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.

Mechanism of extracellular release of human neutrophil calprotectin complex.

Calprotectin is an abundant cytosolic protein complex of human neutrophils with in vitro extracellular antimicrobial activity. Studies suggest that calprotectin may be actively secreted from intact HL-60 cells and that it can be translocated to polymorphonuclear neutrophil (PMN) cell membranes. To examine whether calprotectin is secreted extracellularly, we incubated soluble and particulate stimuli, including live and heat-inactivated Candida albicans, with whole blood and measured calprotectin levels in the plasma. We compared the release of calprotectin to that of lactoferrin, a protein known to be secreted by PMNs. Extracellular lactoferrin was detected after incubation with any of the particulate stimuli. In contrast, a significant increase in extracellular calprotectin was found only after incubation with live C. albicans. Specifically, the increase in extracellular calprotectin correlated directly with a proportional decrease in PMN viability. Our results indicate that human PMN calprotectin is not secreted extracellularly except as a result of cell disruption or death.

Parkinsonism following encephalitis of unknown etiology.

The patient, a clinical case of parkinsonism, was a 32-year-old man, born in 1942, long after the prevalence of von Economo's lethargic encephalitis in Japan. Anatomically, the neurons in the substantia nigra of the mid-brain were extensively degenerated, and presented Alzheimer's neurofibrillary tangles. At the same time, melanin pigment was scattered in the tissue and was phagocytized by glia cells. Perivascular cuffing was observed in the frontal lobe, parietal lobe, temporal lobe, hippocampus, and thalamus as well as in the substantia nigra. Neuronophagia was noted in the thalamic nuclei. The present case was believed to have parkinsonism not clinically or pathologically related to von Economo's encephalitis or to Japanese encephalitis, but following a mild encephalitis of unknown etiology.

Hemochromatosis associated with brain lesions--a disorder of trace-metal binding proteins and/or polymers?

A 68-year-old man, after having been diagnosed as having hepatic disease at about the age of 41 years, had been hospitalized frequently until his death. Blood sugar, iron, and copper had not increased during his illness. Although the diagnosis of liver cirrhosis had been made and he had been receiving therapy, various neurologic symptoms without disturbances of consciousness appeared six months before his death. Autopsy revealed hemochromatosis, liver cirrhosis, and pancreatic fibrosis. A large amount of iron had accumulated in the liver, the pancreas, and the thyroid gland, while considerable numbers of ceroid and lipofuscin pigment granules had accumulated diffusely in the brain. Abnormal astrocytes of the Alzheimer II type were diffusely distributed in the brain and contained no intranuclear glycogen which stained positive with the carmine stain. No spongy changes were seen in the deeper layers of the cerebral cortex. Chemical analyses for trace metals in the brain, liver, and kidneys revealed a large amount of iron and increased copper in the liver, and considerable quantities of copper, manganese, calcium, and mercury in the brain. Because of changes in the erythrocyte sedimentation rate and marked thymol turbidity seen before and after the occurrence of the neurologic symptoms, this man was suspected of having disorders of the trace-metal binding proteins and/or of their polymers.

Beta-sheet antibiotic peptides as potential dental therapeutics.

Small, cysteine-rich, beta-sheet peptide antibiotics are found throughout the Animalia. Though broad spectrum in potential, they may exert selective antimicrobial effects under certain conditions. We have explored the antimicrobial properties of two families of beta-sheet peptide antibiotics, defensins and protegrins, against periodontopathic bacteria. The rabbit defensin NP-1 was active against facultative Gram-negative bacteria associated with early onset periodontitis, including Actinobacillus actinomycetemcomitans and the Capnocytophaga spp. Porcine protegrins showed even greater activity against those organisms, as well as against anaerobic bacteria associated with adult periodontitis, including Porphyromonas gingivalis Prevotella intermedia and Fusobacterium nucleatum. Based on these observations, we believe that protegrin-like beta-sheet peptide antibiotics may be useful dental therapeutics.

Preferential adsorption of human neutrophil myeloperoxidase isoform III by oral bacteria.

Neutrophil myeloperoxidase (MPO) adsorbs to bacteria as a pre-requisite for killing by the MPO/hydrogen-peroxide/chloride system. Three chromatographically distinct isoforms of MPO (MPO I, MPO II, and MPO III) have been isolated from human neutrophils. The purpose of this study was to determine whether oral bacteria--including Actinobacillus actinomycetemcomitans, Capnocytophaga sputigena, Haemophilus aphrophilus, and Eikenella corrodens--differentially adsorb the three major isoforms of MPO from a mixture of MPO I, II, and III, and to assess the effect of pH and normal human serum (NHS) on MPO adsorption. MPO III adsorbed preferentially (i) at high bacterial concentrations, (ii) in the pH range of 6.0-8.0, and (iii) in the presence and absence of NHS. These results support the role of MPO III in the killing of oral bacteria.

Sensitivity of periodontal pathogens to the bactericidal activity of synthetic protegrins, antibiotic peptides derived from porcine leukocytes.

Protegrins, small peptides (1900 to 2160 daltons) isolated from porcine leukocytes, are bactericidal against a broad range of medical pathogens in vitro under conditions which reflect the extracellular milieu. The purpose of this study was to determine whether Gram-negative, facultative periodontal pathogens were sensitive to the protegrins. Synthetic L- and D-enantiomers of protegrin 1 (PG-1 and D-PG-1, respectively), and L-enantiomers of protegrins 2, 3, and 5 (PG-2, PG-3, and PG-5) were tested against Actinobacillus actinomycetemcomitans (three strains) and Capnocytophaga spp. (three strains). Strains of both A. actinomycetemcomitans and Capnocytophaga spp. were sensitive to PG-1, and exhibited ED99 (dose at which 99% killing was observed after 1 hr at 37 degrees C) of 0.5 to 3 microg/mL and 4 to 19 microg/mL, respectively. The D-form and the L-form were equally effective. Serum (above 5% v/v) inhibited the bactericidal effects of 10 microg/mL PG-1, but the inhibitory effect was overcome by concentrations of PG-1 at 100 microg/mL. Different patterns of sensitivity were observed when the effects of PG-1, D-PG-1, PG-2, PG-3, and PG-5 were compared against A. actinomycetemcomitans and the Capnocytophaga. We conclude that protegrins may be useful antimicrobial agents in therapy against periodontal infections.

Differential modulation of adherence of oral streptococci by human neutrophil myeloperoxidase.

In this study, the modulation of adherence of hydrogen peroxide (H2O2)-producing and non-H2O2-producing strains of oral streptococci by the host leukocyte enzyme myeloperoxidase (MPO) was examined. It was found that exposure to MPO decreased adherence of many strains of oral streptococci to saliva-coated hydroxyapatite beads in the presence of exogenous H2O2 and chloride. The MPO-H2O2-Cl-system increased the adherence of one strain. In the absence of exogenous H2O2, the MPO-H2O2-Cl-system decreased the adherence of H2O2-producing strains only. Glucose increased streptococcal H2O2 production and also increased the anti-adhesive activity of MPO in the absence of exogenous H2O2. We conclude that: (1) host leukocytes can modulate the adherence of oral streptococci via MPO; (2) endogenous production of H2O2 by the oral streptococci can provide sufficient substrate H2O2 to drive this system; and (3) MPO will exert differential modulatory effects on the adherence of oral streptococci, based in part upon the level of endogenous H2O2 production and in part upon the particular characteristics of the adhesins of the bacteria.

Induction of activated lymphocyte killing by bacteria associated with periodontal disease.

Complex interactions occur among host defense cells during bacterial infection. Bacteria and bacterial products may enhance or inhibit the effector and regulatory activity of human lymphocytes. Accordingly, we tested the ability of human periodontal pathogens to activate peripheral blood lymphocytes using standard chromium-release assays to measure lymphocyte-mediated cytolysis. Human adherent-cell depleted peripheral blood lymphocytes (PBL) with the addition of glutaraldehyde-fixed bacteria at a 5:1 bacteria:lymphocyte ratio were incubated at 37 degrees C for 24 hr in RPMI 1640 medium. Six of eight bacteria tested significantly augmented lymphocyte killing of the natural killer (NK) cell-sensitive human erythroleukemia cell line K562. E. corrodens, representing activating bacteria, was also able to induce the killing of NK-resistant targets (M14, Raji), comparable with induction by interleukin-2. Lipopolysaccharides extracted from A. actinomycetemcomitans strains, when incubated with PBL, were able to enhance cytotoxicity without the presence of whole bacteria. A majority of cytotoxicity was mediated by NK cells bearing Leu-11 and NKH-1 markers.

Antimicrobial properties of hydrogen peroxide and sodium bicarbonate individually and in combination against selected oral, gram-negative, facultative bacteria.

The topical application of hydrogen peroxide (H2O2) and sodium bicarbonate (NaHCO3), individually and in combination, has been used empirically in the treatment of periodontal diseases. In this study, we examined both minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of these disinfectants individually and in combination against selected facultative, Gram-negative oral bacteria in a microtiter dilution assay. The bacteria studied included Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Eikenella corrodens, and Capnocytophaga gingivalis. These bacteria exhibited MBC (one hr) values ranging from 75 mumol/L to greater than 10 mmol/L and MIC from less than 5 to 500 mumol/L for H2O2. The tested bacteria exhibited MIC values for NaHCO3 of from 23 to 182 mmol/L, and the MBC (one hr) exceeded 728 mmol/L for most of the strains examined. At sublethal (sub-MIC) concentrations, sodium bicarbonate antagonized the ability of H2O2 to inhibit bacterial growth in MIC assays, but sublethal concentrations of H2O2 had no effect on the MIC values of NaHCO3. Lethal concentrations of H2O2 and NaHCO3 exhibited synergistic antimicrobial activity in combination in one-hour bactericidal assays. Since the bactericidal properties of these antimicrobial agents are synergistic, we conclude that it may be rational to use them in combination to treat certain forms of periodontal disease. Also, lower and perhaps safer concentrations of H2O2 can be used in combination with NaHCO3 when oxidative antimicrobial chemotherapy is indicated.

In vitro antimicrobial activity of the human neutrophil cytosolic S-100 protein complex, calprotectin, against Capnocytophaga sputigena.

Calprotectin is a complex of two anionic proteins found in abundance in the cytosol of neutrophils, certain macrophages, and oral epithelial keratinocytes. Bacteria of the genus Capnocytophaga are pathogens of periodontal origin which can cause systemic infection in neutropenic subjects. Recently, it has been observed that Capnocytophaga may be internalized by neutrophils within the cytosol rather than within a membrane-delimited phagosome. The purpose of this study was to test the in vitro antibacterial effect of the cytosolic complex, calprotectin, against Capnocytophaga sputigena. Calprotectin was purified from the cytosol of human neutrophils by gel filtration and anion exchange FPLC, and it exerted potent in vitro antimicrobial effects against C. sputigena. Net bacteriostatic activity was exerted up to 18 h, after which bactericidal effects were observed. Both net bacteriostatic and bactericidal activity occurred at concentrations above 20 micrograms/mL and exhibited identical dose-response characteristics. Particle counts increased in the presence of calprotectin, despite net bacteriostasis as assessed by changes in colony-forming units (CFU). Dose-response characteristics and direct particle counts suggested that net bacteriostatic effects were the result of balanced cell division and death, rather than suspension of cell division. We conclude that calprotectin can be a significant contributor to host defense against infection by Capnocytophaga.

The neutrophil: mechanisms of controlling periodontal bacteria.

The control of potentially periodontopathic microorganisms by host neutrophils is crucial to periodontal health. Neutrophils may use oxidative or nonoxidative mechanisms and either kill bacteria, influence bacterial growth, or modify bacterial colonization in the periodontium. Delivery of antimicrobial substances by neutrophils involves respiratory burst activity, phagocytosis, secretion, or cytolysis/apoptosis. Neutrophils contain a number of antimicrobial components including calprotectin complex, lysozyme, defensins, cofactor-binding proteins, neutral serine proteases, bactericidal/permeability increasing protein, myeloperoxidase, and a NADPH oxidase system. Many of these components are multifunctional and exhibit several mechanisms of antimicrobial activity. When comparisons are made among periodontal bacteria, differences in sensitivity to different components are observed. A hypothesis of specific defense is presented: That specific periodontal diseases can result from the failure of specific aspects of the host immune system (the neutrophil, in particular) in its interaction with specific periodontal pathogens. Failure may be due to phenotypic variation (pleomorphism) within the host or bacterial evasive strategies.

Calprotectin and lactoferrin levels in the gingival crevicular fluid of children.

The purpose of this study was to determine the levels of calprotectin and lactoferrin, 2 microbiostatic proteins, in the gingival crevicular fluid (GCF) of normal children. The children represented a population, primarily underprivileged, seeking care at a regional dental health care center. GCF was collected from Ramfjord teeth (or their deciduous equivalent). GCF volume was quantified by conductance. Calprotectin and lactoferrin levels were quantified by sandwich ELISA, and found to have a mean value of 70.8+/-94.2 microg/mL and 68.2+/-108.7 microg/mL, respectively. The levels of calprotectin and lactoferrin varied directly with one another and inversely with the amount of fluid obtained in a 20-second sampling period. The mean levels were at or above the minimum inhibitory concentration determined in vitro.

Detachment of oral bacteria from saliva-coated hydroxyapatite by human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMN) demonstrated the ability to detach Actinomyces viscosus, A. naeslundii and Streptococcus sanguis from saliva-coated hydroxyapatite beads (SHA). Between 60 to 80% of bacteria were detached within 1 hour at PMN-to-bacteria ratios between 1:10 to 1:22. Detachment was enhanced by treating bacteria with fresh but not heat-inactivated normal human serum. Detachment of serum-treated A. viscosus was inhibited by cytochalasin B, L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK), and deoxyglucose but not colchicine, phenylmethylsulfonyl fluoride (PMSF), N-carbobenzoxy-L-phenylalanine chloromethyl ketone (ZPCK), and sodium azide. In the absence of serum treatment, the detachment of A. viscosus was insensitive to lactose, galactose, and mannose. We conclude that PMN can efficiently detach bacteria from SHA, this detachment is enhanced by serum, and this enhancement is probably dependent upon complement. Additionally, detachment of A. viscosus bound to SHA by PMN (1) does not appear to involve bacterial lectin activity, (2) seems to be dependent upon glycolytic metabolism, microfilament formation, and the activity of a TPCK-sensitive serine protease, and (3) is not sensitive to inhibitors of tubulin polymerization or heme-protein activity.

The distribution of the antimicrobial protein, calprotectin, in normal oral keratinocytes.

Calprotectin is a heterodimeric peptide isolated from neutrophil cytosol that exhibits profound antimicrobial effects. Using monoclonal antibody MAC 387, calprotectin was found to be expressed in oral keratinocytes from normal, non-inflamed oral mucosa. Orthokeratinized sites including the attached gingiva and hard palate expressed low levels of calprotectin with a restricted pattern; immunoreactants were identified only within subcorneal keratinocytes. Parakeratinized mucosa from the lips, soft palate, tongue and buccal mucosa expressed calprotectin in a more widespread, yet variable pattern, immunoreactants being detectable in only a portion of the spinous layer in some cases whereas in others the pattern of expression was more topographically diffuse. Antigen was not detected in basilar and lower strata cells. Both cytoplasmic and nuclear decoration could be identified. The results indicate that oral mucosa harbours an antimicrobial deterrent to micro-organisms that may enhance the physical epithelial barrier of host defence.

Influence of endogenous catalase activity on the sensitivity of the oral bacterium Actinobacillus actinomycetemcomitans and the oral haemophili to the bactericidal properties of hydrogen peroxide.

Actinobacillus actinomycetemcomitans and the genetically-related oral haemophili (Haemophilus segnis, Haemophilus aprhophilus and Haemophilus paraphrophilus) exhibit a range of sensitivities to the lethal effect of hydrogen peroxide (H2O2), A. actinomycetemcomitans being the most resistant. To extend this information, susceptibility to a range of H2O2 concentrations (10(-6)-10(-3) M) was assessed by incubating bacterial suspensions for 1 h at 37 degrees C in the presence of H2O2 and spreading the suspensions on chocolate agar plates to determine the concentration of H2O2 producing a 50 per cent reduction in colony-forming units (LD50). Catalase activity was quantified with a Clark-type oxygen electrode, which polarographically monitored the formation of dissolved oxygen in bacterial suspensions or sonicates following addition of reagent H2O2. Sensitivity to H2O2 did not correlate with catalase activity, either in intact cells or in bacterial sonicates. Specifically, some bacterial strains with undetectable catalase activity were highly resistant to H2O2. Micromolar concentrations of sodium azide which completely inhibited cell-associated catalase activity did not affect the resistance of A. actinomycetemcomitans to H2O2. Thus, the endogenous catalase activity of A. actinomycetemcomitans and certain oral haemophili is not an important determinant of resistance to the bactericidal effects of H2O2.