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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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Journal Article

Animals; Antibody Formation; Bacterial Proteins; Bioware; Disease Models, Animal; DNA, Bacterial; Endonucleases; Female; Mice; Mice, Inbred C57BL; Micrococcal Nuclease; Osteolysis; Osteomyelitis; Prosthesis-Related Infections; Reverse Transcriptase Polymerase Chain Reaction; Staphylococcal Infections; Staphylococcus aureus; Tibia; Xen29

Although osteomyelitis (OM) remains a serious problem in orthopedics, progress has been limited by the absence of an in vivo model that can quantify the bacterial load, metabolic activity of the bacteria over time, immunity, and osteolysis. To overcome these obstacles, we developed a murine model of implant-associated OM in which a stainless steel pin is coated with Staphylococcus aureus and implanted transcortically through the tibial metaphysis. X-ray and micro-CT demonstrated concomitant osteolysis and reactive bone formation, which was evident by day 7. Histology confirmed all the hallmarks of implant-associated OM, namely: osteolysis, sequestrum formation, and involucrum of Gram-positive bacteria inside a biofilm within necrotic bone. Serology revealed that mice mount a protective humoral response that commences with an IgM response after 1 week, and converts to a specific IgG2b response against specific S. aureus proteins by day 11 postinfection. Real-time quantitative PCR (RTQ-PCR) for the S. aureus specific nuc gene determined that the peak bacterial load occurs 11 days postinfection. This coincidence of decreasing bacterial load with the generation of specific antibodies is suggestive of protective humoral immunity. Longitudinal in vivo bioluminescent imaging (BLI) of luxA-E transformed S. aureus (Xen29) combined with nuc RTQ-PCR demonstrated the exponential growth phase of the bacteria immediately following infection that peaks on day 4, and is followed by the biofilm growth phase at a significantly lower metabolic rate (p < 0.05). Collectively, these studies demonstrate the first quantitative model of implant-associated OM that defines the kinetics of microbial growth, osteolysis, and humoral immunity following infection.