Investigation into antibacterial and wound healing properties of platelets lysate against Acinetobacter baumannii and Klebsiella pneumoniae burn wound infections.

BACKGROUND AND AIM: Treatment of burn wound infections has become a global challenge due to the spread of multidrug-resistant bacteria; therefore, the development of new treatment options for the mentioned infections is essential. Platelets have drawn much attention for this purpose because they are a safe and cost-effective source of different antimicrobial peptides and growth factors. The present study evaluated antibacterial effects and wound healing properties of Platelet-derived Biomaterial (PdB) against Acinetobacter baumannii and Klebsiella pneumoniae burn wound infections. METHODS: PdB was prepared through the freezing and thawing process and then, in vitro antibacterial effect was determined by disk diffusion and broth microdilution methods. Afterward, burn wound was inflicted on 56 rats, infected with both bacteria, and topical administration was performed to evaluate antibacterial effects and wound healing properties of PdB. RESULTS: In vitro results showed that PdB inhibited the growth of A. baumannii in the highest dose (0.5), while we did not detect any inhibitory effects against K. pneumoniae. By contrast, PdB significantly inhibited the growth of bacteria in treated animal wounds compared to the control groups (P value < 0.05). Macroscopic assessments pointed to the significant enhancement of wound closure in the treated animals. In addition, histopathological examination demonstrated that treatment of rats with PdB led to a considerable increase in re-epithelialization and attenuated the formation of granulation tissue (P value < 0.05). CONCLUSION: The use of topical PdB is an attractive strategy for treating A. baumannii and K. pneumoniae burn wound infections because it inhibits bacterial growth and promotes wound healing properties.

A surface-exposed neuraminidase affects complement resistance and virulence of the oral spirochaete Treponema denticola.

Neuraminidases (sialidases) catalyse the removal of terminal sialic acid from glycoconjugates. Bacterial pathogens often utilize neuraminidases to scavenge host sialic acid, which can be utilized either as a nutrient or as a decorating molecule to disguise themselves from host immune attacks. Herein, a putative neuraminidase (TDE0471) was identified in Treponema denticola, an oral spirochaete associated with human periodontitis. TDE0471 is a cell surface-exposed exo-neuraminidase that removes sialic acid from human serum proteins; it is required for T.denticola to grow in a medium that mimics gingival crevice fluid, suggesting that the spirochaete may use sialic acid as a nutrient in vivo. TDE0471 protects T.denticola from serum killing by preventing the deposition of membrane attack complexes on the bacterial cell surface. Animal studies revealed that a TDE0471-deficient mutant is less virulent than its parental wild-type strain in BALB/C mice. However, it causes a level of tissue damage similar to the wild type in complement-deficient B6.129S4-C3(tm1) (Crr) /J mice albeit the damage caused by both bacterial strains is more severe in these transgenic mice. Based on these results, we propose that T.denticola has evolved a strategy to scavenge host sialic acid using its neuraminidase, which allows the spirochaete to acquire nutrients and evade complement killing.

In vitro activity of photoactivated disinfection using a diode laser in infected root canals.

OBJECTIVE: To investigate the lethal activity of photoactivated disinfection (PAD) on Enterococcus faecalis (ATCC 29212) and mixed populations of aerobic or anaerobic bacteria in infected root canals using a diode laser after the application of a photosensitizer (PS). MATERIALS AND METHODS: First, the bactericidal activity of a low power diode laser (200 mW) against E. faecalis ATCC 29212 pre-treated with a PS (toluidine blue) for 2 min were examined after different irradiation times (30 s, 60 s and 90 s). The bactericidal activity in the presence of human serum or human serum albumin (HSA) was also examined. Second, root canals were infected with E. faecalis or with mixed aerobic or anaerobic microbial populations for 3 days and then irrigated with 1.5% sodium hypochlorite and exposed to PAD for 60 s. RESULTS: Photosensitization followed by laser irradiation for 60 s was sufficient to kill E. faecalis. Bacteria suspended in human serum (25% v/v) were totally eradicated after 30 s of irradiation. The addition of HSA (25 mg/ml or 50 mg/ml) to bacterial suspensions increased the antimicrobial efficacy of PAD after an irradiation time of 30 s, but no longer. The bactericidal effect of sodium hypochlorite was only enhanced by PAD during the early stages of treatment. PAD did not enhance the activity of sodium hypochlorite against a mixture of anaerobic bacteria. CONCLUSIONS: The bactericidal activity of PAD appears to be enhanced by serum proteins in vitro, but is limited to bacteria present within the root canal.

Effect of ozone on periodontopathogenic species--an in vitro study.

The in vitro study was aimed to determine the effect of ozone on periodontopathogenic microorganisms. Ozone was generated for 6 s-2 × 24 s (corresponding to 0.56 mg-2 × 2.24 mg of ozone) against 23 mainly anaerobic periodontopathogenic species. Agar diffusion test was used as a screening method. Then, the killing activity was tested in a serum-free environment and with 25% v/v inactivated serum. Further, the effect of ozone on bactericidal activity of native serum was analyzed against Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans. Agar diffusion test showed a high efficacy of ozone against microorganisms, especially against Porphyromonas gingivalis. This result was confirmed by the killing tests; most of the strains in a concentration of 10(5) were completely eliminated after twofold 18-s application of ozone. Only four of the six potentially "superinfecting" species (Staphylococcus aureus, Enterococcus faecalis, Enterobacter cloacae, Candida albicans) survived in part. Addition of heat-inactivated serum reduced the killing rate of ozone by 78% after 6-s and by 47% after twofold 18-s exposures; no strain was completely eradicated after any application of ozone. The bactericidal effect of native serum was enhanced after application of ozone; no effect was visible on the included A. actinomycetemcomitans strain which was found to be completely resistant to the bactericidal action of serum. In conclusion, (a) ozone has a strong antibacterial activity against putative periodontopathogenic microorganisms, and (b) the bactericidal effect is reduced in the presence of serum. Ozone may have potential as an adjunctive application to mechanical treatment in periodontitis patients.

Interpain A, a cysteine proteinase from Prevotella intermedia, inhibits complement by degrading complement factor C3.

Periodontitis is an inflammatory disease of the supporting structures of the teeth caused by, among other pathogens, Prevotella intermedia. Many strains of P. intermedia are resistant to killing by the human complement system, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with recombinant cysteine protease of P. intermedia (interpain A) resulted in a drastic decrease in bactericidal activity of the serum. Furthermore, a clinical strain 59 expressing interpain A was more serum-resistant than another clinical strain 57, which did not express interpain A, as determined by Western blotting. Moreover, in the presence of the cysteine protease inhibitor E64, the killing of strain 59 by human serum was enhanced. Importantly, we found that the majority of P. intermedia strains isolated from chronic and aggressive periodontitis carry and express the interpain A gene. The protective effect of interpain A against serum bactericidal activity was found to be attributable to its ability to inhibit all three complement pathways through the efficient degradation of the alpha-chain of C3 -- the major complement factor common to all three pathways. P. intermedia has been known to co-aggregate with P. gingivalis, which produce gingipains to efficiently degrade complement factors. Here, interpain A was found to have a synergistic effect with gingipains on complement degradation. In addition, interpain A was able to activate the C1 complex in serum, causing deposition of C1q on inert and bacterial surfaces, which may be important at initial stages of infection when local inflammatory reaction may be beneficial for a pathogen. Taken together, the newly characterized interpain A proteinase appears to be an important virulence factor of P. intermedia.

Studies on the mechanism of bacterial resistance to complement-mediated killing. II. C8 and C9 release C5b67 from the surface of Salmonella minnesota S218 because the terminal complex does not insert into the bacterial outer membrane.

The mechanism for consumption of terminal complement components and release of bound components from the surface of serum-resistant salmonella minnesota S218 was studied. Consumption of C8 and C9 by S218 occurred through interaction with C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8-deficient serum and washed to remove all C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8- deficient serum and washed to remove al but cell bound C5b67. Rapid release of (125)I C5 and (125)I C7 from the membrane of S218 was dependent on binding of C8 because (125)I C5 and (125)I C7 deposition in C8D serum was stable and was twofold higher in C8D than in PNHA, and addition of purified C8 or C8 and C9 to S218 previously incubated in C8D serum caused rapid release of (125)I C5 and (125)I C7 from the organism. Analysis by sucrose density gradient ultracentrifugation of the fluid phase from the reaction of S218 and 10 percent PNHS revealed a peak consistent with SC5b-9, in which the C9:C7 ratio was 3.3:1, but the NaDOC extracted bound C5b-9 complex sedimented as a broad peak with C9:C7 of less than 1.2:1. Progressive elution of C5b67 and C5b-9 from S218 but not serum-sensitive S. minnesota Re595 was observed with incubation in buffers of increasing ionic strength. Greater than 90 percent of the bound counts of (125)I C5 or (125)I C9 were released from S218 by incubation in 0.1 percent trypsin, but only 57 percent of (125)I C9 were released by treatment of Re595 with trypsin. These results are consistent with the concept that C5b-9 forms on the surface of the serum-sensitive S. minnesota S218 in normal human serum, but the formed complex is released and is not bactericidal for S218 because it fails to insert into hydrophobic outer membrane domains.

Influence of serum on interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with an epithelial cell line.

BACKGROUND AND OBJECTIVE: The purpose of this study was to investigate the influence of serum on the interaction of periodontal pathogens with epithelial cells using an epithelial cell line (KB cells). This is important because serum is a key component of gingival crevicular fluid and may influence inflammatory responses in epithelial cells exposed to periodontal pathogens. MATERIAL AND METHODS: Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 were co-cultured with KB cells either with or without the addition of up to 10% human serum or 50 mg/mL human serum albumin. The numbers of free-floating, adherent and intracellular bacteria were determined up to 18 h after exposure of the epithelial cells to the pathogens. Additionally, the concentrations of interleukin (IL)-6 and IL-8 produced by the epithelial cells in response to exposure to the bacteria were determined. RESULTS: Serum and human serum albumin reduced the number of internalized A. actinomycetemcomitans Y4 organisms in the epithelial cells, increased the levels of IL-6 and IL-8 in the supernatants of infected cells (those with internalized A. actinomycetemcomitans) and influenced non-infected epithelial cells. Increased IL-6 and IL-8 concentrations were also detected in the supernatants of KB cells infected with P. gingivalis ATCC 33277. Interleukin-6 and IL-8 were detectable after addition of serum, probably as a result of inhibition of the activity of P. gingivalis cysteine proteinases by serum. CONCLUSION: Serum promotes the release of the cytokines IL-6 and IL-8 by epithelial cells. This mechanism is influenced by periodontal pathogens and may maintain clinical periodontal inflammation.

Binding of complement inhibitor C4b-binding protein contributes to serum resistance of Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the alpha-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.

The human CXC chemokine granulocyte chemotactic protein 2 (GCP-2)/CXCL6 possesses membrane-disrupting properties and is antibacterial.

Granulocyte chemotactic protein 2 (GCP-2)/CXCL6 is a CXC chemokine expressed by macrophages and epithelial and mesenchymal cells during inflammation. Through binding and activation of its receptors (CXCR1 and CXCR2), it exerts neutrophil-activating and angiogenic activities. Here we show that GCP-2/CXCL6 itself is antibacterial. Antibacterial activity against gram-positive and gram-negative pathogenic bacteria of relevance to mucosal infections was seen at submicromolar concentrations (minimal bactericidal concentration at which 50% of strains tested were killed, 0.063 +/- 0.01 to 0.37 +/- 0.03 muM). In killed bacteria, GCP-2/CXCL6 associated with bacterial surfaces, which showed membrane disruption and leakage. A structural prediction indicated the presence of three antiparallel NH(2)-terminal beta-sheets and a short amphipathic COOH-terminal alpha-helix; the latter feature is typical of antimicrobial peptides. However, when the synthetic derivatives corresponding to the NH(2)-terminal (50 amino acids) and COOH-terminal (19 amino acids, corresponding to the putative alpha-helix) regions were compared, higher antibacterial activity was observed for the NH(2)-terminus-derived peptide, indicating that the holopeptide is necessary for full antibacterial activity. An artificial model of bacterial membranes confirmed these findings. The helical content of GCP-2/CXCL6 in the presence or absence of lipopolysaccharide or negatively charged membranes was studied by circular dichroism. As with many antibacterial peptides, membrane disruption by GCP-2/CXCL6 was dose-dependently reduced in the presence of NaCl, which, we here demonstrate, inhibited the binding of the peptide to the bacterial surface. Compared with CXC chemokines ENA-78/CXCL5 and NAP-2/CXCL7, GCP-2/CXCL6 showed a 90-fold-higher antibacterial activity. Taken together, GCP/CXCL6, in addition to its chemotactic and angiogenic properties, is likely to contribute to direct antibacterial activity during localized infection.

Influence of lipid composition on membrane activity of antimicrobial phenylene ethynylene oligomers.

Host defense peptides (HDPs), part of the innate immune system, selectively target the membranes of bacterial cells over that of host cells. As a result, their antimicrobial properties have been under intense study. Their selectivity strongly depends on the chemical and mostly structural properties of the lipids that make up different cell membranes. The ability to synthesize HDP mimics has recently been demonstrated. To better understand how these HDP mimics interact with bilayer membranes, three homologous antimicrobial oligomers (AMOs) 1-3 with an m-phenylene ethynylene backbone and alkyl amine side chains were studied. Among them, AMO 1 is nonactive, AMO 2 is specifically active, and AMO 3 is nonspecifically active against bacteria over human red blood cells, a standard model for mammalian cells. The interactions of these three AMOs with liposomes having different lipid compositions are characterized in detail using a fluorescent dye leakage assay. AMO 2 and AMO 3 caused more leakage than AMO 1 from bacteria membrane mimic liposomes composed of PE/PG lipids. The use of E. coli lipid vesicles gave the same results. Further changes of the lipid compositions revealed that AMO 2 has selectively higher affinity toward PE/PG and E. coli lipids than PC, PC/PG or PC/PS lipids, the major components of mammalian cell membranes. In contrast, AMO 3 is devoid of this lipid selectivity and interacts with all liposomes with equal ease; AMO 1 remains inactive. These observations suggest that lipid type and structure are more important in determining membrane selectivity than lipid headgroup charges for this series of HDP mimics.

Eugenol inhibited the antimicrobial functions of neutrophils.

Eugenol-containing restorative materials are commonly used for vital pulp therapy. A well-regulated host defense response is pivotal for the success of vital pulp therapy. The present study was to assess the effects of eugenol on the antimicrobial functions of polymorphonuclear leukocytes (neutrophils). Treatment with eugenol (< or = 1.25 mmol/L) for 30 minutes did not significantly affect the viability of neutrophils. However, preincubation of neutrophils with eugenol (1.25 mmol/L and 2.5 mmol/L) abolished their bactericidal activity against oral pathogens Streptococcus mutans and Actinobacillus actinomycetemcomitans. In addition, through the suppression of the extracellular release of myeloperoxidase and the intracellular production of reactive oxygen species, eugenol at sufficient concentrations impaired the activation of neutrophils by cytochalasin B and fMet-Leu-Phe (CB/fMLP). These results suggested that the antimicrobial functions of neutrophils were interfered by eugenol, and the inhibitory effects of eugenol (< or = 1.25 mmol/L) were not due to direct killing of neutrophils.

Effect of 2-hydroxyethyl-methacrylate (HEMA) on the phagocytic and respiratory burst activity of human neutrophils and monocytes.

Neutrophils and monocytes/macrophages (MØ), found in oral mucosa and gingival sulcus, phagocytose and kill bacteria using products produced during a respiratory burst. 2-Hydroxyethyl-methacrylate (HEMA) is a major component released from resin glass ionomer and dental adhesives. Hence, in pulp and gingiva, phagocytes can come into contact with unpolymerized HEMA monomers. The aim of this study was to examine the effects of exposure to HEMA on neutrophil and monocyte bactericidal function. Blood collected from five female volunteers was exposed in vitro to HEMA for 2 h and then phagocytosis, respiratory burst, and cellular integrity were measured using flow cytometry. Respiratory burst was quantified by measuring fluorescent rhodamine 123 generated via oxidation of dihydrorhodamine 123. Cellular membrane integrity was evaluated by staining with propidium iodide. The respiratory burst activity of the neutrophils was significantly decreased by exposure to 7.5 and 15 mM HEMA. No significant effect of HEMA was seen on the number of granulocytes or monocytes capable of performing respiratory burst. Furthermore, there was no significant effect of HEMA on the phagocytic activity of the monocytes or the granulocytes. In conclusion, HEMA did not affect the phagocytosis activity of neutrophils; however, the ability of the cells to kill internalized prey was significantly reduced.

In vitro killing of oral Capnocytophaga by granule fractions of human neutrophils is associated with cathepsin G activity.

The Capnocytophaga are inhabitants of the hypoxic human gingival crevice that are normally prevented by neutrophils from causing periodontal and systemic infection. To identify potential nonoxidative bactericidal mechanisms against Capnocytophaga within human neutrophils, gel filtration chromatography was used to fractionate neutrophil granule extracts. Seven granule fractions, designated A through G, were obtained. The Capnocytophaga were most sensitive to killing by fraction D. Fraction D exhibited substantial bactericidal activity under aerobic and anaerobic conditions. The bactericidal activity associated with ion-exchange subfractions D8-D11, which contained primarily cathepsin G as assessed by enzymatic activity, amino acid composition, and NH2-terminal sequence. Heat-inactivation, diisopropylfluorophosphate, PMSF, and N-benzyloxycarbonylglycylleucylphenylalanyl-chloromethyl ketone inhibited bactericidal activity against Capnocytophaga sputigena but not Escherichia coli. We conclude that (a) human neutrophil cathepsin G is an important antimicrobial system against the Capnocytophaga, (b) the bactericidal activity of cathepsin G against Capnocytophaga is oxygen independent, and (c) an intact enzyme active site is involved in the killing of C. sputigena but not E. coli. We suggest that human neutrophil cathepsin G is an important antimicrobial system against certain oral bacteria and that cathepsin G kills bacteria by two distinct mechanisms.

Regulation of human monocyte/macrophage function by extracellular matrix. Adherence of monocytes to collagen matrices enhances phagocytosis of opsonized bacteria by activation of complement receptors and enhancement of Fc receptor function.

In inflammation monocytes emigrate from the peripheral circulation into an extravascular area rich in extracellular matrix proteins. In this milieu, phagocytes ingest and kill invading pathogens. In the present studies, we found that monocytes adhered to type I collagen gels phagocytized 2.5-12-fold more opsonized Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae than plastic-adherent monocytes. The rate of phagocytosis and the number of bacteria ingested by collagen-adherent monocytes was equal to, or greater than, the number of bacteria ingested by 7-d cultured macrophages (M phi). Although both collagen- and plastic-adherent monocytes were bactericidal for E. coli and S. aureus, more bacteria were killed by collagen-adherent monocytes by virtue of their enhanced phagocytic capacity. Cultured M phi only were bacteriostatic. Adherence of monocytes to collagen gels activated C receptors (CR) types 1 and 3 for phagocytosis, and enhanced Fc receptor (FcR)-mediated phagocytosis. Collagen- and plastic-adherent monocytes produced equivalent amounts of superoxide anion in response to phorbol myristate acetate and opsonized zymosan. Thus, the enhanced phagocytosis and killing of opsonized bacteria by collagen-adherent monocytes appear to be by regulation of the function of membrane CR and FcR, without apparent enhancement of the respiratory burst. These data suggest that adherence of monocytes to the extracellular matrix during inflammation may rapidly activate these cells for enhanced phagocytic bactericidal activity.

Leukocytic function in hypogammaglobulinemia.

The phagocytic, bactericidal, and metabolic capabilities of circulating blood leukocytes from three adults (two males, one female) with hypogammaglobulinemia and recurrent pneumonia, chronic sinusitis, and intestinal giardiasis were studied. These functions were found to be normal when leukocytes from the patients were incubated in media containing normal human serum. Phagocytosis of Staphylococcus albus and polystyrene balls by both patient and normal leukocytes was diminished when the cells were incubated in hypogammaglobulinemic plasma. A similar defect in opsonization by patient plasma was also noted for pneumococci, Escherichia coli and variably with Staphylococcus aureus. Both patient and normal sera had equivalent levels of heat-labile S. albus opsonins; normal serum, however, contained heat-stable S. albus-specific absorbable opsonins in significantly greater quantities to account for its superior opsonic capacity. The addition of commercial gamma globulin or purified IgG to hypogammaglobulinemic sera restored full S. albus opsonic activity. The relevancy of these observations to the impaired host defenses in these patients will be discussed.

Liposomes or traditional adjuvants: induction of bactericidal activity by the macrophage infectivity potentiator protein (Mip) of Neisseria meningitidis.

Currently, one of the main approaches to achieve a vaccine for serogroup B Neisseria meningitidis is based on outer membrane proteins with low antigenic variability among strains. Since these proteins tend to be minor components of the outer membrane, recombinant production is required to obtain them in sufficient amounts for evaluation and development of vaccines. In this study, we analysed the ability of recombinant macrophage infectivity potentiator (rMip) protein to induce protective bactericidal activity in mice. The rMip protein was cloned from N. meningitidis strain H44/76 and was used to immunise mice, and the sera obtained were tested against the homologous and several heterologous N. meningitidis strains. The sera were obtained using the rMip alone, with adjuvant Al(OH)3 , or after inclusion into liposomes. Bactericidal activity was variable depending on the strain, although high titres were seen against strains H44/76 and NmP27. Liposomes enhanced fourfold the reactivity against the homologous strain. The results presented suggest that the rMip protein should be considered a promising candidate for the improvement of future protein-based vaccines.

Production of protective antibodies against Porphyromonas gingivalis strains by immunization with recombinant gingipain domains.

We studied the effect of antibodies against Porphyromonas gingivalis gingipain domains, preparing them against three recombinant fragments of RgpA (catalytic domain, r-Rgp CAT; hemagglutinin domains, r-Rgp 44 and r-Rgps 15-27) and one fragment of Kgp (catalytic domain, r-Kgp CAT). Enhancement of opsonization and killing by human polymorphonuclear leukocytes were measured in the noninvasive FDC 381 and invasive W50 strains of P. gingivalis. Anti-r-Rgp 44 was the most effective in both strains of P. gingivalis. The present findings lead us to recommend RgpA 44 as a candidate immunogen for vaccines against P. gingivalis.

Oxidative function of neutrophils in periodontitis patients with type 1 diabetes mellitus.

BACKGROUND: The involvement of reactive oxygen species in periodontal diseases is unclear. The aim of the present study was to explore the oxidative function of neutrophilic leukocytes of the peripheral venous blood in patients with severe periodontitis and type 1 diabetes mellitus and in the control subjects with healthy periodontal tissues and without systemic diseases. MATERIALS AND METHODS: The leukocytes for the present investigation were obtained from peripheral venous blood of 38 patients with severe periodontitis and type 1 diabetes mellitus and 27 control subjects. The maximal luminol-dependent chemiluminescence and peak time values of neutrophils stimulated with non-opsonized Escherichia coli ATCC 25922 and Staphylococcus aureus 256 were measured. RESULTS: The maximal luminol-dependent chemiluminescence of neutrophils in patients with severe periodontitis, stimulated with non-opsonized Escherichia coli and Staphylococcus aureus, is mainly lower than that in the control group subjects (p < 0.001). Luminol-dependent chemiluminescence of neutrophils reached its maximal value at the same time in both the diseased and the healthy patients. CONCLUSION: In periodontitis, local non-opsonized bacteria might stimulate neutrophilic leukocytes to release oxygen species. However, these cells in patients with periodontitis are characterized by a lower intensity of luminol-dependent chemiluminescence (p < 0.001) compared to analogous findings in people with healthy periodontal tissues. This might indicate insufficient microbicidal activity of these cells in patients with periodontitis.

Polyanionic agents do not inhibit phagosome-lysosome fusion in cultured macrophages.

The survival of some intracellular pathogens within macrophages may be aided by an ability of the organism to antagonize, from within the entrapping phagosome, its fusion with lysosomes. On the other hand, certain polyanionic agents have been implicated in imposing a similar block to fusion from the lysosomal domain--because the transfer of various foreign markers from lysosomes to newly formed phagosomes is remarkably inhibited in these polyanion-containing cells. Based on an analysis of various observations and our own recent data, we propose that the polyanionics do not, in fact, prevent phagosome-lysosome fusion but, instead, physically entrap the usual markers in a gelatinous matrix within the lysosomes. This view accounts for many paradoxical consequences of polyanionic accumulation and for the curiously normal behavior of macrophages that are presumed to be suffering from such a crucial intracellular dysfunction.

Horse complement protein C9: primary structure and cytotoxic activity.

Lack of hemolytic activity of horse serum is an inherent property of horse C9. To understand the molecular reasons for this deficiency we have cloned C9 cDNA from a horse liver cDNA library and have sequenced the cDNA yielding the complete coding sequence for horse C9. Purification of C9 from horse plasma and microsequencing established the N-terminus of the mature protein and verified that the correct horse C9 cDNA clone had been isolated. The deduced amino acid sequence corresponds to a mature protein of 526 amino acids that is 77% identical to human C9. It has the same domain structure as human C9 and contains 22 cysteines and four invariant tryptophans. The few differences include the N-terminus, which is an unblocked glycine in horse C9 but pyroglutamine in human C9, and three potential N-glycosylation sites compared to two in human C9. The N-terminal difference is unimportant since microsequencing of bovine C9, which is strongly hemolytic, established that it also has an unblocked glycine identical to horse C9. There are no obvious structural differences apparent that could resolve the differences in hemolytic potency between the two molecules. Aside from a few conservative replacements, both C9 sequences are identical between positions 250 and 360. This region includes the membrane interaction domain in C9 and the postulated transmembrane segment that is thought to constitute the wall of a putative transmembrane pore and, therefore, should be required for cytotoxicity. In agreement with this prediction we have observed that, in contrast to the marked decrease in hemolytic activity, horse C9 is very efficient in killing a variety of Gram-negative bacteria. These results demonstrate that horse C9 is a structurally competent molecule with efficient cytotoxic activity. Its inability to lyse erythrocytes may be related to the action of control proteins on target cell membranes.