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Biomedical subjects

H R Creamer

Publications and source records attributed to H R Creamer.

At least 19 recordsLinked to original sources
PubMed1993

Phorbol myristate acetate induction of chemotactic migration of human polymorphonuclear neutrophils.

The in vitro migration of human polymorphonuclear neutrophils (PMNs) was studied employing an enzymatic assay of cell migration with phorbol myristate acetate (PMA) as the test stimulant. Our data clearly show that PMA in concentrations between 1 and 100 ng/ml in the lower wells of blind-well chambers induced chemotactic migration. Chemokinesis (increased migration) was not induced when PMA was present in both the upper and lower chambers (i.e., in a nongradient mode). Clearly our data indicate that PMA is chemotactic for human PMNs and, coupled with published studies of the effect of PMA on PMNs, suggest activation of an intracellular gradient of membrane-associated protein kinase C as a possible new mechanism for the induction of oriented migration of PMNs. Such a mechanism may be generalized to include membrane-soluble materials (e.g., inflammatory mediators, microbial products), which establish internal gradients of activated PKC rather than via the "classic" agonist-surface receptor mechanism, providing an alternative pathway for the induction of leukocyte chemotaxis.

Chemotaxis, Leukocyte↗
PubMed1991

Concurrent lipopolysaccharide enhances chemotactic response of human polymorphonuclear leukocytes to bacterial chemotaxin.

Polymorphonuclear neutrophil (PMN) function is thought to be critical in resistance to infectious agents and this implies that the PMN must be able to migrate into, and to function in, environments that may have high levels of bacterial lipopolysaccharide (LPS). Therefore, we have evaluated the effect of LPS on the in vitro migration of PMNs. Our data reveal that the human PMN is resistant to the deleterious effects of high levels of LPS, that in high concentrations LPS is, itself, a direct chemoattractant for PMNs, and that PMN migration toward a bacterial chemotaxin is enhanced if LPS is also present. Such capabilities suggest that the PMN may be uniquely qualified to migrate into microenvironments that are rich in LPS.

Cell Migration Inhibition↗
PubMed1991

A novel hypothesis concerning the mechanisms of activation, and of control, of periodontal bone loss.

Chronic inflammatory periodontitis fulfills the classical definition of an infectious disease in that it is a disease of the host caused by the activities of one or more parasites. Typically, the etiology of an infectious disease has been defined as the specific microbe which incites the disease process, even though the quality and nature of host responses to the pathogen may underlie much of the pathology seen. This approach in the study of the etiology of chronic periodontitis has not resulted in the identification of a single 'periodontopathogen', but rather is leading to the realization that multiple sets of microbes may induce the same endpoint, albeit some possibly more efficiently than others. The premise of this paper is that a different view of the literature in the area, with the primary emphasis on the mechanisms of damage and resistance to periodontitis, reveals a probable commonality, rather than a plethora of diseases. The concept of a mechanism-based etiology, rather than of a microbe-based one, deserves consideration for this complex, host-parasite interaction. The novel hypothesis presented here is that the common virulence factor of chronic periodontitis is lipopolysaccharide (LPS), that the central damaging mediator is a cyclooxygenase product of arachidonic acid (probably prostaglandin E2), and that the critical resistance mechanism that limits disease activity is the effective, peripheral neutralization of LPS by emigrated polymorphonuclear neutrophils.

Alveolar Bone Loss↗
PubMed1991

Suppression of human neutrophil functions by tetracyclines.

Tetracycline inhibition of neutrophil-associated collagenolysis has been the focus of a number of investigations. Evidence has suggested that this inhibition results from the ability of this family of antimicrobial drugs to bind divalent cations such as Ca2+ and Zn2+, two cations that are required for full expression of activity of metalloproteinases such as collagenase and gelatinase. Data presented in this study demonstrate that tetracyclines can also inhibit neutrophil-mediated RBC lysis, superoxide anion synthesis, degranulation and migration. To some extent, tetracycline inhibition of neutrophil functions is mimicked by the Ca2+ binding agents, EDTA and TMB-8. However, Ca2+ enrichment restored full function to EDTA- and TMB-8-treated cells but not to tetracycline-treated neutrophils. This suggests that Ca2+ binding plays a role but is not the critical effect leading to tetracycline suppression of neutrophil functions. It has been suggested that tetracyclines can suppress leukocyte-associated tissue damage. Host tissues are protected from neutrophil-mediated damage by two mechanisms: 1. Neutrophil granule-associated enzymes are secreted in an inactive state; and, 2. tissues are protected from these enzymes by a potent inhibitor shield. Neutrophils can bypass these protective elements by activating enzymes and by destroying the shield through the synthesis of oxygen radicals. Therefore, tetracyclines may suppress neutrophil-mediated tissue damage by inhibiting their migration and degranulation and, potentially more importantly, by suppressing synthesis of oxygen radicals.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

An enzyme-assessed microplate-assay for neutrophil adherence. I. IgA-induced adherence of human PMNs.

The binding of PMNs to extracellular matrix and cells is crucial to PMN host defense. Adherence mechanisms and the many families of molecules involved are major areas of study. We present here details of an enzyme-assessed microtiter plate assay for neutrophil adherence. This assay uses low numbers of cells (50,000/well) and permits analysis of several hundred wells in a short period of time, by using an ELISA reader. With this assay we observed 5- to 10-fold increases in the number of adherent human PMNs in response to nanogram amounts of LPS or as little as 5.0 micrograms/ml of aggregated IgA. Although fluoride blocked the LPS-induced adherence response, IgA-induced cell binding was largely unaffected.

Cell Adhesion↗
PubMed1989

Effect of fluoride on movement of concanavalin A-acceptor molecules of human neutrophils.

The effects of fluoride (F) on neutrophil protuberance formation and induced Con A acceptor molecule migration were assessed microscopically. Below 5 mM, F had little effect on acceptor migration, while it markedly inhibited formation of colchicine-induced protuberances. The anion also increased the rate at which preformed protuberances regressed. Since protuberance formation is enhanced by disassembly of microtubules, these data suggest that F promotes and/or stabilizes microtubule assembly. Microtubule assembly is favored by binding of GTP to tubulin subunits, while GDP binding favors disassembly of microtubules. Since F binds with GDP, forming a new complex that mimics GTP, the anion would be expected to enhance microtubule assembly. Over the same F concentration range, the anion failed to inhibit acceptor polarization, but did inhibit cytochalasin B-enhanced dispersion of prepolarized Con A acceptors, implying that, at low concentrations, F also affected microfilament cycling. Concentrations of F in excess of 5 mM inhibited acceptor migration as well as protuberance formation. At 20 mM, the anion abolished both events, yet at this same concentration F induced neutrophil superoxide generation and degranulation, suggesting that acceptor migration is not a prerequisite for these two neutrophil effector activities.

Actin Cytoskeleton↗
PubMed1989

Fluoride activation of neutrophils: similarities to formylmethionyl-leucyl-phenylalanine.

Fluoride induced degranulation of both primary and specific granules from neutrophils pretreated with cytochalasin B. There was a similarity in the dependency on extracellular Ca2+ for fluoride- and for FMLP-stimulated O2- generation and degranulation. Pertussis toxin, but not cholera toxin, inhibited FMLP and fluoride activation of neutrophils, while neither toxin affected PMA activation of these cells. These results suggest that fluoride and FMLP activate neutrophils through a common Ca2+-dependent and pertussis toxin-sensitive pathway.

Calcium↗
PubMed1989

Aggregated human colostral sIgA stimulates delayed, non-complement-dependent, NBT reduction by human neutrophils.

Antibodies often alert polymorphonuclear neutrophils (PMNs) to the presence of pathogens. In a study to learn if secretory immunoglobulins can carry out this function, we observed that as little as 4 micrograms/ml of secreted human immunoglobulin A from colostrum (sIgA), in the absence of antigen, stimulated human PMNs to reduce nitroblue tetrazolium (NBT). NBT reduction was inhibited 71% by superoxide dismutase. Active complement pathways were not required since comparable activity was obtained in the presence of heat-inactivated serum. Aggregated forms of sIgA were much more stimulatory than nonaggregated dimeric sIgA. Such interaction between PMNs and sIgA could act in situ to enhance protection against infections of exposed body sites or could initiate inflammatory tissue damage.

Complement Activation↗
PubMed1987

The concentration of lipopolysaccharide on individual root surfaces at varying times following in vivo root planing.

Material with endotoxin activity has been detected in extracts prepared from pooled, periodontally involved teeth, and it has been shown that root planing in vivo reduces the level of such material. However, questions concerning the concentration of endotoxin on the diseased surfaces of individual teeth and questions concerning how rapidly individual root planed tooth surfaces retoxify in vivo have not been addressed previously. Citric acid extracts were prepared from individual, periodontally diseased teeth that had been extracted either from the oral cavity without prior root planing or at varying times up to 12 weeks following root planing. Using a chromogenic Limulus Amebocyte Lysate (LAL) assay, we were able to quantitate the amount of endotoxin associated with diseased root surfaces of individual teeth. We concluded that the extracted material contained endotoxin since it activated LAL and since the LAL-activation was heat-stable, acid-stable and neutralizeable by polymyxin B. The levels of endotoxin found on the root surfaces of these individual, periodontally involved teeth at varying times following in vivo root planing support the following conclusions: the concentration of endotoxin present on diseased root surfaces is markedly reduced, but not eliminated, by in vivo root planing, significant retoxification of root planed surfaces occurs within a relatively short time period after root planing and biological responses to such toxification conceivably may lead to subsequent phases having reduced levels of endotoxin.

Adult↗
PubMed1986

Role of extracellular calcium in neutrophil responsiveness to chemotactic tripeptides.

Cellular stores of Ca2+, but not extracellular Ca2+, are required for effective FMLP stimulation of neutrophil O2- production and degranulation. Neutrophils transferred from Ca2+-containing to Ca2+-free medium gradually lose their responsiveness to FMLP, such that after 40 min in the Ca2+-free environment they have lost 60-70% of their initial responsiveness to FMLP. The loss in responsiveness is reflected both in an increase in lag interval and decrease in velocity of O2-synthesis. The rate of decline in responsiveness to FMLP is greatly accelerated when neutrophils incubated in the presence of A23187 and Ca2+-free medium, while the rate of loss of responsiveness to FMLP is not affected by EGTA but the extent of loss is increased. Gradual recovery of FMLP-induced O2- generation occurs when cells are transferred from Ca2+-free to Ca2+-containing medium. PMA-induced neutrophil O2- generation is not influenced by the presence or absence of extracellular Ca2+. It is our view that the rise or fall of neutrophil responsiveness reflects repletion or depletion of cellular stores of Ca2+ essential for stimulus-effector coupling and that the role of extracellular Ca2+ is subservient to maintenance of these stores.

Calcimycin↗
PubMed1986

Modulation of the kinetics of induced neutrophil superoxide generation by fluoride.

Fluoride (F-) is unique in that, depending upon concentration, it can either stimulate or inhibit the synthesis of the neutrophil's major intracellular microbicidal product, superoxide anion (O2-). While a number of studies have delved into F- induction of O2- generation, little has been reported on the effect of F- on the synthesis of O2- induced by other agents. In this report, we show that F- inhibits the activation and activity of neutrophils induced either by formyl-methionyl-leucylphenylalanine (FMLP) or by phorbol myristate acetate (PMA). The extent of increase in lag period and decrease in velocity of O2- synthesis was found to be directly related to the concentrations of F- and of hydrogen ion in the medium and to the length of contact time between the neutrophil and F-. Decreasing the pH of the medium, while maintaining a constant concentration of F-, disproportionately increased the inhibitory effect of the anion over the effect of pH alone. Fluoride inhibited the responsiveness of neutrophils to FMLP more than it did their responsiveness to PMA. A gradual but partial reversal of F-'s inhibition of O2- synthesis could be achieved by transferring inhibited cells to F(-)-free medium. Our study shows that the suppression of both FMLP- and PMA-induced synthesis of O2- by neutrophils is most dramatic at a reduced pH and is probably related to the intracellular accumulation of F-.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1984

Activated rat neutrophils. A sequential quantitative assay for aggregation and NBT reduction.

We present here a rapid, sensitive, and convenient approach for the analysis of activated Lewis rat PMNs based on detecting separately, or in tandem, PMN aggregation and PMN reduction of nitroblue tetrazolium (NBT). These responses are quantitated using an ELISA scanner which can rapidly measure optical densities of cell cultures in microtiter plates. Aggregation induced by as little as 0.005 micrograms/ml of phorbol myristate acetate (PMA), 0.01 micrograms/ml lipopolysaccharide (LPS), or a 1:160 dilution of lymphokine-containing rat serum can be detected employing this approach. NBT reduction was induced by as little as 0.01 micrograms/ml PMA. Blocking studies employing 2-deoxyglucose, iodoacetamide, and polymyxin B gave the expected results and confirmed that these assays detect cellular responses to soluble stimuli. Using this technology the effects of PMA and LPS on rat peritoneal exudate PMNs were evaluated. Rat PMNs appeared less sensitive to LPS than human PMNs and also reduced NBT more slowly following stimulation with PMA. Because of the slowness in NBT reduction following stimulation, NBT reduction can be evaluated, in tandem, after measuring aggregation. The simplicity of this system, coupled with the speed with which large numbers of microcultures can be read and the low number of cells required, make this approach for studying responses especially attractive.

Animals↗
PubMed1983

Endogenous component chemotactic assay (ECCA).

We have developed a chemotactic assay in which migrated cells are quantitated by measuring levels of an endogenous cellular component. The endogenous component chemotactic assay (ECCA) employs standard double-membrane, blind-well methodologies but is unique in that leukocyte migration is quantitated by measuring lactic dehydrogenase (LDH) activity endogenous to cells that have migrated. This approach avoids the tedium of microscopic counting as well as the problems associated with cell-labeling techniques. Using the ECCA technique we have shown: (1) that N-formylmethionyl-leucyl-phenylalanine (fMLP) is both chemokinetic and chemotactic for human polymorphonuclear neutrophils (PMNs); (2) that both incubation time and starting PMN density affect the proportion of cells that migrate; (3) that approximately 30% of the available PMNs eventually migrate; and (4) that PMN "fall off" from membranes, readily detectable by this assay, is affected by starting PMN density, incubation period, and nature of the attractant. The technique as presented can detect migration when a starting cell density as low as 7 x 10(4) PMNs/well is employed and can be made more sensitive by increasing the period over which LDH is allowed to act. Considerable potential exists to further apply the ECCA concept to the study of the migration of subpopulations of cells in mixtures by assaying for distinguishing endogenous cellular markers.

Buffers↗
PubMed1982

A rapid quantitative assay for activated neutrophils.

We present here a rapid, sensitive, and convenient assay for activated human PMNs based on detecting the decreased optical density (OD) of aggregated cell suspensions. This quantitative assay uses an ELIZA machine to measure OD changes, with time, of activated cells (5 X 10(5) cells/well) in microtiter plates. The assay is sensitive, detecting aggregation induced by as little as 0.0001 microgram/ml of LPS, or lymphokines in Con-A-activated supernatant diluted 1/500. The assay permits analysis of 400 separate PMN suspensions on the same day starting with less than 80 ml of blood.

Cell Aggregation↗