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Biomedical subjects

J P Atkinson

Publications and source records attributed to J P Atkinson.

At least 181 records · Page 10Linked to original sources
PubMed1982

Hemolytic and antigenic measurements of complement. A comparison of serum and plasma samples in normal individuals and patients.

Over a 2-year period we evaluated 10 patients with a discrepancy between functional and antigenic assays in the routinely employed clinical assays for measuring serum complement concentrations. These differences were shown to be secondary to cold-dependent activation of the classical complement pathway in vitro, in some cases by cryoglobulins and in others by unknown means. Plasma samples were procured with commonly used anticoagulants (EDTA, citrate, or heparin) that prevent in vitro complement activation. To assess whether plasma samples were suitable for complement determination, a comparison of serum vs. plasma samples for THC, C4, C2, C3, factor B, and C6 levels in normals and patient populations was undertaken. Only modest differences were found between serum and plasma samples for these functional and antigenic assays except for heparinized samples in which determinations by rate nephelometry produced falsely elevated C4, C3, and factor B antigenic levels. Thus plasma samples, especially EDTA or citrate, are suitable for complement determinations and could be used either routinely or, more selectively, in patients in whom there is a discrepancy between functional and antigenic determinations.

Adult↗
PubMed1981

Isolation of a biologically active macrophage receptor for the third component of complement.

C3b, the major cleavage fragment of the third component of complement (C3), has been demonstrated to bind to a specific receptor on various mammalian cells. Tissue-bound C3b receptors have also been demonstrated, most conclusively in renal glomeruli. On interaction with C3b, C3b receptors are thought to initiate cellular functions such as phagocytosis and to determine the fate of complement-fixing soluble and particulate immune complexes. We report here the isolation by affinity chromatography of a macrophage glycoprotein with an apparent molecular weight (MW) of approximately 64,000 that has properties expected of the C3b receptor. It is a cell-surface macromolecule (labelled with 125I and lactoperoxidase) which, in its isolated state, retains the ability to bind both C3 and C3b.

Animals↗
PubMed1981

Cold dependent activation of complement in systemic lupus erythematosus. A unique cause for a discrepancy between clinical and laboratory parameters.

A 31-year-old woman with systemic lupus erythematosus (SLE) was studied because she developed markedly reduced total hemolytic complement activity at a time when her disease was clinically inactive. Functional assays demonstrated reduced activities of C1, C4, and C2, but normal concentrations of C3 and the terminal (C5-9) components were present. Antigenic concentrations of C1, C4, and C2 were normal. Plasma or serum obtained from blood allowed to clot at 37 degrees C had normal complement activity. Complement activity was depleted when the patient's serum was incubated in the cold. At reduced temperatures, the patient's serum (or purified IgG) depleted complement activity of normal human sera. A second patient with SLE was also demonstrated to have this same phenomenon. Cryoglobulins were not detectable in these patients. These data indicate that in vivo the patients' complement was normal and that the observed in vitro reduction was caused by cold dependent activation of the classical pathway.

Adolescent↗
PubMed1981

Metabolism of exogenous arachidonic acid by murine macrophage-like tumor cell lines.

Murine macrophage-like cell lines, J774.2, P388D1, RAW264.7 and PU-5-1R, were incubated with exogenous arachidonic acid (AA). The major metabolites were identified by comigration with known standards in TLC and HPLC and by characteristic behavior following reduction. During a 30 min incubation J774.2 cells metabolized exogenous 14C-AA (10 microM) to PGE2 (14.8%), 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) (13.0%), thromboxane B2 (TXB2) (7.4%), PGD2 (4.4%) and PGF2 alpha (3.0%). The remainder was incorporated into phospholipids (39.0%), triglycerides (6.1%), and as yet unidentified metabolites (8.2%). No PGF1 alpha was found. Metabolism of exogenous AA was rapid, being less than 90% completed at 3.5 min. Metabolism of exogenous AA is not increased by the simultaneous addition of macrophage stimuli including the cation ionophore A-23187, particulate phagocytic stimuli and endotoxin. The synthesis of cyclooxygenase products was inhibited by low doses of indomethacin (ID50=0.6 microM) while the synthesis of TXB2 and HHT was selectively inhibited by benzylimidazole (ID50=9.5 microM). Identification of a probable lipoxygenase product is being pursued. The synthesis of this product is not inhibited by indomethacin and migrates with an Rf value close to 5,12-diHETE in TLC. P388D1 and RAW264.7 cells metabolize exogenous AA to the same products as J774.2, but in different proportions, while PU-5-1R does not produce cyclooxygenase metabolites to any appreciable extent.

Animals↗
PubMed1981

Characterization of ligand-binding activity of isolated murine Fc gamma receptor.

Fc gamma-binding macromolecules were isolated from 2 murine macrophage-like cell lines by affinity chromatography using various immunoglobulins coupled to Sepharose. P388D1 and J744.2 cells were radiolabeled with 125I using a modified lactoperoxidase method, washed cells were solubilized using NP-40, and the solubilized cell lysates were incubated at 4 degrees C with immunoadsorbents. Fc gamma receptor-like material was obtained from the IgG-Sepharose columns by elution at 4 degrees C with 0.5 N acetic acid containing 1% NP-40 into buffer for neutralization. Purified material thus obtained retained its ligand-binding activity, since approximately 30 to 60% rebound to IgG-Sepharose. Upon analysis by SDS polyacrylamide gel electrophoresis, putative Fc gamma receptor had an apparent m.w. of 50 to 65,000. Neither a comparable SDS-PAGE band nor receptor activity was isolated from a 3rd murine cell line, which lacks the Fc gamma receptor. Putative receptor obtained by elution from IgG2a-Sepharose rebound equally well to IgG2a-, IgG1- and IgG2b-Sepharose, but did not rebind to IgG3-, F(ab')2-, or BSA-Sepharose. Similarly, Fc gamma-binding macromolecules eluted from IgG2b-Sepharose rebound only to IgG2b-, IgG2a- and IgG1-Sepharose rebound only to IgG2b-, IgG2a- and IgG1-Sepharose. The rebinding of either receptor preparation was inhibited in a dose-dependent manner by both monomeric IgG2a and monomeric IgG2b, and myeloma protein from each subclass inhibited to the same extent. Therefore, the mouse Fc gamma receptor in its isolated state does not appear to discriminate between monomeric IgG2a and IgG2b.

Animals↗
PubMed1980

A murine C4 molecule with reduced hemolytic efficiency.

C4 functional activity and antigenic levels were determined in H-2-congenic mouse strains. In strains with the H-2w7 haplotype, the C4 hemolytic activity per unit of residual Ss antigenic activity, after depletion of the nonfunctional Slp-positive molecules was 25-33% that found with other H-2 haplotypes. This reduced hemolytic efficiency was not the result of either a more labile C4 molecule or of the presence of inhibitors. Moreover, other strains with comparable antigenic concentrations of Ss (C4) and Slp has three- to fourfold higher levels of C4 hemolytic activity. Based on these data and previously reported structural differences between C4 molecules from the H-2w7 haplotype compared with other standard H-2 haplotypes, the reduced hemolytic efficiency of this molecule is probably secondary to alterations in the structure of its alpha-chain.

Animals↗
PubMed1980

Genetic and structural characterization of H-2-controlled allelic forms of murine C4.

The patterns of expression of several types of genetic variation in murine C4 were investigated. Dominance was assessed for S-region-determined differences in molecular weight of the C4 alpha-chain from Sw7 strains relative to all other strains examined and in C4 hemolytic titers. Codominant expression in F1 hybrids was shown for both of the above variations. Comparison of tryptic peptide profiles of radiolabeled C4 beta-chains encoded by different S regions revealed differences in their primary structures. The demonstration of codominant expression for the variant C4 traits examined and the evidence from tryptic peptide maps for haplotype differences in C4 primary structure strongly support the hypothesis that polymorphism of murine C4 is controlled by structural genes which are located in the S region.

Alleles↗
PubMed1980

Improved cell surface radioiodination of macrophages.

Rabbit alveolar macrophages at high cell concentrations are inefficiently radiolabeled by the usual lactoperoxidase-catalyzed iodination method. Soluble macromolecules secreted by macrophages were found to inhibit the radioiodination of macrophages and other cells. A modified procedure is described which minimizes the presence of such inhibitory material and thereby considerably improves the radioiodination efficiency for both alveolar macrophages and the macrophage-like P388D1 cell line.

Animals↗
PubMed1980

Stimulus-specific deactivation of chemotactic factor-induced cyclic AMP response and superoxide generation by human neutrophils.

The responses of isolated human peripheral neutrophils to either simultaneous or sequential additions of two chemotactic factors were studied. Simultaneous additions of formyl-methionyl-leucyl-phenylalanine (10-100 nM) and the fifth component of complement, C5a (1-10 microliters/ml), evoked partially additive responses of membrane depolarization as measured by the fluorescent dye 3,3'-dipropyl-thiocarbocyanine, a transient elevation of intracellular cyclic AMP (cAMP), and superoxide (O2-) generation as assessed by ferricytochrome c reduction. Preincubation of the cells with either formyl-methionyl-leucyl-phenylalanine or C5a alone caused dose-dependent inhibition of the depolarization, the cAMP increase, and O2- release induced by a subsequent exposure to an optimal dose of the same stimulus, i.e., deactivation occurred. In contrast, when cells were treated with one chemotactic factor and then exposed to the other stimulus, the cells exhibited a normal response of peak depolarization, the rise in cAMP, and O2-0 production i.e., cross-deactivation failed to occur. The results imply that deactivation of these phenomena is stimulus specific. Further, these observations are consistent with the hypothesis that cross-deactivation of chemotaxis is mediated by one or more processes that are irrelevant to O2- generation, and that occur distal to the depolarization and cAMP steps in the sequence of neutrophil activation: possibly microtubule polymerization and orientation.

Anaphylatoxins↗
PubMed1980

Superoxide generation and granule enzyme release induced by ionophore A23187. Studies on the early events of neutrophil activation.

The responses of isolated human peripheral neutrophils to activation by the divalent cation ionophore A23187 (4 x 10(-8) M to 2 x 10(-5) M) were studied over the course of 15 min at 37 degrees C. Dose-dependent increases were noted in the following A23187-induced functions: (1) membrane depolarization, as assessed by a fluorescent cyanine dye, (2) altered membrane permeability to 22Na+ and 45Ca2+, (3) increased intracellular levels of cAMP, (4) O2-generation, and (5) granule enzyme release. Membrane depolarization and the cAMP response preceded the onset of O2- generation and exocytosis, which began after a latency period of approximately 30 sec. All five functions induced by 2 x 10(-5) M A23187 required approximately 1 mM Ca0 for optimal expression and declined substantially as Ca0 was reduced towards a nominal O mM. In contrast, the Na+ ionophore gramicidin D (0.1 to 10 microgram/ml) caused cell depolarization and increased 22Na+ ionophore gramicidin D (0.1 to 10 microgram/ml) caused cell depolarization and increased 22Na+ uptake but did not stimulate 45Ca2+ uptake, the cAMP response, O2- generation, or exocytosis of granule constituents. The observations dissociate membrane depolarization and altered Na+ permeability, on the one hand, from a Ca2+ influx, cAMP response, O2- release, and exocytosis on the other. These studies, then, are consistent with a hypothesis that a Ca2+ influx is required for the biologic expressions of cell activation.

Anti-Bacterial Agents↗
PubMed1980

Cyclic AMP concentrations in human peripheral blood lymphocytes. Changes in association with cell purification.

In studies over a several year period of cAMP in human peripheral blood lymphocytes, a wide range of concentrations has been from low values (1 to 5 pmole/10(7) cells) to high values (80 to 160). Evaluation of this variation indicated that between cell separation and completion of the experiment, there eas a progressive decrease in lymphocyte cAMP as determined by RIA or immunocytochemistry. After a 2 hr incubation at 37 degrees C, a new steady state was reached at 5% to 30% of the initial concentration. This phenomenon was unique to lymphocytes and not preventable by manipulation of the incubation conditions. The cause of the cAMP reduction was shown to be decreasing AC activity. These data suggested the possibility that AC was activated during cell isolation. However, the phenomenon was present in cells not exposed to dextran or Ficoll-Hypaque, and simulation of conditions employed during purification did not result in increased cAMP. Consequently, high-lymphocyte cAMP concentrations may be the normal levels in vivo.

Adenylyl Cyclases↗
PubMed1980

Purification of Fc gamma receptor from rabbit alveolar macrophages that retains ligand-binding activity.

The Fc gamma receptor of rabbit alveolar macrophages was purified by affinity chromatography by using rabbit gamma-globulin (Rab gamma G) coupled to Sepharose. Macrophage preparations were efficiently labeled with 125I by using a modified lactoperoxidase method. After incubation of NP-40 cell lysates with Rab gamma G-Sepharose, elution at 4 degrees C with 0.5 N acetic acid containing 1% NP-40 and rapid neutralization allowed recovery of active Fc gamma receptor. Purified Fc gamma receptor retained its ligand-binding activity, since approximately 41 to 72% of labeled material specifically rebound to Rab gamma G-Sepharose. Active receptor also rebound to human IgG- and rat IgG-sepharose. Active Fc gamma receptor did not bind to Sepharose coupled to rabbit Fab, rabbit F(ab)'2 or human F(ab)'2 fragments, nor to Sepharose coupled to chicken IgG. Analysis of Fc gamma receptor by SDS polyacrylamide gels demonstrated a broad peak of radioactivity in the apparent m.w. range of 50,000 to 70,000 in 5.6% acrylamide gels and 35,000 to 55,000 in 9% gels. Labeled receptor with similar structural characteristics and ligand-binding activity was also obtained from highly purified adherent cell populations and from macrophages biosynthetically-labeled with [14C]glucosamine in culture.

Animals↗