Release of a wound-healing agent from PLGA microspheres in a thermosensitive gel.

The purpose of this research was to develop a topical microsphere delivery system in a thermosensitive 20% poloxamer 407 gel (Pluronic F127) to control release of KSL-W, a cationic antimicrobial decapeptide, for a period of 4-7 days for potential application in combat related injuries. KSL-W loaded microsphere formulations were prepared by a solvent extraction-evaporation method (water-oil-water), with poly (D,L-lactic-co-glycolic acid) (PLGA) (50 : 50, low-weight, and hydrophilic end) as the polymeric system. After optimization of the process, three formulations (A, B, and C) were prepared with different organic to water ratio of the primary emulsion while maintaining other components and manufacturing parameters constant. Formulations were characterized for surface morphology, porous nature, drug loading, in vitro drug release, and antimicrobial activity. Microspheres containing 20% peptide with porous surfaces and internal structure were prepared in satisfactory yields and in sizes varying from 25 to 50 µm. Gels of 20% Pluronic F127, which were liquid at or below 24.6°C and formed transparent films at body temperature, were used as carriers for the microspheres. Rheological studies showed a gelation temperature of 24.6°C for the 20% Pluronic F127 gel alone. Gelation temperature and viscosity of formulations A, B, and C as a function of temperature were very close to those of the carrier. A Franz diffusion cell system was used to study the release of peptide from the microspheres suspended in both, phosphate-buffered saline (PBS) and a 20% Pluronic F127 gel. In vitro release of greater than 50% peptide was found in all formulations in both PBS and the gel, and in one formulation there was a release of 75% in both PBS and the gel. Fractions collected from the release process were also tested for bactericidal activity against Staphylococcus epidermidis using the broth microdilution method and found to provide effective antimicrobial activity to warrant consideration and testing in animal wound models for treating combat-related injuries.

Pathogenicity of exopolysaccharide-producing Actinomyces oris isolated from an apical abscess lesion.

AIM: To demonstrate a capacity for producing exopolysaccharides (EPSs) and an ability to form biofilm on abiotic materials of Actinomyces oris strain K20. METHODOLOGY: The productivity of EPSs and the ability to form biofilm of strain K20 were evaluated by measuring viscosity of spent culture media and by scanning electron microscopy (SEM) and the biofilm assay on microtitre plates, respectively. High-performance liquid chromatography was used to determine the chemical composition of the viscous materials. To examine the role of the viscous materials attributable to the pathogenicity in this organism, the ability of strain K20 to induce abscess formation was compared in mice to that of ATCC 27044. RESULTS: The viscosity of the spent culture media of K20 was significantly higher than that of ATCC 27044. Strain K20 showed dense meshwork structures around the cells and formed biofilms on microtitre plates, whereas ATCC 27044 did not. Chemical analysis of the viscous materials revealed that they were mainly composed of neutral sugars with mannose constituting 77.5% of the polysaccharides. Strain K20 induced persistent abscesses in mice lasting at least 5 days at a concentration of 10(8) cells mL(-1), whereas abscesses induced by ATCC 27044 healed and disappeared or decreased in size at day 5. CONCLUSIONS: Strain K20 produced EPSs, mainly consisting of mannose, and formed biofilms. This phenotype might play an important role for A. oris to express virulence through the progression of apical periodontitis.

Antimicrobial peptides containing unnatural amino acid exhibit potent bactericidal activity against ESKAPE pathogens.

A series of 36 synthetic antimicrobial peptides containing unnatural amino acids were screened to determine their effectiveness to treat Enterococcus faecium, Staphylococcus aureus, Klebsiella pnemoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE) pathogens, which are known to commonly infect chronic wounds. The primary amino acid sequences of these peptides incorporate either three or six dipeptide units consisting of the unnatural amino acids Tetrahydroisoquinolinecarboxylic acid (Tic) and Octahydroindolecarboxylic acid (Oic). The Tic-Oic dipeptide units are separated by SPACER amino acids with specific physicochemical properties that control how these peptides interact with bacterial cell membranes of different chemical compositions. These peptides exhibited minimum inhibitory concentrations (MIC) against these pathogens in the range from >100 to 6.25 µg/mL. The observed diversity of MIC values for these peptides against the various bacterial strains are consistent with our hypothesis that the complementarity of the physicochemical properties of the peptide and the lipid of the bacteria's cell membrane determines the resulting antibacterial activity of the peptide.

Antimicrobial decapeptide KSL-W attenuates Candida albicans virulence by modulating its effects on Toll-like receptor, human ß-defensin, and cytokine expression by engineered human oral mucosa.

We investigated the toxicity of synthetic antimicrobial decapeptide KSL-W on normal human gingival epithelial cell cultures, its effect on Candida albicans adhesion and growth, and the activation of epithelial cell innate immunity. Our results indicate that KSL-W had no toxic effect on cell adhesion or growth, suggesting its safe use with human cells. Pre-treating C. albicans with KSL-W attenuated the yeast's virulence as demonstrated by its reduced adhesion and growth on engineered human oral mucosa epithelium and the subsequent decreased expression of some innate defense molecules by targeted epithelial cells. Indeed, the expression of Toll-like receptors and human ß-defensins was reduced in tissues infected with KSL-W-treated Candida. Proinflammatory cytokine secretion (IL-1ß and IL-6) by the epithelial cells was also regulated by KSL-W in a manner similar to that of antifungal molecule amphotericin B. These findings therefore show that KSL-W is safe for use with human cells and is able to attenuate Candida virulence by modulating its effects on host innate immunity. This study proposes the potential application of KSL-W peptide as an alternative antifungal agent.

The effect of lactoferrin on oral bacterial attachment.

INTRODUCTION: Lactoferrin (Lf), an iron-binding salivary glycoprotein, plays an important role in human innate defense against local mucosal infection. We hypothesized that Lf interferes with initial oral bacterial attachment to surfaces by iron sequestration, so inhibiting subsequent biofilm formation. The objective was to investigate the effect of Lf on the early stages of single-species and multi-species oral biofilm development. METHODS: Streptococcus gordonii, Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis were used in this study. Glass disks of a two-track flow cell coated with flowing artificial saliva (0.3 ml/min) with and without Lf (100 microg/ml) were used for studying bacterial attachment (3 h, 37 degrees C). Attachment was also examined by incubating single or multiple species of test bacteria (10(7) colony-forming units/ml) with Lf-coated (20-100 microg/ml) and uncoated glass slides. The effects of beta-lactoglobulin, 2,2'-dipyridyl (25-100 microg/ml), an iron chelator, and FeCl3 on attachment were also examined. RESULTS: Lf inhibited the initial attachment of S. gordonii (50.3%, P < 0.05) but not that of F. nucleatum and P. gingivalis. However, the attachment of a dual-species biofilm containing S. gordonii (i.e. S. gordonii/F. nucleatum or S. gordonii/P. gingivalis) was significantly reduced (48.7% or 62.1%, respectively, P < 0.05) in the presence of Lf. beta-Lactoglobulin did not affect the attachment of S. gordonii. In the presence of 100 microm 2,2'-dipyridyl, attachment of S. gordonii was reduced by 53.87%. No reduction in attachment was noted in S. gordonii pretreated with Lf (100 microg/ml) and FeCl3 (20-200 microm). CONCLUSION: Lf suppresses initial attachment of S. gordonii and S. gordonii coaggregates by iron sequestration. This may lead to subsequent inhibition of oral biofilm development.

The anti-endotoxic effects of the KSL-W decapeptide on Escherichia coli O55:B5 and various oral lipopolysaccharides.

BACKGROUND AND OBJECTIVE: Host responses following the recognition of bacterial lipopolysaccharide can range from acute inflammation to septic shock. The aim of this study was to evaluate the ability of the KSL-W decapeptide to bind to and block the endotoxic effects of lipopolysaccharide. MATERIAL AND METHODS: An enzyme-linked immunosorbent assay-based binding assay using fluorescently labeled KSL-W to detect adsorbed Escherichia coli O55:B5 lipopolysaccharide was employed. A commercially available recombinant Factor C lipopolysaccharide detection assay, hemagglutination of rabbit erythrocytes as well as E-selectin expression in human umbilical vein endothelial cells were used to assess the anti-endotoxic effects after KSL-W exposure to E. coli lipopolysaccharide as well as to oral lipopolysaccharide samples. RESULTS: Lipopolysaccharide-binding assays using E. coli O55:B5 lipopolysaccharide revealed both a higher maximal binding range (532-713 microM) and a half-maximum binding concentration (70-185 microM) for the KSL-W peptide when compared with its analog control. Significant inhibition of E-selectin expression in human umbilical vein endothelial cells (p < 0.0001) as well as hemagglutination of rabbit erythrocytes occurred after the interaction of KSL-W with E. coli lipopolysaccharide. Recombinant Factor C enzyme detection inhibition revealed dose-dependent inhibition values ranging from 1.0-51.8 microM. which were dependent upon the type of lipopolysaccharide sample tested. CONCLUSION: These results demonstrate that for the concentrations tested, the KSL-W decapeptide was nontoxic to mammalian cells and could bind to and block the host recognition and response towards enteric, as well as oral, lipopolysaccharide samples.

Targeted profiling of oral bacteria in human saliva and in vitro biofilms with quantitative real-time PCR.

An in vitro plaque model based on the use of human salivary bacteria and tooth-like surfaces was previously developed for studying the formation of oral biofilm and its use for pre-clinical testing of candidate antimicrobial or antiplaque agents. In this study, a quantitative Taqman PCR assay (QPCR) was developed to compare the bacterial compositions of in vitro biofilms to parent saliva samples, and to determine the relative contributions of different species in the formation of the oral biofilm. In addition, the growth inhibition of saliva-derived plaque was evaluated by chlorhexidine. With this assay, which consisted of primer/probe sets targeting either 16S rDNA sequences present in public databases or cloned ribosomal intergenic spacer region (ISR) sequences, 15 oral bacteria derived from saliva as well as those that were responsible for biofilm formation in an in vitro plaque model were rapidly identified and quantified. Among the target organisms were Actinobacillus actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Lactobacillus acidophilus, Micromonas micros, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, Streptococcus sobrinus, Tannerella forsythensis, and Veillonella parvula. Primer and probe sets developed were both sensitive and specific. The relative profiles of a number of bacteria in 45-h-old biofilms were determined and, when compared to saliva samples, it was found that most of the bacteria identified in saliva also populated the in vitro plaque, including some anaerobes. Brief exposure of biofilms to chlorhexidine resulted in significant losses in viability. This new broad spectrum QPCR assay in combination with the in vitro plaque model will be of significant value in the quantitative study of the microbial composition of human saliva, saliva-derived plaque, and pre-clinical evaluation of potential antimicrobial and antiplaque molecules.

Control of oral biofilm formation by an antimicrobial decapeptide.

Oral biofilms are mixed-species microbial communities, and their uncontrolled outgrowth can express as oral diseases. Antimicrobial peptides represent alternative classes of antimicrobials that exhibit selectivity for prokaryotes. We wanted to test the effect of a synthetic decapeptide antimicrobial, KSL, on the development of oral biofilms formed by isolated human salivary bacteria. We used differential interference contrast microscopy, coupled with a dual-flow cell system, to determine the effect of KSL on oral biofilm development. We used reductions of viable counts and confocal microscopy to assess the bactericidal activity of KSL on mature oral biofilms. KSL effectively blocked biofilm development. A significant effect on the viability of mature biofilms was observed when KSL was used in the presence of a surface-active agent, or after biofilms were mechanically disrupted. This study shows that KSL may be a useful adjunct for conventional oral hygiene to prevent plaque-mediated dental diseases.

A novel exopolysaccharide from a clinical isolate of Prevotella nigrescens: purification, chemical characterization and possible role in modifying human leukocyte phagocytosis.

Prevotella nigrescens, a gram-negative black-pigmented anaerobic rod, has frequently been isolated from periodontitis and periapical periodontitis lesions. We have isolated an exopolysaccharide-producing P. nigrescens, strain 22, from a chronic periodontitis lesion. The purpose of this study was to determine the chemical composition and function of the exopolysaccharide associated with this clinical isolate. The chemical composition and structure of the purified exopolysaccharide from strain 22 were determined by high performance liquid chromatography and methylation analysis. To define the biological function of this exopolysaccharide, a chemically induced exopolysaccharide nonproducing mutant, strain 328, which was derived from strain 22, was established. The biological effects of exopolysaccharide were determined by comparing the ability of strain 22, strain 328 or heat-killed strain 22 to form abscesses in mice and to interfere with the phagocytic activity of peripheral blood polymorphonuclear leukocytes. Chemical analysis showed that isolated exopolysaccharide consisted of mannose (521.6 microg/mg), glucose (25.6 microg/mg), fructose (65.8 microg/mg), galactose (12.5 microg/mg), arabinose (6.2 microg/mg), xylose (3.2 microg/mg), rhamnose (6.1 microg/mg), and ribose (0.6 microg/mg). Methylation analysis of exopolysaccharide indicated that the linkages of mannose were primarily (1-->2, 1-->6) (1-->2) (1-->6), and (1-->3). Strain 22 and, to a lesser extent, its heat-killed counterpart induced greater abscess formation in mice than strain 328, even though the enzymatic profile of strain 22 was similar to that of strain 328. The ability of strain 328 to induce abscess formation was restored by adding the purified exopolysaccharide isolated from strain 22 to the cell suspension of strain 328. Exopolysaccharide alone failed to induce abscess formation in mice. Further, strain 328 but not the untreated or heat-killed strain 22, was phagocytosed by polymorphonuclear leukocytes both in the presence and in the absence of opsonic factors. The results suggest that these polysaccharides isolated from strain 22, which primarily consisted of mannose, may play a key role in the development of the chronic inflammatory lesion from which this strain was isolated.

Inhibitory effect of green tea catechins on cysteine proteinases in Porphyromonas gingivalis.

The purpose of this study was to examine the effects of catechins and their derivatives on the activities of Arg-gingipain (Rgp) and Lys-gingipain (Kgp) in Porphyromonas gingivalis. Catechin derivatives, which included (-)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and (-)-catechin gallate, significantly inhibited the Rgp activity. The 50% inhibitory concentrations (IC50s) of these catechin derivatives for Rgp ranged from 3 to 5 microm. While (-)-epigallocatechin and (-)-gallocatechin moderately inhibited Rgp activity (IC50s, 20 microm), (-) -epicatechin, (+)-catechin, and gallic acid were not effective, with IC50s greater than 300 microm. Further, some of the catechin derivatives tested also inhibited the Kgp activity, though to a lesser extent than inhibition of the Rgp activity. These findings suggest that green tea catechins may have the potential to reduce periodontal breakdown resulting from the potent proteinase activity of P. gingivalis.

Susceptibility of oral bacteria to an antimicrobial decapeptide.

Naturally occurring antimicrobial peptides have emerged as alternative classes of antimicrobials. In general, these antimicrobial peptides exhibit selectivity for prokaryotes and minimize the problems of engendering microbial resistance. As an alternative method to search for more effective broad-spectrum peptide antimicrobials, investigators have developed peptide libraries by using synthetic combinatorial technology. A novel decapeptide, KKVVFKVKFK (KSL), has been identified that shows a broad range of antibacterial activity. The purpose of this study was to test the efficacy of this antimicrobial peptide in killing selected strains of oral pathogens and resident saliva bacteria collected from human subjects. Cytotoxic activity of KSL against mammalian cells and the structural features of this decapeptide were also investigated, the latter by using two-dimensional NMR in aqueous and DMSO solutions. MICs of KSL for the majority of oral bacteria tested in vitro ranged from 3 to 100 microg ml(-1). Minimal bactericidal concentrations of KSL were, in general, within one to two dilutions of the MICs. KSL exhibited an ED(99) (the dose at which 99 % killing was observed after 15 min at 37 degrees C) of 6.25 microg ml(-1) against selected strains of Lactobacillus salivarius, Streptococcus mutans, Streptococcus gordonii and Actinobacillus actinomycetemcomitans. In addition, KSL damaged bacterial cell membranes and caused 1.05 log units reduction of viability counts of saliva bacteria. In vitro toxicity studies showed that KSL, at concentrations up to 1 mg ml(-1), did not induce cell death or compromise the membrane integrity of human gingival fibroblasts. NMR studies suggest that KSL adopts an alpha-helical structure in DMSO solution, which mimics the polar aprotic membrane environment, whereas it remains unstructured in aqueous medium. This study shows that KSL may be a useful antimicrobial agent for inhibiting the growth of oral bacteria that are associated with caries development and early plaque formation.

Inhibitory effects of green tea catechins on protein tyrosine phosphatase in Prevotella intermedia.

Members of the Prevotella intermedia group possess protein tyrosine phosphatase (PTPase). The purpose of this study was to investigate the effects of catechin derivatives from Japanese green tea on the activity of PTPase in P. intermedia and related organisms. Multilocus enzyme electrophoresis of alkaline phosphatase derived from P. intermedia, Prevotella nigrescens, Prevotella pallens and Porphyromonas gingivalis revealed a species-specific migration pattern. Among the tea catechin derivatives tested, (-)-epigallocatechin gallate (EGCg), similar to orthovanadate, a specific inhibitor for PTPase, was effective in inhibiting the PTPase activity in P. intermedia at 0.5 microm, and related species at 5 microm. The results suggested that the inhibitory effect observed is due to the presence of galloyl moiety in the structure. In contrast, neither the green tea catechins nor orthovanadate inhibited the phosphatase activity in P. gingivalis, suggesting that this organism possessed a different family of alkaline phosphatase.

Prevotella intermedia native plasmid can be mobilized by an Escherichia coli conjugal IncP plasmid.

We have determined the nucleotide sequence of a small Prevotella intermedia cryptic plasmid, pYHBi1, which consisted of sequences that were highly homologous to the amino acid sequence of the replication and mobilization proteins found in related organisms. We have also demonstrated that chimeric plasmids derived from this P. intermedia native plasmid can be mobilized between Escherichia coli strains by using a broad-host-range E. coli conjugative plasmid, IncP plasmid RP4. The results suggest that pYHBi1 possesses gene(s) responsible for conjugal transfer.

Effects of porphyrins and inorganic iron on the growth of Prevotella intermedia.

We demonstrated earlier that hemin-iron-containing compounds which include hemin, human hemoglobin, bovine hemoglobin, and bovine catalase stimulate the growth of Prevotella intermedia [Leung, Subramaniam, Okamoto, Fukushima, Lai, FEMS Microbiol. Lett. 162 (1998) 227-233]. However, the contributions of tetrapyrrole porphyrin ring in these hemin-iron sources as well as inorganic iron for the growth of this organism have not been determined. The purpose of this study was to examine the effects of porphyrins, host iron-binding proteins, and various inorganic iron sources on the growth of hemin-iron depleted P. intermedia. Protoporphyrin IX and protoporphyrin IX-zinc, either in the presence or absence of supplemented ferrous or ferric iron, promoted the growth of P. intermedia at a rate that was comparable to that of the hemin control. On the other hand, neither the host iron proteins, transferrin and lactoferrin, nor the inorganic iron sources which included ferrous chloride, ferric chloride, ferric citrate, ferric nitrate, and ferric ammonium citrate at concentrations up to 200 microM stimulated the growth of hemin-iron-restricted P. intermedia. The results suggest that P. intermedia only use iron in a specific form and that the porphyrin-ring structure is essential for the growth of P. intermedia as in the case of other related organisms.

Intracerebral haemorrhage and Qigong.

We report on a 65-year-old woman who presented with acute right-sided weakness because of an intracerebral (thalamic) haemorrhage. As a Qigong enthusiast with a long-standing history of hypertension, she developed a stroke syndrome soon after practising Qigong one morning. Following neurological recovery, the patient exhibited erratic blood pressure responses while practising Qigong, despite the fact that resting blood pressure was normal. The haemodynamic responses to exercise are discussed and a review of the therapeutic implications of practising Qigong is presented.