of 1 mM GppNHp decreased the percentage of binding to the slowly dissociating, high affinity binding component from 84 to 58%. These results suggest that multiple states of the C5a receptor exist.

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The effect of recombinant human C5a (rhC5a) on the synthesis of interleukin 1 beta (IL-1 beta) was investigated in human monocytes, isolated by leukapheresis and countercurrent elutriation. rhC5a induced IL-1 beta mRNA synthesis in a dose- and time-dependent manner. Maximal induction was achieved at 3 h with rhC5a concentrations of 200 to 500 ng/ml. The maximal rhC5a-stimulated mRNA induction was about 75% of that observed using LPS as the stimulus (300 ng/ml). The inducing activity of rhC5a could be neutralized by preincubation with a polyclonal anti-C5a antibody. On the other hand rhC5a in optimal concentrations only weakly stimulated IL-1 beta protein synthesis, as measured by a two-site directed enzyme-linked immunoassay. When compared on Northern blots, a slightly reduced mobility of C5a-stimulated IL-1 beta mRNA was observed relative to LPS-stimulated RNA. To exclude the possibility that structural defects in the IL-1 beta mRNA are responsible for the weak translational efficiency after C5a stimulation a primer extension experiment was performed. No difference in the length of the extended fragment was detected between LPS and C5a-stimulated RNA, suggesting that the 5'-regions of the RNAs are identical. When LPS- or C5a-stimulated RNA was used to program IL-1 beta synthesis in an in vitro translation system from rabbit reticulocytes, no difference in translational efficiency was observed. Our results indicate that in human monocytes two signals for IL-1 beta gene expression are necessary, a signal for transcriptional activation and another signal to induce translation of the mRNA.

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The effect of recombinant human C5a (rhC5a) on the synthesis of interleukin-1 beta (IL 1 beta) was investigated in highly purified human monocytes, isolated by leukapheresis and counter-current elutriation. RhC5a induced IL 1 beta mRNA synthesis in a dose- and time-dependent manner; maximal induction was achieved at 3 h with rhC5a concentrations of 200 to 500 ng/ml. The IL 1 beta mRNA induction with rhC5a was about 75% of the response observed using lipopolysaccharide (LPS) as the stimulus (300 ng/ml). On the other hand, rhC5a in optimal concentrations only weakly stimulated IL 1 beta protein synthesis, as measured by a two-site-directed enzyme-linked immunoassay. When LPS- or C5a-stimulated RNA was used to program IL 1 beta synthesis in an in vitro translation system from rabbit reticulocytes, no difference in translational efficiency was observed. Our results indicate that, in human monocytes, two signals for IL 1 beta gene expression are necessary, one signal for transcriptional activation and a second signal to induce translation of the mRNA.

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Superoxide dismutase (SOD)-inhibitable reduction of cytochrome c is the basis for a widely used assay to measure superoxide production. We report novel modifications leading to a dual wavelength, high throughput simultaneous kinetic and endpoint microplate assay with high reproducibility. Neutrophils were isolated using a modified elutriation procedure to minimize priming and adherence during isolation. Cytochrome c reduction was measured in a microplate reader using 96-well polystyrene plates with a modified (Plastek A*) surface to prevent the adherence and consequent activation of PMNs. Comparison of Plastek A* treated and untreated plates revealed a statistically significant difference in basal as well as stimulated levels of superoxide production. Absorption measurements were made at both 550 nm, the absorption maximum of reduced cytochrome c, and 557 nm, an isosbestic point. A significant increase in both well-to-well reproducibility and sensitivity (detection limit) was realized by using the normalized 550-557 nm difference values compared to the 550 nm absorbance values alone. These modifications represent an improved method for handling and assessing the function of superoxide production, providing greater experimental reproducibility and lessening the perturbations caused by the microplate.

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Polymorphonuclear leukocytes (PMNs) have been implicated in postischemic myocardial injury and associated derangements in contractile function. To examine the direct effects of PMNs on cardiac function, isolated right ventricular papillary muscles of the rabbit were exposed to increasing concentrations of purified rabbit PMNs in the presence of cimetidine. PMNs induced a significant concentration-dependent decrease in contractile function, where 5 x 10(5) PMNs/ml reduced contractile force to 75 +/- 2.1% of control (vs. 95 +/- 5% for time control; P less than 0.005). Similar decreases were also observed for peak positive and negative first derivatives of contractile force. The degree of PMN-induced contractile dysfunction correlated with the activity of the PMNs in an aggregation assay (r = 0.82, P less than 0.01). The loss of contractile function in response to PMNs was attenuated by catalase, which metabolizes H2O2, but not by superoxide dismutase, a scavenger of the superoxide anion. PMNs can convert H2O2 to either the hypochlorite anion or the hydroxyl radical, which are removed by methionine or mannitol, respectively. However, these scavengers did not ameliorate the PMN-induced loss of cardiac function. Exposure of papillary muscles to H2O2 resulted in a concentration-dependent decrease in contractile function where 100 microM reduced contractile force to 78 +/- 4%, an effect prevented by catalase. Thus PMNs reduce the contractile function of isolated papillary muscles probably by the release of H2O2.

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The effects of [K+]o on taurine release from glial cells were studied with primary cultures of cerebellar astrocytes and with LRM55 cells, a continuous glial cell line. The characteristics of K(+)-stimulated taurine release were virtually identical in the 2 cell types. Both cerebellar astrocytes and LRM55 cells released taurine when stimulated with high-K+ medium prepared by isosmotically substituting KCl for NaCl, but neither cell type released taurine when stimulated with hyperosmotic high-K+ medium prepared by adding solid KCl to control medium. The membrane potential of LRM55 cells was measured by intracellular recording and was insensitive to changes in [K+]o below 20 mM. LRM55 cells released taurine when stimulated with nondepolarizing concentrations of K+ (13-22 mM) if the isosmotically prepared high-K+ medium was used, but the cells did not release taurine when treated with a depolarizing concentration of K+ (50 mM) if hyperosmotic high-K+ medium was used. The time course of K(+)-stimulated taurine release was quite slow, having a time to peak of 10-15 min. Small changes (2.5-10%) in the osmolarity of the medium strongly affected taurine release by cerebellar astrocytes and LRM55 cells. K(+)-stimulated taurine release from both cell types was inhibited when the osmolarity was increased with sucrose or NaCl and was enhanced when the osmolarity was reduced. Similarly, baseline taurine release was suppressed by small elevations in osmolarity and increased by reduced osmolarity.(ABSTRACT TRUNCATED AT 250 WORDS)

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We have studied the topography of interaction of a family of fluorescent formyl peptides containing four (CHO-Met-Leu-Phe-Lys-fluorescein), five (CHO-Met-Leu-Phe-Phe-Lys- fluorescein), and six (CHO-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and CHO-Met-Leu-Phe-Phe-Phe-Lys- fluorescein) amino acids with their receptor using spectroscopic methods adapted to small sample volumes. Only the fluorescent peptides containing four and five amino acids were quenched upon binding to the receptor, indicating physical contact of the chromophore with the receptor. In contrast, only the hexapeptides were accessible to antibodies to fluorescein. Taken together, these results suggest that the carboxy terminus of the tetrapeptide or the pentapeptide is protected in the receptor binding pocket while the fluorescein on the carboxy terminus of either hexapeptide is exposed and recognized by the antibody to fluorescein. These results indicate that the binding pocket accommodates at least five but no more than six amino acids.

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The migration of polymorphonuclear leucocytes (PMN) to extravascular sites and the interaction with chemotactic substances at such locations is called exudation. Since stimulation of PMN in vitro modifies the characteristics of PMN, we asked the question whether similar modifications take place during in-vivo exudation. We found that resting guinea-pig peritoneal exudate PMN were hyperpolarized in comparison to blood PMN of the same species. Guinea-pig and human exudate PMN responded to lower N-formyl-methionylleucylphenylalanine (fmet-leu-phe) concentrations than blood PMN and exhibited a larger membrane depolarization. Furthermore, experiments with the fluorescence-activated cell sorter revealed increased forward light scatter from resting exudate PMN compared to blood PMN. Experiments with the fluorescence-activated cell sorter using the fluoresceinated ligand fmet-leu-phe-lysin-fluorescein (fmet-leu-phe-lys-F) indicated that the priming of exudate PMN was associated with increased fmet-leu-phe-lys-F binding on the individual cells. In contrast, phorbol myristate acetate (PMA) did not induce an increased membrane depolarization response in human and guinea-pig exudate PMN. With this stimulus, the only sign of priming was the shorter activation time in exudate PMN compared to blood PMN. Thus, in-vivo exudation modifies the characteristics of resting and stimulated PMN. The priming is different for different stimuli. Increased responsiveness to fmet-leu-phe may be due to fmet-leu-phe receptor upregulation. The distinct characteristics of exudate PMN that we describe may allow definition of clinical situations in which PMN are primed in vivo.

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Since neutrophil cytoplasts lacking nucleus and granules were first prepared by centrifuging neutrophils over a discontinuous Ficoll gradient containing cytochalasin B, several functional deficits have been reported in these cytoplasts. Although these functional deficits have been considered to originate from the absence of organelles, cell damage during preparation could not be excluded. Therefore, in the following experiments the Ficoll gradient was modified to isolate both cytoplasts and karyogranuloplasts, which have a nucleus and granules and represent the cell after loss of the cytoplast. Electron microscopy and analysis of marker proteins and cell volume showed that karyogranuloplasts were distinct from neutrophils. The phorbol myristate acetate (PMA) or N-formylmethionylleucylphenylalanine (FMLP)-induced O2- release, corrected by surface area, was in the following order: neutrophils greater than cytoplasts greater than karyogranuloplasts. Both aggregation and membrane potential depolarization were maximal in neutrophils, intermediate in karyogranuloplasts, and lowest in cytoplasts when either PMA or FMLP was used as a stimulant. Extracellular release of the granule contents (degranulation) was triggered by FMLP in both neutrophils and karyogranuloplasts. Cytochalasin B pretreatment greatly enhanced FMLP-induced O2- release, degranulation, aggregation, and depolarization of membrane potential in neutrophils and karyogranuloplasts, but not in cytoplasts. The ability of cytochalasin B to potentiate FMLP-triggered cell function probably depends on granules or cell organelles which are depleted in cytoplasts. Chemokinesis and chemotaxis were impaired in both karyogranuloplasts and cytoplasts. Specific FML[3H]P binding was greater in karyogranuloplasts than in cytoplasts. Cellular actin content, measured by the DNase I inhibition assay, was abundant in cytoplasts and was extremely low in karyogranuloplasts. Karyogranuloplasts retain various neutrophil functions, except for chemotaxis, and provide an important control when studying the role of cell organelles in cytoplast function.

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Simultaneous measurements of the calcium rise, membrane potential change, and 90 degrees light scatter (shape change) responses exhibited by neutrophils upon activation, can be obtained with identical result as that obtained when independently performing each measurement. The putative intracellular mediator diacylglycerol depolarizes membrane potential and causes a decrease in light scatter. Leukotriene B4 causes a rise in calcium and a decrease in light scatter. The chemotactic peptide, N-formylmethionyl-leucyl-phenylalanine, causes a depolarization of membrane potential, a calcium rise, and a decrease in light scatter. The fura 2 measurements of intracellular free calcium indicate that the calcium concentration of unstimulated cells is much lower than previously thought based on quin 2 studies.

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Polymorphonuclear neutrophils (PMNs) were isolated from 24 healthy adults 20 to 61 years of age and the proportion of cells that demonstrated depolarization responses to the synthetic chemotaxin N-formyl-methionyl-leucyl-phenylalanine (FMLP) were enumerated using the lipophilic fluorescent cyanine dye 3,3'-di-pentyl-oxacarbocyanine [di-O-C(5)(3)] and flow cytometry. The membrane potential responses were correlated to the cells' respiratory burst capabilities as measured by nitroblue tetrazolium (NBT) reduction and/or superoxide production in response to FMLP; 40.2% +/- 15.1% (mean +/- SD) of cells depolarized to FMLP. The mean SE for duplicate determinations 1 hour apart was 3.8% (range 0.4% to 13.6%, n = 15). There was no correlation between the percentage of depolarizing PMNs and the yield of cells, the percentage of immature cells, or the circulating WBC count. There was no difference in the average age of men (34.9 +/- 9.9 years, n = 11) v women (33.8 +/- 8.5, n = 13) (mean +/- SD) studied, or in the percentage of depolarizing PMNs when men and women were compared (42.2 +/- 10.6% v 43.1 +/- 13.3%, respectively). However, there was a significant increase in the percentage of depolarizing PMNs with increasing age of women (r = .61, P less than .025) but not men (r = .03, P greater than .05). Analysis of variance revealed significantly greater person-to-person variability in the membrane potential response than in day-to-day variability in the same person (P less than .0005). The percentage of depolarizing PMNs in response to FMLP was significantly correlated with the percentage of NBT-positive cells from both purified PMNs and from whole blood (r = .849, P less than .0005, r = .857, P less than .05, respectively), and with the amount of superoxide produced, expressed as a percentage of that amount produced by cytochalasin B (cyto-B)-pretreated cells (r = .565, P less than .01). The data indicate that PMNs from healthy adults demonstrate a heterogeneous membrane potential response to the chemotaxin FMLP that correlates with the cells' oxidative responsiveness and that intersubject differences can be detected. In addition, the proportion of responsive PMNs increases with increasing age in women.

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An IgG1 monoclonal antibody, 31D8, that recognizes normal neutrophil (PMN) membranes, was used to study PMN from patients with chronic myelogenous leukemia (CML). Nineteen patients with Philadelphia chromosome positive CML were followed over a ten-month period and compared with 23 normals, six patients with leukemoid reactions, and eight patients with phagocytic cell defects. The percentage of PMN binding of 31D8 among normal subjects was variable about a normal distribution with an average of 95 +/- 2% of cells binding 31D8. In contrast, there were two groups of CML patients: in 14 patients 88 +/- 3% PMN bound 31D8 while in the remaining five patients only 6 +/- 6% PMN bound 31D8. PMN 31D8 binding was normal in the control patient groups. Control antibodies 7C3 (binds to PMN precursors) and OKM1 (binds to the CR3 (iC3b) receptor) bound normally to CML neutrophils. Functionally, CML cells had normal chemotaxis to several stimuli and normal superoxide generation to phorbol myristate acetate. However, superoxide production in response to fmet-leu-phe was significantly less in 31D8 negative CML PMN than both 31D8 positive CML PMN and normal PMN which contained 85% 31D8 positive and 15% 31D8 negative PMN. Clinically, 2 of 14 CML patients with 31D8 positive PMN were in blast crisis (one extramedullary) at the time of study and the other 12 patients remained clinically stable in the chronic phase during the ten months of study. In contrast, one of five patients with 31D8 negative PMN was in blast crisis at the time of study and all four of the remaining patients progressed to either the accelerated phase or blast crisis. Three of these patients died of their disease eight to ten months after their initial study. Thus, failure of CML cells to bind 31D8 may be useful for predicting which patients are likely to progress to the accelerated phase or blast crisis.

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