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Highly branched poly(N-isopropylacrylamide) (HB-PNIPAM), functionalized with vancomycin at the chain ends, acted as a bacterial adhesive and was incorporated into polyurethane foams to form semi-in...

Highly branched poly(

Branched poly(N-isopropylacrylamide) was functionalized with Amphotericin B (AmB) at the chain ends to produce an antifungal material. The polymer showed antifungal properties against AmB-sensitive strains of Candida albicans, Fusarium keratoplasticum and Aspergillus flavus (minimal inhibitory concentration ranged from 5 to 500 µg ml−1) but was not effective against an AmB resistant strain of C. albicans nor against C. tropicalis. The polymer end groups bound to the AmB target, ergosterol, and the fluorescence spectrum of a dye used as a solvatochromic probe, Nile red, was blue shifted indicating that segments of the polymer became desolvated on binding. The polymer was less toxic to corneal and renal epithelial cells and explanted corneal tissue than the free drug. Also, the polymer did not induce reactive oxygen species release from peripheral blood mononuclear cells, nor did it cause a substantial release of the proinflammatory cytokines, tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), (at 0.5 mg ml−1).

Branched poly(N-isopropylacrylamide) was functionalized with Amphotericin B (AmB) at the chain ends to produce an antifungal material. The polymer showed antifungal properties against AmB-sensitive strains of Candida albicans, Fusarium keratoplasticum and Aspergillus flavus (minimal inhibitory concentration ranged from 5 to 500 µg ml−1) but was not effective against an AmB resistant strain of C. albicans nor against Candida tropicalis. The polymer end groups bound to the AmB target, ergosterol, and the fluorescence spectrum of a dye used as a solvatochromic probe, Nile red, was blue shifted indicating that segments of the polymer became desolvated on binding. The polymer was less toxic to corneal and renal epithelial cells and explanted corneal tissue than the free drug. Also, the polymer did not induce reactive oxygen species release from peripheral blood mononuclear cells, nor did it cause a substantial release of the proinflammatory cytokines, tumour necrosis factor-α and interleukin-1β (at 0.5 mg ml−1).

Microbial keratitis can arise from penetrating injuries to the cornea. Corneal trauma promotes bacterial attachment and biofilm growth, which decrease the effectiveness of antimicrobials against microbial keratitis. Improved therapeutic efficacy can be achieved by reducing microbial burden prior to antimicrobial therapy. This paper assesses a highly-branched poly(N-isopropyl acrylamide) with vancomycin end groups (HB-PNIPAM-van), for reducing bacterial attachment and biofilm formation. The polymer lacked antimicrobial activity against Staphylococcus aureus, but significantly inhibited biofilm formation (p = 0.0008) on plastic. Furthermore, pre-incubation of S. aureus cells with HB-PNIPAM-van reduced cell attachment by 50% and application of HB-PNIPAM-van to infected ex vivo rabbit corneas caused a 1-log reduction in bacterial recovery, compared to controls (p = 0.002). In conclusion, HB-PNIPAM-van may be a useful adjunct to antimicrobial therapy in the treatment of corneal infections.

Vascular endothelial growth factor (VEGF) stimulates endothelial cells to migrate, proliferate and form new blood vessels. However direct delivery of VEGF has not become clinically adopted as a means of stimulating blood vessel formation and wound healing because of its relatively poor stability and its production of immature blood vessels. A simpler way of stimulating production of VEGF in situ is explored in this study following reports of deoxy sugars involved in inducing VEGF production. The pro-angiogenic effect of L and D isomers of deoxy sugars (ribose, fucose and rhamnose) loaded into biodegradable chitosan/collagen hydrogels was examined using a chick chorionic allantoic membrane assay. The L-sugars were all pro-angiogenic but only the 2-deoxy-D-ribose had strong effects on angiogenesis. Furthermore, these sugars could not be metabolised by four strains of Staphylococcus aureus, as a metabolic substrate for growth, although some of these could be metabolised by another typical pathogen, Pseudomonas aeruginosa. The effects of 2-deoxy-D-ribose in a chitosan/collagen hydrogel on wound healing were also assessed. This biomaterial doubled the rate of cutaneous wound healing in rats associated with an increase in vascularisation detected by staining for CD34 positive cells.

The behavior of a linear copolymer of N-isopropylacrylamide with pendant vancomycin functionality was compared to an analogous highly branched copolymer with vancomycin functionality at the chain ends. Highly branched poly(N-isopropylacrylamide) modified with vancomycin (HB-PNIPAM-van) was synthesized by functionalization of the HB-PNIPAM, prepared using reversible addition-fragmentation chain transfer polymerization. Linear PNIPAM with pendant vancomycin functionality (L-PNIPAM-van) was synthesized by functionalization of poly(N-isopropylacrylamide-co-vinyl benzoic acid). HB-PNIPAM-van aggregated S. aureus effectively, whereas the L-PNIPAM-van polymer did not. It was found that when the HB-PNIPAM-van was incubated with S. aureus the resultant phase transition provided an increase in the intensity of fluorescence of a solvatochromic dye, nile red, added to the system. In contrast, a significantly lower increase in fluorescence intensity was obtained when L-PNIPAM-van was incubated with S. aureus. These data showed that the degree of desolvation of HB-PNIPAM-van was much greater than the desolvation of the linear version. Using microcalorimetry, it was shown that there were no significant differences in the affinities of the polymer ligands for d-Ala-d-Ala and therefore differences in the interactions with bacteria were associated with changes in the probability of access of the polymer bound ligands to the d-Ala-d-Ala dipeptide. The data support the hypothesis that generation of polymer systems that respond to cellular targets, for applications such as cell targeting, detection of pathogens etc., requires the use of branched polymers with ligands situated at the chain ends.

Porphyromonas gingivalis is a Gram-negative, keystone pathogen in periodontitis that leads to tissue destruction and ultimately tooth loss. The organism is able to infect oral epithelial cells and two-dimensional (monolayer) cultures have been used to investigate this process. However, recently there has been interest in the use of three-dimensional, organotypic mucosal models to analyse infection. These models are composed of collagen-embedded fibroblasts overlain with multilayers of oral epithelial cells. In this study we report for the first time significant differences in the response of oral mucosal models to P. gingivalis infection when compared to monolayer cultures of oral epithelial cells. Intracellular survival (3-fold) and bacterial release (4-fold) of P. gingivalis was significantly increased in mucosal models compared with monolayer cultures, which may be due to the multi-layered nature and exfoliation of epithelial cells in these organotypic models. Furthermore, marked differences in the cytokine profile between infected organotypic models and monolayer cultures were observed, particularly for CXCL8 and IL6, which suggested that degradation of cytokines by P. gingivalis may be less pronounced in organotypic compared to monolayer cultures. These data suggest that use of oral mucosal models may provide a greater understanding of the host responses to P. gingivalis invasion than simple monolayer cultures.

In view of the growing worldwide rise in microbial resistance, there is considerable interest in designing new antimicrobial copolymers. The aim of the current study was to investigate the relationship between antimicrobial activity and copolymer composition/architecture to gain a better understanding of their mechanism of action. Specifically, the antibacterial activity of several copolymers based on 2-(methacryloyloxy)ethyl phosphorylcholine [MPC] and 2-hydroxypropyl methacrylate (HPMA) toward Staphylococcus aureus was examined. Both block and graft copolymers were synthesized using either atom transfer radical polymerization or reversible addition-fragmentation chain transfer polymerization and characterized via (1)H NMR, gel permeation chromatography, rheology, and surface tensiometry. Antimicrobial activity was assessed using a range of well-known assays, including direct contact, live/dead staining, and the release of lactate dehydrogenase (LDH), while transmission electron microscopy was used to study the morphology of the bacteria before and after the addition of various copolymers. As expected, PMPC homopolymer was biocompatible but possessed no discernible antimicrobial activity. PMPC-based graft copolymers comprising PHPMA side chains (i.e. PMPC-g-PHPMA) significantly reduced both bacterial growth and viability. In contrast, a PMPC-PHPMA diblock copolymer comprising a PMPC stabilizer block and a hydrophobic core-forming PHPMA block did not exhibit any antimicrobial activity, although it did form a biocompatible worm gel. Surface tensiometry studies and LDH release assays suggest that the PMPC-g-PHPMA graft copolymer exhibits surfactant-like activity. Thus, the observed antimicrobial activity is likely to be the result of the weakly hydrophobic PHPMA chains penetrating (and hence rupturing) the bacterial membrane.

Outer membrane protein A (OmpA) is a key outer membrane protein found in Gram‐negative bacteria that contributes to several crucial processes in bacterial virulence. In Porphyromonas gingivalis, OmpA is predicted as a heterotrimer of OmpA1 and OmpA2 subunits encoded by adjacent genes. Here we describe the role of OmpA and its individual subunits in the interaction of P. gingivalis with oral cells. Using knockout mutagenesis, we show that OmpA2 plays a significant role in biofilm formation and interaction with human epithelial cells. We used protein structure prediction software to identify extracellular loops of OmpA2, and determined these are involved in interactions with epithelial cells as evidenced by inhibition of adherence and invasion of P. gingivalis by synthetic extracellular loop peptides and the ability of the peptides to mediate interaction of latex beads with human cells. In particular, we observe that OmpA2‐loop 4 plays an important role in the interaction with host cells. These data demonstrate for the first time the important role of P. gingivalis OmpA2 extracellular loops in interaction with epithelial cells, which may help design novel peptide‐based antimicrobial therapies for periodontal disease.

Porphyromonas gingivalis , a key periodontal pathogen, is capable of invading a variety of cells, including oral keratinocytes, by exploiting host cell receptors, including alpha-5 beta-1 (α5β1) integrin. Previous studies have shown that P. gingivalis accelerates the cell cycle and prevents apoptosis of host cells, but it is not known whether the cell cycle phases influence bacterium-cell interactions. The cell cycle distribution of oral keratinocytes was characterized by flow cytometry and BrdU (5-bromo-2-deoxyuridine) staining following synchronization of cultures by serum starvation. The effect of cell cycle phases on P. gingivalis invasion was measured by using antibiotic protection assays and flow cytometry, and these results were correlated with gene and surface expression levels of α5 integrin and urokinase plasminogen activator receptor (uPAR). There was a positive correlation ( R = 0.98) between the number of cells in S phase and P. gingivalis invasion, the organism was more highly associated with cells in S phase than with cells in G 2 and G 1 phases, and S-phase cells contained 10 times more bacteria than did cells that were not in S phase. Our findings also show that α5 integrin, but not uPAR, was positively correlated with cells in S phase, which is consistent with previous reports indicating that P. gingivalis invasion of cells is mediated by α5 integrin. This study shows for the first time that P. gingivalis preferentially associates with and invades cells in the S phase of the cell cycle. The mechanism of targeting stable dividing cells may have implications for the treatment of periodontal diseases and may partly explain the persistence of this organism at subgingival sites.

Tannerella forsythia is an important pathogen in periodontal disease. Previously, we showed that its sialidase activity is key to utilization of sialic acid from a range of human glycoproteins for biofilm growth and initial adhesion. Removal of terminal sialic acid residues often exposes β-linked glucosamine or galactosamine, which may also be important adhesive molecules. In turn, these residues are often removed by a group of enzymes known as β-hexosaminidases. We show here that T. forsythia has the ability to cleave glucosamine and galactosamine from model substrates and that this activity can be inhibited by the hexosaminidase inhibitor PugNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate). We now demonstrate for the first time that β-hexosaminidase activity plays a role in biofilm growth on glycoprotein-coated surfaces because biofilm growth and initial cell adhesion are inhibited by PugNAc. In contrast, adhesion to siallo-glycoprotein-coated surfaces is unaltered by PugNAc in the absence of sialidase activity (using a sialidase-deficient mutant) or surprisingly on the clinically relevant substrates saliva or serum. These data indicate that β-hexosaminidase activity has a significant role in biofilm formation in combination with sialidase activity in the biofilm lifestyle of T. forsythia.

Biocompatible hydrogels have many applications, ranging from contact lenses to tissue engineering scaffolds. In most cases, rigorous sterilization is essential. Herein we show that a biocompatible diblock copolymer forms wormlike micelles via polymerization-induced self-assembly in aqueous solution. At a copolymer concentration of 10.0 w/w %, interworm entanglements lead to the formation of a free-standing physical hydrogel at 21 °C. Gel dissolution occurs on cooling to 4 °C due to an unusual worm-to-sphere order-order transition, as confirmed by rheology, electron microscopy, variable temperature (1)H NMR spectroscopy, and scattering studies. Moreover, this thermo-reversible behavior allows the facile preparation of sterile gels, since ultrafiltration of the diblock copolymer nanoparticles in their low-viscosity spherical form at 4 °C efficiently removes micrometer-sized bacteria; regelation occurs at 21 °C as the copolymer chains regain their wormlike morphology. Biocompatibility tests indicate good cell viabilities for these worm gels, which suggest potential biomedical applications.

The major bacterial pathogens associated with periodontitis includeTannerella forsythia. We previously discovered that sialic acid stimulates biofilm growth ofT. forsythia, and that sialidase activity is key to utilization of sialoconjugate sugars and is involved in host–pathogen interactionsin vitro. The aim of this work was to assess the influence of the NanH sialidase on initial biofilm adhesion and growth in experiments where the only source of sialic acid was sialoglycoproteins or human oral secretions. After showing thatT. forsythiacan utilize sialoglycoproteins for biofilm growth, we showed that growth and initial adhesion with sialylated mucin and fetuin were inhibited two- to threefold by the sialidase inhibitor oseltamivir. A similar reduction (three- to fourfold) was observed with ananHmutant compared with the wild-type. Importantly, these data were replicated using clinically relevant serum and saliva samples as substrates. In addition, the ability of thenanHmutant to form biofilms on glycoprotein-coated surfaces could be restored by the addition of purified NanH, which we show is able to cleave sialic acid from the model glycoprotein fetuin and, much less efficiently, 9-O-acetylated bovine submaxillary mucin. These data show for the first time that glycoprotein-associated sialic acid is likely to be a keyin vivonutrient source forT. forsythiawhen growing in a biofilm, and suggest that sialidase inhibitors might be useful adjuncts in periodontal therapy.

Wound infection is a common complication often resulting in delayed healing with adverse clinical and financial consequences. Current antimicrobial treatments are far from ideal, side effects can include both bacterial resistance and toxicity. As a result, a great deal of effort over the last 20 years has been spent on investigating new forms of antimicrobial dressings. Here, we report the unexpected antimicrobial activity of a relatively new biocompatible thermo-responsive PHPMA–PMPC–PHPMA triblock copolymer gelator [where PHPMA denotes poly(2-hydroxypropyl methacrylate) and PMPC denotes poly(2-(methacryloyloxy)ethyl phosphorylcholine)]. In a radial diffusion assay, a 20% w/v copolymer gel produced an inhibitory zone up to six times greater than the corresponding control against Staphylococcus aureus. Similarly, in a broth inhibition assay the same copolymer reduced bacterial growth by 45% compared with control experiments conducted in the absence of any copolymer. Moreover, addition of the copolymer to a 3D-infected skin model reduced bacterial recovery by 38% compared to that of controls over 24–48 h. This is particularly relevant since these antimicrobial triblock copolymers were recently shown to be non-toxic when exposed to a tissue-engineered skin model. This antimicrobial activity was also successfully immobilised by grafting PMPC–PHPMA diblock copolymer brushes onto silicon wafers. Our results indicate that both PMPC–PHPMA diblock and PHPMA homopolymer brushes exhibit antimicrobial activity. Our hypothesis for the mode of action is that the moderately hydrophobic PHPMA chains penetrate the bacterial membrane, causing leakage of the cell contents. In summary, these gels and surfaces offer a promising new approach to antimicrobial dressings.

As our knowledge of host-microbial interactions within the oral cavity increases, future treatments are likely to be more targeted. For example, efforts to target a single species or key virulence factors that they produce, while maintaining the natural balance of the resident oral microbiota that acts to modulate the host immune response would be an advantage. Targeted approaches may be directed at the black-pigmented anaerobes, Porphyromonas gingivalis and Prevotella intermedia, associated with periodontitis. Such pigments provide an opportunity for targeted phototherapy with high-intensity monochromatic light. Functional inhibition approaches, including the use of enzyme inhibitors, are also being explored to control periodontitis. More general disruption of dental plaque through the use of enzymes and detergents, alone and in combination, shows much promise. The use of probiotics and prebiotics to improve gastrointestinal health has now led to an interest in using these approaches to control oral disease. More recently the potential of antimicrobial peptides and nanotechnology, through the application of nanoparticles with biocidal, anti-adhesive and delivery capabilities, has been explored. The aim of this review is to consider the current status as regards non-conventional treatment approaches for oral infections with particular emphasis on the plaque-related diseases.

Bacterial colonisation and biofilm formation on the surface of urinary catheters is a common cause of nosocomial infection, and as such is a major impediment to their long-term use. Understanding the mechanisms of biofilm formation on urinary catheters is critical to their control and will aid the future development of materials used in their manufacture. In this report we have used proteomic analysis coupled with immunoassays to show that the major outer membrane protein (OmpA) of Escherichia coli is overexpressed during biofilm formation. A series of synthetic hydrogels being developed for potential use as catheter coatings were used as the substrata and OmpA expression was increased in biofilms on all these surfaces, as well as being a feature of both a laboratory and a clinical strain of E. coli. Up-regulation of OmpA may, therefore, be a common feature of E. coli biofilms. These findings present OmpA as a potential target for biofilm inhibition and may contribute to the rational design of biofilm inhibiting hydrogel coatings for urinary catheters.

Background/aims: This study aimed to investigate the concentration of the cytokine interleukin (IL)-1β and its receptor antagonist IL-1ra in gingival crevicular fluid (GCF) in patients with adult periodontitis who were heavy smokers compared with non-smokers. Method: GCF samples were collected from two groups of subjects: smokers and non-smokers. Thirty-nine GCF samples were harvested from 13 subjects with moderate to severe adult periodontitis who were heavy smokers. A further 30 GCF samples were harvested from 10 subjects with moderate to severe adult periodontitis who were non-smokers. Subjects were selected from both genders and none had any relevant systemic illness, were pregnant, had recent medication or had received any periodontal therapy in the preceding 3 months. One deep bleeding site, one deep non-bleeding site and one healthy site were investigated in each subject. Clinical measurements were recorded for each site, after obtaining a GCF sample using a Periopaper® strip. IL-1β and IL-1ra were quantified using new commercially available ELISA kits (Quantikine™), and could be detected in all samples. Results: For smokers, the mean concentrations for IL-1β were 2714.5 (SD 4416.2) pg/µL for healthy sites, 37.0 (SD 57.2) pg/µL for non-bleeding periodontitis sites and 24.5 (SD 29.2) pg/µL for bleeding periodontitis sites. The concentrations of IL-1β for non-smokers for the same category of sites were 393.8 (SD 867.1), 74.2 (SD 107.0) and 73.1 (SD 61.0) pg/µL, respectively. The mean concentrations of IL-1ra for smokers were 5.8 × 105 (SD 9.7) pg/µL for healthy sites, 2.2 × 105 (SD 0.15) pg/µL for deep non-bleeding sites and 0.19 × 105 (SD 0.07) pg/µL for deep bleeding sites. The concentrations for non-smokers were: 4.1 × 1010 (SD 3.8), 18.1 × 105 (SD 20.4) and 3.2 × 105 (SD 2.3) pg/µL, respectively. Significance levels of P

Oral colonising bacteria are highly adapted to the various environmental niches harboured within the mouth, whether that means while contributing to one of the major oral diseases of caries, pulp infections, or gingival/periodontal disease or as part of a commensal lifestyle. Key to these infections is the ability to adhere to surfaces via a range of specialised adhesins targeted at both salivary and epithelial proteins, their glycans and to form biofilm. They must also resist the various physical stressors they are subjected to, including pH and oxidative stress. Possibly most strikingly, they have developed the ability to harvest both nutrient sources provided by the diet and those derived from the host, such as protein and surface glycans. We have attempted to review recent developments that have revealed much about the molecular mechanisms at work in shaping the physiology of oral bacteria and how we might use this information to design and implement new treatment strategies.

The gram-negative anaerobe Porphyromonas gingivalis colonizes the gingival crevice and is etiologically associated with periodontal disease that can lead to alveolar bone damage and resorption, promoting tooth loss. Although susceptible to antibiotics, P. gingivalis can evade antibiotic killing by residing within gingival keratinocytes. This provides a reservoir of organisms that may recolonize the gingival crevice once antibiotic therapy is complete. Polymersomes are nanosized amphiphilic block copolymer vesicles that can encapsulate drugs. Cells internalize polymersomes by endocytosis into early endosomes, where they are disassembled by the low pH, causing intracellular release of their drug load. In this study, polymersomes were used as vehicles to deliver antibiotics in an attempt to kill intracellular P. gingivalis within monolayers of keratinocytes and organotypic oral mucosal models. Polymersome-encapsulated metronidazole or doxycycline, free metronidazole, or doxycycline, or polymersomes alone as controls, were used, and the number of surviving intracellular P. gingivalis was quantified after host cell lysis. Polymersome-encapsulated metronidazole or doxycycline significantly (P

Tannerella forsythia is a key contributor to periodontitis, but little is known of its virulence mechanisms. In this study we have investigated the role of sialic acid in biofilm growth of this periodontal pathogen. Our data show that biofilm growth of T. forsythia is stimulated by sialic acid, glycolyl sialic acid, and sialyllactose, all three of which are common sugar moieties on a range of important host glycoproteins. We have also established that growth on sialyllactose is dependent on the sialidase of T. forsythia since the sialidase inhibitor oseltamivir suppresses growth on sialyllactose. The genome of T. forsythia contains a sialic acid utilization locus, which also encodes a putative inner membrane sialic acid permease (NanT), and we have shown this is functional when it is expressed in Escherichia coli . This genomic locus also contains a putatively novel TonB-dependent outer membrane sialic acid transport system (TF0033-TF0034). In complementation studies using an Escherichia coli strain devoid of its outer membrane sialic acid transporters, the cloning and expression of the TF0033-TF0034 genes enabled an E. coli nanR nanC ompR strain to utilize sialic acid as the sole carbon and energy source. We have thus identified a novel sialic acid uptake system that couples an inner membrane permease with a TonB-dependent outer membrane transporter, and we propose to rename these novel sialic acid uptake genes nanO and nanU , respectively. Taken together, these data indicate that sialic acid is a key growth factor for this little-characterized oral pathogen and may be key to its physiology in vivo .

We recently described the Palate Lung Nasal Clone (PLUNC) family of proteins as an extended group of proteins expressed in the upper airways, nose and mouth. Little is known about these proteins, but they are secreted into the airway and nasal lining fluids and saliva where, due to their structural similarity with lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein, they may play a role in the innate immune defence. We now describe the generation and characterisation of novel affinity-purified antibodies to SPLUNC2, and use them to determine the expression of this, the major salivary gland PLUNC. Western blotting showed that the antibodies identified a number of distinct protein bands in saliva, whilst immunohistochemical analysis demonstrated protein expression in serous cells of the major salivary glands and in the ductal lumens as well as in cells of minor mucosal glands. Antibodies directed against distinct epitopes of the protein yielded different staining patterns in both minor and major salivary glands. Using RT-PCR of tissues from the oral cavity, coupled with EST analysis, we showed that the gene undergoes alternative splicing using two 5′ non-coding exons, suggesting that the gene is regulated by alternative promoters. Comprehensive RACE analysis using salivary gland RNA as template failed to identify any additional exons. Analysis of saliva showed that SPLUNC2 is subject to N-glycosylation. Thus, our study shows that multiple SPLUNC2 isoforms are found in the oral cavity and suggest that these proteins may be differentially regulated in distinct tissues where they may function in the innate immune response.

The melanocortin peptides α-MSH, Lys-Pro-Val and Lys-Pro- d -Val are known to be potent anti-inflammatory agents; however their role as antibacterial peptides is less clear. The aim of this study was to determine whether these peptides displayed antibacterial properties, and specifically whether the Lys-Pro- d -Val tripeptide was more potent than Lys-Pro-Val, consistent with their anti-inflammatory actions. α-MSH, Ac-Lys-Pro- d -Val-NH 2 and Ac-Lys-Pro-Val-NH 2 were found to be antibacterial against both Gram-positive and Gram-negative bacteria ( Staphylococcus aureus and Escherichia coli ) over a broad range of concentrations compared to a control peptide, Ac-Ala-Ala-Ala-NH 2 . However, the relative potency of α-MSH, Ac-Lys-Pro- d -Val-NH 2 , Ac-Lys-Pro-Val-NH 2 did not differ. Furthermore, it was found that the cationic charge on the lysine residue was not required for activity as a variant peptide Ac-Ala-Pro- d -Val-NH 2 was also antibacterial. We therefore describe a novel X-Pro- d / l -Val peptide sequence with similarity to the short melanocortin peptides, which possess antibacterial activity. The combined anti-inflammatory and antibacterial action of such peptides may also have potential value therapeutically.

Summary Streptococcus sanguis is the most common oral bacterium causing infective endocarditis and its ability to adhere to platelets, leading to their activation and aggregation, is thought to be an important virulent factor. Previous work has shown that S. sanguis can bind directly to platelet glycoprotein (GP) Ib but the nature of the adhesin was unknown. Here, we have shown that a high molecular weight glycoprotein of S. sanguis mediates adhesion to glycocalacin. The bacterial glycoprotein was purified from cell extracts by chromatography on GPIb- and wheatgerm agglutinin affinity matrices and its interaction with GPIb was shown to be sialic acid-dependent. We designated the glycoprotein serine-rich protein A (SrpA). An insertional inactivation mutant lacking the SrpA of S. sanguis showed significantly reduced binding to glycocalacin, reduced adherence to platelets and a prolonged lag time to platelet aggregation. In addition, under flow conditions, platelets rolled and subsequently adhered on films of wild-type S. sanguis cells at low shear (50/s) but did not bind to films of the SrpA mutant. Platelets did not bind to wild-type bacterial cells at high shear (1500/s). These findings help to understand the mechanisms by which the organism might colonize platelet-fibrin vegetations.

Functionalised thin films of copolymers of acrylic acid and 1,7-octadiene have been deposited by plasma-co-polymerisation to create a range of surfaces of well-defined chemistry, containing carboxyl functional groups. X-Ray photoelectron spectroscopy (XPS) has been used to characterise the deposits. Adsorption of immunoglobulin G from single solution has been studied by indirect, antibody detection (ELISA), and by direct, radio-labelling and fluorescent detection methods. In addition, XPS analysis of the surface adsorbed protein was also performed. Direct measurement techniques showed an apparent decrease in protein binding on carboxylic acid functionalised surfaces, whilst antibody detection showed an increase in binding with increased carboxylic acid content of the surface. This difference has been ascribed to orientational/conformational and/or packing differences of the proteins on the different surfaces, leading to an increased biological (“functional”) activity on the carboxylic acid functionalised surfaces. Adsorption of IgG from serum has also been probed by ELISA. An increase in IgG binding with surface carboxylic acid content was measured, indicating a similar effect when IgG is adsorbed from a complex protein mixture.

Polymeric functional thin films have been deposited from plasmas of allyl alcohol, acrylic acid, allylamine and octa-1,7-diene onto polystyrene microwells and aluminium foil, and analysed by X-ray photoelectron spectroscopy (XPS) and water contact angle measurement. The films were found to be conformal and pin-hole free. Advancing and receding water contact angles measured in air showed that the surfaces had a range of hydrophilicities. The adsorption of human vitronectin, human immunoglobulin G (IgG) and heat-denatured bovine type II collagen to the different plasma polymer surfaces from single solutions was compared by enzyme linked immunosorbent assay (ELISA). Results demonstrate that the adsorption of proteins depends not only on the chemistry of the surface, but also on the nature of the protein. Vitronectin adsorbed most extensively to the acrylic acid-deposited surface, while immunoglobulin G adsorbed more readily to the allylamine deposited surface. The functionalised surfaces performed poorly in terms of collagen binding, with much higher levels of adsorption to the hydrocarbon (octa-1,7-diene) plasma polymer, and the uncoated polystyrene control wells. The amount of adsorbed protein detected on a surface is often explained in terms of surface hydrophilicity/hydrophobicity. The results of this study show that adsorption of these proteins is not simply a matter of wettability, but relates more to the chemical functionality of the surface, which in turn affects a number of surface properties, including wettability, surface charge and pKa.

We describe a series of experiments designed to investigate the conformational transition that highly-branched polymers with ligands undergo when interacting with bacteria, a process that may provide a new sensing mechanism for bacterial detection. Fluorescent highly-branched poly(N-isopropyl acrylamide)s (HB-PNIPAM) were prepared by sequential self-condensing radical copolymerizations, using anthrylmethyl methacrylate (AMMA) and fluorescein-O-acrylate (FA) as fluorescent comonomers and 4-vinylbenzyl pyrrole carbodithioate as a branch forming monomer. Differences in reactivity necessitated to first copolymerize AMMA then react with FA in a separate sequential monomer feed step. Modifications of the chain ends produced vancomycin-functional derivatives (HB-PNIPAM-Van). The AMMA and FA labels allow probing of the conformational behaviour of the polymers in solution via Förster resonance energy transfer experiments. It was shown that interaction of this polymer's end groups with Staphylococcus aureus induced a macromolecular collapse. The data thus provide conclusive evidence for a conformational transition that is driven by binding to a bacterium.

Aims The aims of this study were to determine the pattern of use and re-use of matrix bands in general practice in Scotland, to demonstrate which type of matrix band is most commonly used and to examine infection control measures of relevance to the safe use and re-use of matrix bands. Materials and methods Subjects: 621 of Scotland's 1,849 general dental practitioners were randomly selected. Data collection: A 19-item self-reported questionnaire was mailed in June 1999 with a follow-up mailing sent in August 1999. Analysis: Data analysis involved descriptive statistics and cross-tabulation. Where appropriate, differences between categories were tested for significance by a Chi-square test. Results A total of 479 questionnaires were returned, representing a response rate of 77%. Reported compliance with routine glove wearing was high (91%). Most dentists (92%) provided training on instrument cleaning and sterilisation for their dental nurses. Ultrasonic baths were used by 59% of practitioners; the remainder soaked or manually scrubbed instruments to remove debris before autoclaving. The Siqveland matrix was the matrix of choice for 96% of respondents. 7% provided a new matrix band for each patient. Most (64%) changed bands only when they were bent or damaged; 29% changed them daily or weekly. Deterrents to use of a new band for each patient were cost (39%) and time (52%). A total of 54% of respondents considered matrix band replacement unnecessary between patients. Conclusions The Siqveland matrix band is the most popular among the study group of dental practitioners. Re-use of matrix bands is common. Guidelines for the safe re-use of matrix bands are required.

Aims To use a sensitive forensic test to measure blood contamination of used Siqveland matrix bands following routine cleaning and sterilisation procedures in general dental practice. Materials and methods Sixteen general dental practices in the West of Scotland participated. Details of instrument cleaning procedures were recorded for each practice. A total of 133 Siqveland matrix bands were recovered following cleaning and sterilisation and were examined for residual blood contamination by the Kastle-Meyer test, a well-recognised forensic technique. Results Ultrasonic baths were used for the cleaning of 62 (47%) bands and retainers and the remainder (53%) were hand scrubbed prior to autoclaving. Overall, 21% of the matrix bands and 19% of the retainers gave a positive Kastle-Meyer test, indicative of residual blood contamination, following cleaning and sterilisation. In relation to cleaning method, 34% of hand-scrubbed bands and 32% of hand-scrubbed retainers were positive for residual blood by the Kastle-Meyer test compared with 6% and 3% respectively of ultrasonically cleaned bands and retainers (P < 0.001). Conclusions If Siqveland matrix bands are re-processed in the assembled state, then adequate pre-sterilisation cleaning cannot be achieved reliably. Ultrasonic baths are significantly more effective than hand cleaning for these items of equipment.