lactoferrin and hypothiocyanite-ion

The aerobic Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen responsible for life-threatening acute and chronic infections in humans. As part of chronic infection P. aeruginosa forms biofilms, which shield the encased bacteria from host immune clearance and provide an impermeable and protective barrier against currently available antimicrobial agents. P. aeruginosa has an absolute requirement for iron for infection success. By influencing cell-cell communication (quorum sensing) and virulence factor expression, iron is a powerful regulator of P. aeruginosa behaviour. Consequently, the imposed perturbation of iron acquisition systems has been proposed as a novel therapeutic approach to the treatment of P. aeruginosa biofilm infection. In this review, we explore the influence of iron availability on P. aeruginosa infection in the lungs of the people with the autosomal recessive condition cystic fibrosis as an archetypal model of chronic P. aeruginosa biofilm infection. Novel therapeutics aimed at disrupting P. aeruginosa are discussed, with an emphasis placed on identifying the barriers that need to be overcome in order to translate these promising in vitro agents into effective therapies in human pulmonary infections.

Topics: Anti-Infective Agents; Biofilms; Chelating Agents; Cystic Fibrosis; Gene Expression Regulation, Bacterial; Homeostasis; Humans; Iron; Lactoferrin; Lung; Pseudomonas aeruginosa; Pseudomonas Infections; Quorum Sensing; Respiratory Tract Infections; Thiocyanates

Innate human salivary defence proteins, lysozyme, lactoferrin and peroxidase, are known to exert a wide antimicrobial activity against a number of bacterial, viral and fungal pathogens in vitro. Therefore, these proteins, alone or in combinations, have been incorporated as preservatives in foods and pharmaceuticals as well as in oral health care products to restore salivas' own antimicrobial capacity in patients with dry mouth. These antimicrobials used in oral health care products, such as dentifrices, mouth-rinses, moisturizing gels and chewing gums, have been purified from bovine colostrum. In this review I critically evaluate the clinical efficacy and safety of this kind of preventive approach against various oral diseases and symptoms.

Topics: Animals; Anti-Infective Agents, Local; Bacteria; Cattle; Colostrum; Complex Mixtures; Dentifrices; Drug Combinations; Drug Design; Female; Glucose Oxidase; Humans; Lactoferrin; Lactoperoxidase; Mouthwashes; Muramidase; Polymers; Pregnancy; Proteins; Saliva, Artificial; Salivary Proteins and Peptides; Thiocyanates; Xerostomia

This study evaluates the effects of two oral hygiene products containing nonimmunoglobulin antimicrobial agents on whole saliva and on subjective oral symptoms in patients with xerostomia. Twenty patients used a lactoperoxidase-system-containing toothpaste (Biotene) combined with the use of a mouthrinse (Biotene), comprising also lysozyme and lactoferrin, for 4 weeks. Saliva samples were collected at base line, after 4 weeks' use of the products, and at the end of a 4-week washout period. Samples were analyzed for selected biochemical and microbiologic factors. The effects on subjective oral symptoms were also recorded. A 4-week daily use of toothpaste and mouthrinse relieved the symptoms of oral dryness in 16 patients. The levels of salivary hypothiocyanite, lysozyme, lactoferrin, or myeloperoxidase activity did not change, but there was a significant decrease in salivary pH (P < 0.05), total peroxidase activity (P < 0.05), and total protein content (P = 0.01). In patients with the lowest salivary flow rates (n = 5) a significant (P > or = 0.04) increase was detected in salivary hypothiocyanite concentrations. No major changes occurred in salivary microflora. The products relieved subjective oral symptoms in most xerostomic patients, but this was not necessarily related to the presence of antimicrobial agents.

Topics: Adult; Aged; Anti-Infective Agents, Local; Bacteria, Anaerobic; Candida; Colony Count, Microbial; Dentifrices; Female; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Lactoferrin; Lactoperoxidase; Male; Middle Aged; Mouthwashes; Muramidase; Palliative Care; Peroxidases; Saliva; Salivary Proteins and Peptides; Thiocyanates; Xerostomia

To determine the antimicrobial activity of ALX-009, a combination of bovine lactoferrin and hypothiocyanite, in sputum against Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc), key pathogens causing infection in the lungs of cystic fibrosis (CF) patients.. The antimicrobial activity of ALX-009 against clinical respiratory P. aeruginosa isolates was determined by time-kill assay. Sputum from CF patients was treated with ALX-009, either alone or in combination with tobramycin, and the effect on P. aeruginosa, Bcc and total sputum density was determined.. Time-kill assay indicated that ALX-009 was bactericidal at 24 h against 4/4 P. aeruginosa isolates under aerobic conditions, and against 3/4 isolates under anaerobic conditions. ALX-009 was also bactericidal against P. aeruginosa in sputum samples at 6 h (n = 22/24 samples) and 24 h (n = 14/24 samples), and demonstrated significantly greater activity than tobramycin at both timepoints. Activity against Bcc in sputum samples (n = 9) was also demonstrated, but the magnitude of change in Bcc density was less than for P. aeruginosa. To determine the effect of treating sputum with two doses of ALX-009, similar to current regimens for inhaled antibiotics, aliquots of a further 10 sputum samples positive for P. aeruginosa were treated with one (t = 0 h) or two doses (t = 0 h, t = 12 h) of ALX-009; treatment with two doses resulted in bactericidal activity in 7/10 samples at 34 h compared with only 3/10 samples when treatment was with one dose.. ALX-009 demonstrates promise as a novel antimicrobial that could be used to decrease P. aeruginosa density in the lungs of people with CF.

Topics: Anti-Infective Agents; Burkholderia cepacia complex; Cystic Fibrosis; Humans; Lactoferrin; Microbial Sensitivity Tests; Microbial Viability; Pseudomonas aeruginosa; Sputum; Thiocyanates

Chelating iron may be a promising new therapy to eliminate Pseudomonas aeruginosa biofilms in the lungs of cystic fibrosis (CF) patients. Here, we investigate whether ALX-109 [a defined combination of an investigational drug containing lactoferrin (an iron-binding glycoprotein) and hypothiocyanite (a bactericidal agent)], alone and in combination with tobramycin or aztreonam, reduces P. aeruginosa biofilms grown on human CF airway epithelial cells.. P. aeruginosa (PAO1 and six clinical isolates of Pseudomonas) biofilms grown at the apical surface of confluent monolayers of CF airway epithelial cells were treated with ALX-109, either alone or in combination with tobramycin or aztreonam. Bacterial cfu remaining after treatment were determined by plate counting.. ALX-109 alone reduced PAO1 biofilm formation, but had no effect on established biofilms. ALX-109 enhanced the ability of tobramycin and aztreonam to inhibit PAO1 biofilm formation and to reduce established PAO1 biofilms. ALX-109 and tobramycin were additive in disrupting established biofilms formed by six clinical isolates of P. aeruginosa obtained from the sputum of CF patients. Mucoid P. aeruginosa isolates were most susceptible to the combination of ALX-109 and tobramycin. In addition, ALX-109 also enhanced the ability of aztreonam to reduce established PAO1 biofilms.. Inhalation therapy combining hypothiocyanite and lactoferrin with TOBI(®) (tobramycin) or Cayston(®) (aztreonam) may be beneficial to CF patients by decreasing the airway bacterial burden of P. aeruginosa.

Topics: Anti-Bacterial Agents; Aztreonam; Biofilms; Cells, Cultured; Colony Count, Microbial; Drug Combinations; Drug Synergism; Epithelial Cells; Humans; Lactoferrin; Microbial Viability; Pseudomonas aeruginosa; Thiocyanates; Tobramycin

Previous studies of the possible associations of salivary antimicrobial agents with dental caries have given controversial results, obviously mainly because almost all studies have been cross-sectional. Our aim was to find out, in a two-year longitudinal follow-up study, the associations among selected salivary non-immune and immune antimicrobial variables, cariogenic bacteria, and caries increment. The study population was comprised of 63 subjects, all of whom had their 13th birthday during the first study year. In addition to a comprehensive dental examination at baseline and after 2 yrs, paraffin-stimulated whole saliva samples were collected in a standardized way at six-month intervals. Saliva samples were analyzed for flow rate, buffer effect, lysozyme, lactoferrin, total peroxidase activity, hypothiocyanite, thiocyanate, agglutination rate, and total and specific anti-S. mutans IgA and IgG, as well as for numbers of total and mutans streptococci, lactobacilli, and total anaerobic bacteria. Cluster analysis and Spearman-Rank correlation coefficients were used to explore possible associations between and among the studied variables. During the two-year period, a statistically significant increase was observed in flow rate, thiocyanate, agglutination rate, anti-S. mutans IgA antibodies, lactobacilli, and total anaerobes, whereas lysozyme, lactoferrin, and total and anti-S. mutans IgG antibodies declined significantly. Based on various analyses, it can be concluded that, at baseline, total IgG and hypothiocyanite had an inverse relationship with subsequent two-year caries increment, anti-S. mutans IgG antibodies increased with caries development, and mutans streptococci and lactobacilli correlated positively with both baseline caries and caries increment. Total anaerobic microflora was consistently more abundant among caries-free individuals. In spite of the above associations, we conclude that none of the single antimicrobial agents as such has sufficiently strong power to have diagnostic significance in vivo with respect to future caries.

Topics: Adolescent; Agglutination; Anti-Bacterial Agents; Antibodies, Bacterial; Bacteria, Anaerobic; Buffers; Child; Cluster Analysis; Cohort Studies; Colony Count, Microbial; Cross-Sectional Studies; Dental Caries; DMF Index; Female; Finland; Follow-Up Studies; Humans; Immunoglobulin A, Secretory; Immunoglobulin G; Lactobacillus; Lactoferrin; Longitudinal Studies; Male; Muramidase; Peroxidases; Saliva; Secretory Rate; Streptococcus mutans; Thiocyanates

Many studies have attempted to relate levels of antimicrobial proteins in saliva to oral health; results have been inconsistent, and one reason might be inconsistency of measures of plaque and saliva within subjects. This study investigated associations between plaque and salivary variables in longitudinal data. Whole saliva, and 8-h plaque pooled from buccal first permanent molars, was obtained from 32 dental students on Tuesdays from 3:00-6:00 p.m. over 4 weeks. Salivary flow rate was determined, and samples were assayed for lysozyme, lactoferrin, total peroxidase, myeloperoxidase, OSCN-, sIgA and total protein. Colonies on mitis-salivarius agar were assigned to Streptococcus sanguis, Strep. mutans or Strep. salivarius on the basis of morphology, supplemented by the API Rapid Strep identification system. Consistency of values within subjects across weeks was evaluated by repeat-measures analysis of variance and intraclass correlation; data were transformed to reduce skewness. Pearson's r was used to determine associations between plaque and salivary variables. Significant intraclass correlations (alpha = 0.05) were found for all salivary variables except myeloperoxidase, and for total flora, total streptococci, Strep. sanguis and Strep. sanguis as a proportion of total streptococci. Significant Pearson correlations with Strep. sanguis as a proportion of total streptococci were found for total protein (r = -0.24), sIgA (r = -0.22), lactoferrin (r = -0.19) and OSCN- (r = 0.20) when data from all weeks were pooled (n = 128). Strep. sanguis proportions tended to be low in subjects with high values for salivary proteins; the range of proportions was wider in subjects with low salivary values. These findings suggest some consistency of weekly values for many plaque and salivary variables. They also support previous cross-sectional data which suggested that salivary antimicrobial proteins may have some effect on plaque composition. This study was made before recent revisions in streptococcal taxonomy, and further research is needed to clarify interactions of salivary proteins with currently defined species.

Topics: Bacteria; Circadian Rhythm; Colony Count, Microbial; Cross-Sectional Studies; Dental Plaque; Female; Humans; Immunoglobulin A, Secretory; Lactoferrin; Longitudinal Studies; Male; Muramidase; Peroxidase; Peroxidases; Saliva; Salivary Proteins and Peptides; Secretory Rate; Streptococcus; Streptococcus mutans; Streptococcus sanguis; Thiocyanates

Resting and stimulated whole saliva was collected from 94 children aged 12-14 years and analyzed for thiocyanate, hypothiocyanite, 'free' and 'total' lysozyme, lactoferrin and secretory IgA. Clinical assessments of the amounts of plaque and gingival inflammation were made, and plaque was collected for determination of dry weight. An inverse relationship was observed between salivary thiocyanate concentrations in both resting and stimulated saliva and the amounts of plaque and gingival inflammation in these subjects (p < 0.05). Lactoferrin concentration in stimulated saliva was directly related to the amounts of plaque and gingivitis (p < 0.05). 'Total' lysozyme concentration in stimulated saliva was directly related to the amount of plaque (p < 0.05), and the 'free' lysozyme concentration in the same saliva was directly related to the amount of gingivitis (p < 0.05). The direct relationship observed between clinical measurements and both lysozyme and lactoferrin concentrations in saliva may have been due to contributions from gingival crevicular fluid. Cluster analysis identified three groups of subjects with different profiles in resting whole saliva, and in particular with different levels of secretory IgA. A statistically significant difference was observed in the quantity of plaque collected from subjects in two of these groups (p < 0.05). These results from cluster analysis using resting whole saliva from children confirmed the findings of a previous study with young adults.

Topics: Adolescent; Child; Cluster Analysis; Dental Plaque; Gingivitis; Humans; Immunoglobulin A, Secretory; Lactoferrin; Multivariate Analysis; Muramidase; Saliva; Salivary Proteins and Peptides; Secretory Rate; Thiocyanates

We have studied the possible relationship between indigenous salivary antimicrobial agents, indigenous mutans streptococci and the capability of added mutans streptococci to grow in saliva. Stimulated whole saliva was collected from 19 healthy donors. Saliva samples were sterilized, supplemented with glucose and inoculated with Streptococcus mutans or Streptococcus sobrinus. The mixtures were incubated for 20 h followed by counting of viable cells. Saliva samples were analysed, both before and after sterilization, for indigenous antimicrobial agents and the bacterial flora. The subjects could be divided into two groups: those (n = 9) whose saliva promoted and those (n = 10) whose saliva inhibited the growth of the inoculated streptococci. A statistically significant correlation (+0.82, p < 0.001) was found between the numbers of viable cells of S. mutans and S. sobrinus after incubation in saliva. The sterilization procedure reduced the content of all antimicrobial proteins. Salivary antimicrobial factors, or levels of indigenous mutans streptococci, did not differ between the two groups. We conclude that none of the individual salivary antimicrobial factors alone can explain the large individual differences in growth-promoting or growth-inhibiting patterns of saliva on S. mutans and S. sobrinus. Inter-individually, saliva either supports or inhibits the growth of mutans streptococci, indicating a similar response of these two species in relation to the properties of saliva.

Topics: Adult; Anti-Bacterial Agents; Antibodies, Bacterial; Female; Humans; Hydrogen-Ion Concentration; Immunoglobulin A, Secretory; Immunoglobulin G; Lactoferrin; Male; Muramidase; Peroxidase; Peroxidases; Saliva; Sterilization; Streptococcus mutans; Streptococcus sobrinus; Thiocyanates

Samples of resting and stimulated whole saliva and stimulated parotid saliva were collected from 40 young adults. One week later, after 48 h on a standardized diet without oral hygiene, all available plaque was collected for dry weighing. An inverse relationship was found between the 'free' lysozyme concentration in stimulated parotid saliva and plaque dry weight (r = -0.46, p less than 0.01). There were no other statistically significant correlation coefficients between concentrations of individual salivary constituents and plaque dry weight. However, cluster analysis of constituents in resting whole saliva revealed three groups of subjects with different salivary profiles, and in particular with different concentrations of both IgA and hypothiocyanite. Subsequent analysis revealed differences in plaque dry weight between the groups, demonstrating the potential biological significance of cluster membership based on salivary factors.

Topics: Adult; Anti-Infective Agents; Dental Plaque; Female; Humans; Immunoglobulin A, Secretory; Lactoferrin; Male; Muramidase; Parotid Gland; Regression Analysis; Saliva; Salivary Proteins and Peptides; Secretory Rate; Thiocyanates; Time Factors

The possible association between salivary non-immunoglobulin (lysozyme, lactoferrin, hypothiocyanite, agglutinins) or immunoglobulin (total IgA, anti-Streptococcus mutans IgA) antimicrobial factors, and the prevalence of dental caries was studied in 59 young adults. These antimicrobial factors were also analysed in relation to the salivary levels of mutans streptococci (MS). The amount of MS correlated significantly (+0.31, p less than 0.05) with the number of initial caries lesions (Di) but not with other caries indices (DMFT, DMFS, DS). The group with no Di (N = 17) had significantly (p less than 0.05) more hypothiocyanite (HOSCN/OSCN-) and anti-S. mutans IgA antibodies in whole saliva than those with initial caries lesions (N = 42). None of the antimicrobial factors alone showed any significant association with salivary MS counts. Our results suggest that HOSCN/OSCN- and anti-S. mutans IgA may be involved in the prevention of the early phases of dental caries.

Topics: Adult; Agglutinins; Anti-Bacterial Agents; Antibodies, Bacterial; Colony Count, Microbial; Dental Caries; DMF Index; Female; Humans; Immunoglobulin A, Secretory; Lactoferrin; Male; Saliva; Salivary Proteins and Peptides; Streptococcus mutans; Thiocyanates

We have studied the effects of iron-free lactoferrin (apo LF) and lactoperoxidase system (lactoperoxidase, LP/SCN-/H2O2), separately and together, on the viability of Streptococcus mutans (serotype c) in vitro. The bacteria were incubated in buffered KCl (pH 5.5) with and without the above components which were used at concentrations normally present in human saliva. Both apo LF and LP-system had a bactericidal effect against S. mutans at low pH. Together they showed an additive, but not a synergistic, antibacterial effect against S. mutans. Apo LF enhanced the LP enzyme activity but decreased the yield of the antimicrobial component, hypothiocyanite (HOSCN/OSCN-), when incorporated into the reaction mixtures. This decrease, which was most pronounced at low pH, was due to an LP-independent reaction between apo LF and HOSCN/OSCN-. Our study indicates that the LP-system and apo LF can be combined to combat oral S. mutans.

Topics: Anti-Bacterial Agents; Dose-Response Relationship, Drug; Drug Interactions; Lactoferrin; Lactoperoxidase; Streptococcus mutans; Thiocyanates

Antimicrobial agents (antibody and non-antibody) present in human saliva protect oral tissues by a variety of mechanisms, such as prevention of bacterial adhesion, agglutination of micro-organisms, and inhibition of multiplication and metabolism. However, studies in which the concentrations of various salivary antimicrobial agents have been correlated to the presence and severity of oral diseases--of dental caries, in particular--have produced controversial data, and it seems evident, also on the basis of the present study, that no single salivary antimicrobial factor (except flow rate) affects oral health to a significant degree. In the present study, we report the levels of some selected salivary antimicrobial agents in predentate and dentate human infants, with a comparison to the levels found in young adults' saliva. Salivary lysozyme, peroxidase, and hypothiocyanite concentrations were already at the adult level at the time when the primary teeth erupt, whereas immunoglobulin (IgA, IgG, and IgM), lactoferrin, myeloperoxidase, and thiocyanate concentrations were significantly lower in children than in adults. Dentate children had more IgG, thiocyanate, and protein in whole saliva than did predentate children.

Topics: Adult; Aging; Bacterial Physiological Phenomena; Child, Preschool; Humans; Immunoglobulin A, Secretory; Immunoglobulin G; Infant; Isoenzymes; Lactoferrin; Muramidase; Oral Health; Peroxidase; Peroxidases; Saliva; Salivary Proteins and Peptides; Thiocyanates; Tooth Eruption