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Notes: Abstract Previous studies have indicated that in certain types of chronic inflammatory periodontal diseases, polymorphonuclear leucocytes (PMN) functions are impaired. In view of the damage oxygen-free radicals may cause to the periodontal tissues, the present study focussed on superoxide (SO) formation and luminol-dependent chemiluminescence (LDCL) by peripheral PMN cells in rapidly progressive periodontitis patients (RPP). PMN cell preparations were stimulated by either opsonized bacteria or phorbol myristate acetate (PMA). The results indicate that PMN cells from RPP patients, stimulated by opsonized bacteria, have significantly enhanced SO formation and LDCL response as compared to healthy subjects. The hyperactivity was cell-associated. In the presence of PMA, no significant differences were detected between the groups. The results suggest that PMN cells from RPP patients are functionally activated, and produce elevated levels of oxygen radicals. These oxygen radicals may play a role in the pathogenesis of RPP.

Notes: Abstract Poly-l-histidine (PHSTD) of molecular weight 26,000 induced the generation of large amounts of Superoxide (O 2 − ) and hydrogen peroxide (H2O2) in human neutrophils (PMNs). Despite its low solubility at neutral pH, PHSTD was bound very rapidly to the PMN surfaces. Maximal generation of O 2 − took place with 4–5 ×10−6 M of PHSTD, starting after a lag of about 25 sec and proceeding for 15–17 min at a rate of 150 nmol/107 PMNs/min, suggesting that this polycation is one of the most potent stimulators of O 2 − generation known, PHSTD was found to be non-toxic for PMNs even at millimolar concentrations. Generation of O 2 − by PHSTD depended on extracellular calcium; it was inhibited by calcium channel blockers and by trifluoperazine, and it triggered a sharp rise in intracellular calcium as determined by the Quin 2 fluorescence technique. The generation of both O 2 − and H2O2 by PHSTD was partially inhibited by cytochalasin B or (CYB, CYE). On the other hand, CYB markedly enhanced the generation of both O 2 − and H2O2 following stimulation of PMNs either by PHSTD, polyarginine, histone, or by antibody-opsonized group A streptococci. Electron microscopic analysis and NBT reduction tests revealed that both PHSTD and PHSTD-opsonized streptococci were avidly phagocytosed by PMNs. Since CYB totally inhibited internalization of both PHSTD and the PHSTD-opsonized streptococci, it was suggested that these agents stimulated oxygen radical generation mainly on the leukocyte surfaces. Complexes (CX) formed between PHSTD and polyanethole sulfonate (a strong polyanion) or between histone and the polyanion mimicked immune CX in their ability to trigger the generation of large amounts of O 2 − which were inhibited by CYB. Generation of O 2 − and chemiluminescence either by PHSTD or by PHSTD-opsonized streptococci were markedly inhibited by poly-l-glutamate, suggesting that PHSTD acted as a cationic agent which interacted via electrostatic forces with some negatively charged sites in the leukocyte membrane. Generation of H2O2 by PHSTD was also markedly inhibited by deoxyglucose, KCN, DASA, as well as by the lipoxygenase inhibitors nordihydroguaiaretic acid, phenidone, and propylgallate. On the other hand, cyclooxygenase inhibitors such as aspirin, indomethacin, and piroxicam were inactive, suggesting that arachidonic acid metabolism via lipoxygenase pathway might have been involved in the activation by PHSTD of the NADPH oxidase in PMNs. PHSTD may mimic the effects of antibodies both as an opsonin and as a potent stimulator of the respiratory burst in PMNs and may thus serve as a model for further study of leukocyte-bacteria interactions in infectious and inflammatory sites and of the pathogenicity of immune complexes.

Notes: Abstract Various cationic polyelectrolytes (poly-α-amino acids and histones), lectins, the chemotactic peptide, f-methionyl-leucyl-phenylalanine (fMLP), the calcium ionophore A23187, and phorbol myristate acetate (PMA) were investigated regarding their capacity to induce luminol-dependent chemiluminescence (LDCL) and superoxide production by human blood leukocytes. Although when tested individually, poly-l-arginine (PARG), phytohemagglutinin (PHA), concanavalin A (Con A), or fMLP induced only a low to moderate LDCL response, very intense synergistic CL reactions were obtained by mixtures of PARG + PHA, PARG + Con A, PARG + PHA + fMLP, Ca2 + ionophore + PARG + PHA + fMLP, and PARG + PMA. The sequence of addition of the various agents to WBC in the presence of luminol absolutely determined the intensity of the LDCL signals obtained, the highest reactions being achieved when the WBC were preincubated for 2–3 rain with A23187 followed by the sequential addition of fMLP, PARG, and PHA. These “multiple hits” induced CL reactions which were many times higher than those obtained by each factor alone. On the other hand, neither poly-L-lysine, poly-L-ornithine, poly-L-histidine, nor poly-L-asparagine, when employed at equimolar concentrations, cooperated efficiently with PHA and fMLP to trigger synergistic LDCL response s in leukocytes. Concomitantly with the induction of LDCL, certain ligand mixtures also triggered the production of superoxide. The LDCL which was induced by the “cocktail” of agents was markedly inhibited by sodium azide (93% inhibition), but to a lesser extent by catalase (10% inhibition) or by snperoxide dismutase (20%-60% inhibition). On the other hand, scavengers of singlet oxygen and OH' (sodium benzoate, histidine) did not affect the synergistic LDCL responses induced by these multiple ligands. Cytochalasin B also markedly inhibited the LDCL responses induced either by soluble stimuli or by streptococci preopsonized either with histone or with polyanethole sulfonate. The LDCL responses which were induced by mixtures of PARG and concanavalin A were also strongly inhibited by mannose, a-methyl mannoside, and poly-L-glutamic acid: The data suggest that the LDCL responses induced by the soluble ligands involved a myeloperoxidase-catalyzed reaction. The possible employment of “cocktails” of ligands to enhance the bactericidal effects of PMNs, macrophages, and natural killer cells on microbial cells and mammalian targets is discussed.

Notes: Abstract Human blood leukocytes generate intense luminol-dependent chemiluminescence (LDCL) following stimulation by streptococci and by Gram negative rods which had been preopsonized by cationic polyelectrolytes (histone, poly L-arginine-PARG, poly L-histidine-PHSTD). Streptococci but not Gram negative rods or hyaluronic acid-rich streptococci (group C) also induced intense LDCL following opsonization with the anionic polyelectrolytes-dextran sulfate or polyanethole sulfonate (liquoid) suggesting that the outer surfaces of different bacteria bound anionic polyelectrolytes to different extents. Both normal and immune serum, synovial fluids and pooled human saliva inhibited the LDCL responses induced by streptococci preopsonized with poly cations. On the other hand, bacteria which had been first preopsonized by the various body fluids and then subjected to a second opsonization by cationic ligands (“sandwiches”), induced a very intense LDCL response in leukocytes. Streptococci which had been preopsonized by PARG, histone or by PHSTD also triggered superoxide generation by blood leukocytes, which was markedly enhanced by a series of cytochalasins. PHSTD alone induced the formation of very large amounts of superoxide. Paradoxically, the same concentrations of cytochalasins B or C which markedly boosted the generation of superoxide following stimulation of leukocytes with soluble or particulate ligands, had a strong inhibitory effect on the generation of LDCL. On the other hand cycochalasins failed to inhibit LDCL which had been induced by phorbol myristate acetate (PMA). Peritoneal macrophages which had been harvested from C. parvum-stimulated mice, generated more LDCL and superoxide following stimulation by PARG than macrophages obtained from proteose peptone-stimulated mice. Macrophages which had been activated either by proteose peptone or by C. parvum and cultivated for 2 hours on teflon surfaces, generated much more LDCL than macrophages which had been cultivated for 24 hours on teflon surfaces. Both cationic and anionic polyelectrolytes mimic the effects of antibodies as activators of the oxygen burst in blood leukocytes and in macrophages. Such polyelectrolytes can serve as models to further study leukocyte-bacteria interactions in infectious and inflammatory sites.

Notes: Abstract Human blood leukocytes generated large amounts of superoxide O2 − following stimulation by certain “cocktails” of soluble agents consisting of poly-L-arginine (PARG), phytohemagglutinin, the chemotactic peptide formylmethionyl-leucyl-phenylalanine and polyanethole sulfanote (liquoid). A variety of cytochalasins, which markedly boosted O2 − generation by the soluble cocktails, markedly depressed luminol-dependent chemiluninescence (LDCL) which had been induced either by opsonized streptococci or by soluble agents. Glutathione, which totally reversed the inhibition of LDCL induced by cytochalasin A, failed to reverse the inhibition of LDCL induced by cytochalasin B. Generation of O2 − by all the soluble agents employed, except PMA, was strongly inhibited either by the omission of extracellular calcium and magnesium or by treatment with the calcium blocker TMB-8. Generation of O2 − was enhanced following stimulation of leukocytes with soluble agents if the cells had been exposed to slightly hypotonic buffers. Leukocytes, which had been preincubated for short periods (5 min) with PARG, saponin, digitonin, or lysolecithin(LL) and which lost their viability, and their O2 − and LDCL-generating capacities following stimulation by soluble agents containing cytochalasin B, nevertheless regained these activities by the addition of NADPH. It is suggested that the lytic agents induced the leakage out of NADPH rather than acting as inactivators of the oxidase in the leukocyte membranes. Prolonged incubation of leukocytes with lytic agents failed to allow restoration, by NADPH, of the generation of SOD-inhibitable O2 − generation. Since PARG acted both as a cytolytic agent and as a inducer of O2 − generation, we postulate that lytic agents might also act as “primers” of the nascent membrane oxidase which could, however, be further potentiated and activated by soluble agents acting in “multiple hits,” PARG could be totally replaced either by LL or by digitonin in the generation of O2 − provided that both PHA and cytochalasin B were present in the reaction mixtures. We suggest that the various ingredients of the soluble “cocktails” may help to assemble components of the NADPH oxidase. Such an assembly and regulations are prerequisite for stimulation of the NADPH oxidase and the generation of oxygen radicals in leukocytes.