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Journal Article

Aminolevulinic Acid; Animals; Biofilms; Bioware; Cell Survival; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Implants, Experimental; Light; Luminescent Measurements; Methylene Blue; Osteomyelitis; Photochemotherapy; Photosensitizing Agents; Rats; Rats, Sprague-Dawley; Staphylococcus aureus; Xen29

Osteomyelitis can lead to severe morbidity and even death resulting from an acute or chronic inflammation of the bone and contiguous structures due to fungal or bacterial infection. Incidence approximates 1 in 1000 neonates and 1 in 5000 children in the United States annually and increases up to 0.36% and 16% in adults with diabetes or sickle cell anaemia, respectively. Current regimens of treatment include antibiotics and/or surgery. However, the increasing number of antibiotic resistant pathogens suggests that alternate strategies are required. We are investigating photodynamic therapy (PDT) as one such alternate treatment for osteomyelitis using a bioluminescent strain of biofilm-producing staphylococcus aureus (S. aureus) grown onto kirschner wires (K-wire). S. aureus-coated K-wires were exposed to methylene blue (MB) or 5-aminolevulinic acid (ALA)-mediated PDT either in vitro or following implant into the tibial medullary cavity of Sprague-Dawley rats. The progression of S. aureus biofilm was monitored non-invasively using bioluminescence and expressed as a percentage of the signal for each sample immediately prior to treatment. S. aureus infections were subject to PDT 10 days post inoculation. Treatment comprised administration of ALA (300 mg kg(-1)) intraperitoneally followed 4 h later by light (635 +/- 10 nm; 75 J cm(-2)) delivered transcutaneously via an optical fiber placed onto the tibia and resulted in significant delay in bacterial growth. In vitro, MB and ALA displayed similar cell kill with > or =4 log(10) cell kill. In vivo, ALA-mediated PDT inhibited biofilm implants in bone. These results confirm that MB or ALA-mediated PDT have potential to treat S. aureus cultures grown in vitro or in vivo using an animal model of osteomyelitis.

Journal Article

Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bioware; Cathelicidins; Chronic Disease; Drug Carriers; Genetic Engineering; Male; Rats; Rats, Inbred F344; Surgical Flaps; Wound Infection; Xen29

BACKGROUND The success of antimicrobial therapy has been impaired by the emergence of resistant bacterial strains. Antimicrobial peptides are ubiquitous proteins that are part of the innate immune system and are successful against such antibiotic-resistant microorganisms. The authors have previously demonstrated the feasibility of protein delivery via microvascular free flap gene therapy and here they examine this approach for recalcitrant infections. METHODS The authors investigated the production of the human cathelicidin antimicrobial peptide-LL37, delivered by ex vivo transduction of the rodent superficial inferior epigastric free flap with Ad/CMV-LL37. The vascular permeabilizing agent vascular endothelial growth factor (VEGF) was co-administered during ex vivo transduction with adenoviral vectors in an attempt to augment transduction efficiency. A rodent model of chronic wound/foreign body infection seeded with bioluminescent Staphylococcus aureus was used to assess the biological efficacy of delivering therapeutic antimicrobial genes using this technology. RESULTS The authors were successful in demonstrating significant LL37 expression, which persisted for 14 days after ex vivo transduction with Ad/CMV-LL37. Transduction efficiency was significantly improved with the co-administration of 5 micrograms of VEGF during transduction without significantly increasing systemic dissemination of adenovirus or systemic toxicity. They were able to demonstrate in the rodent model of chronic wound/foreign body infections a significant reduction in bacterial loads from infected catheters following transduction with Ad/CMV-LL37 and increased bacterial clearance. CONCLUSION This study demonstrates for the first time that microbicidal gene therapy via microvascular free flaps is able to clear chronic infections such as occurs with osteomyelitis resulting from trauma or an infected foreign body [corrected]

Journal Article

Animal Structures; Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Proteins; Bioware; Cell Wall; Colony Count, Microbial; Female; Humans; Mice; Mice, Inbred BALB C; Neutrophils; Opsonin Proteins; Proteome; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staphylococcal Infections; Staphylococcal Vaccines; Staphylococcus aureus; Vaccines, Subunit; Vaccines, Synthetic; Whole Body Imaging; Xen29

Staphylococcus aureus is an important human pathogen with increasing clinical impact due to the extensive spread of antibiotic-resistant strains. Therefore, development of a protective polyvalent vaccine is of great clinical interest. We employed an intravenous immunoglobulin (IVIG) preparation as a source of antibodies directed against anchorless S. aureus surface proteins for identification of novel vaccine candidates. In order to identify such proteins, subtractive proteome analysis (SUPRA) of S. aureus anchorless cell wall proteins was performed. Proteins reacting with IVIG but not with IVIG depleted of S. aureus-specific opsonizing antibodies were considered vaccine candidates. Nearly 40 proteins were identified by this preselection method using matrix-assisted laser desorption ionization--time of flight analysis. Three of these candidate proteins, enolase (Eno), oxoacyl reductase (Oxo), and hypothetical protein hp2160, were expressed as glutathione S-transferase fusion proteins, purified, and used for enrichment of corresponding immunoglobulin Gs from IVIG by affinity chromatography. Use of affinity-purified anti-Eno, anti-Oxo, and anti-hp2160 antibodies resulted in opsonization, phagocytosis, and killing of S. aureus by human neutrophils. High specific antibody titers were detected in mice immunized with recombinant antigens. In mice challenged with bioluminescent S. aureus, reduced staphylococcal spread was measured by in vivo imaging. The recovery of S. aureus CFU from organs of immunized mice was diminished 10- to 100-fold. Finally, mice immunized with hp2160 displayed statistically significant higher survival rates after lethal challenge with clinically relevant S. aureus strains. Taken together, our data suggest that anchorless cell wall proteins might be promising vaccine candidates and that SUPRA is a valuable tool for their identification.

Journal Article

Animals; Arthritis, Experimental; Bioware; Chemotaxis, Leukocyte; Humans; Inflammation; Mice; Mice, Transgenic; Neutrophils; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Protein Transport; PTEN Phosphohydrolase; Xen29

Neutrophils encounter and 'prioritize' many chemoattractants in their pursuit of bacteria. Here we tested the possibility that the phosphatase PTEN is responsible for the prioritization of chemoattractants. Neutrophils induced chemotaxis by two separate pathways, the phosphatidylinositol-3-OH kinase (PI(3)K) phosphatase and tensin homolog (PTEN) pathway, and the p38 mitogen-activated protein kinase pathway, with the p38 pathway dominating over the PI(3)K pathway. Pten(-/-) neutrophils could not prioritize chemoattractants and were 'distracted' by chemokines when moving toward bacterial chemoattractants. In opposing gradients, PTEN became distributed throughout the cell circumference, which inhibited all PI(3)K activity, thus permitting 'preferential' migration toward bacterial products via phospholipase A(2) and p38. Such prioritization was defective in Pten(-/-) neutrophils, which resulted in defective bacterial clearance in vivo. Our data identify a PTEN-dependent mechanism in neutrophils to prioritize, 'triage' and integrate responses to multiple chemotactic cues.

Journal Article

Bioware; Xen29

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