Journal Article

Xen5, Xen 5, Pseudomonas aeruginosa Xen 5, Animals; Fullerenes/*chemistry; Male; Mice; Mice, Inbred BALB C; Photochemotherapy/*methods; Photosensitizing Agents/*chemistry; Pseudomonas Infections/*drug therapy; Pseudomonas aeruginosa/drug effects; Wound Infection/*drug therapy

AIMS: Fullerenes are under intensive study for potential biomedical applications. We have previously reported that a C60 fullerene functionalized with three dimethylpyrrolidinium groups (BF6) is a highly active broad-spectrum antimicrobial photosensitizer in vitro when combined with white-light illumination. We asked whether this high degree of in vitro activity would translate into an in vivo therapeutic effect in two potentially lethal mouse models of infected wounds. MATERIALS & METHODS: We used stable bioluminescent bacteria and a low light imaging system to follow the progress of the infection noninvasively in real time. An excisional wound on the mouse back was contaminated with one of two bioluminescent Gram-negative species, Proteus mirabilis (2.5 x 10(7) cells) and Pseudomonas aeruginosa (5 x 10(6) cells). A solution of BF6 was placed into the wound followed by delivery of up to 180 J/cm(2) of broadband white light (400-700 nm). RESULTS: In both cases there was a light-dose-dependent reduction of bioluminescence from the wound not observed in control groups (light alone or BF6 alone). Fullerene-mediated photodynamic therapy of mice infected with P. mirabilis led to 82% survival compared with 8% survival without treatment (p < 0.001). Photodynamic therapy of mice infected with highly virulent P. aeruginosa did not lead to survival, but when photodynamic therapy was combined with a suboptimal dose of the antibiotic tobramycin (6 mg/kg for 1 day) there was a synergistic therapeutic effect with a survival of 60% compared with a survival of 20% with tobramycin alone (p < 0.01). CONCLUSION: These data suggest that cationic fullerenes have clinical potential as an antimicrobial photosensitizer for superficial infections where red light is not needed to penetrate tissue.

Journal Article

Xen14, Xen 14, E. coli Xen14, IVIS, Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides/chemical synthesis/*pharmacology; Bacterial Infections/*metabolism/microbiology/prevention & control; Cell Line; Cells, Cultured; Chemokine CCL2/metabolism; Chemokine CCL7/metabolism; Chemokine CXCL1/metabolism; Chemokines/*metabolism; Female; Humans; Interleukin-8/metabolism; Leukocytes/cytology/*metabolism; Leukocytes, Mononuclear/cytology/drug effects/metabolism; Macrophages/cytology/drug effects/metabolism; Mice; Mice, Inbred C57BL; Molecular Sequence Data; NF-kappa B/metabolism; Phosphatidylinositol 3-Kinases/metabolism; Phosphorylation/drug effects; Staphylococcal Infections/microbiology/prevention & control; Staphylococcus aureus/drug effects; p38 Mitogen-Activated Protein Kinases/metabolism

With the rapid rise in the incidence of multidrug resistant infections, there is substantial interest in host defense peptides as templates for production of new antimicrobial therapeutics. Natural peptides are multifunctional mediators of the innate immune response, with some direct antimicrobial activity and diverse immunomodulatory properties. We have previously developed an innate defense regulator (IDR) 1, with protective activity against bacterial infection mediated entirely through its effects on the immunity of the host, as a novel approach to anti-infective therapy. In this study, an immunomodulatory peptide IDR-1002 was selected from a library of bactenecin derivatives based on its substantially more potent ability to induce chemokines in human PBMCs. The enhanced chemokine induction activity of the peptide in vitro correlated with stronger protective activity in vivo in the Staphylococcus aureus-invasive infection model, with a >5-fold reduction in the protective dose in direct comparison with IDR-1. IDR-1002 also afforded protection against the Gram-negative bacterial pathogen Escherichia coli. Chemokine induction by IDR-1002 was found to be mediated through a Gi-coupled receptor and the PI3K, NF-kappaB, and MAPK signaling pathways. The protective activity of the peptide was associated with in vivo augmentation of chemokine production and recruitment of neutrophils and monocytes to the site of infection. These results highlight the importance of the chemokine induction activity of host defense peptides and demonstrate that the optimization of the ex vivo chemokine-induction properties of peptides is a promising method for the rational development of immunomodulatory IDR peptides with enhanced anti-infective activity.

Journal Article

Xen14, Xen 14, E. coli Xen14, IVIS, Animals; Anti-Infective Agents/*pharmacology/therapeutic use; Bacteria/*drug effects/pathogenicity; Bacterial Infections/drug therapy/*etiology; Biocompatible Materials/*adverse effects/chemistry; Biofilms; Coated Materials, Biocompatible/chemistry; Fluorescent Dyes/chemistry/metabolism; Humans; Image Enhancement/methods; Light; Luminescent Measurements/instrumentation/*methods; Luminescent Proteins/metabolism; Microscopy, Fluorescence/instrumentation/*methods; Prosthesis-Related Infections/drug therapy/microbiology; Sensitivity and Specificity

This review presents the current state of Bioluminescence and Fluorescent Imaging technologies (BLI and FLI) as applied to Biomaterial-Associated Infections (BAI). BLI offers the opportunity to observe the in vivo course of BAI in small animals without the need to sacrifice animals at different time points after the onset of infection. BLI is highly dependent on the bacterial cell metabolism which makes BLI a strong reporter of viable bacterial presence. Fluorescent sources are generally more stable than bioluminescent ones and specifically targeted, which renders the combination of BLI and FLI a promising tool for imaging BAI. The sensitivity and spatial resolution of both imaging tools are, however, dependent on the imaging system used and the tissue characteristics, which makes the interpretation of images, in terms of the location and shape of the illuminating source, difficult. Tomographic reconstruction of the luminescent source is possible in the most modern instruments, enabling exact localization of a colonized implant material, spreading of infecting organisms in surrounding tissue and immunological tissue reactions. BLI studies on BAI have successfully distinguished between different biomaterials with respect to the development and clearance of BAI in vivo, simultaneously reducing animal use and experimental variation. It is anticipated that bio-optical imaging will become an indispensable technology for the in vivo evaluation of antimicrobial coatings.

Journal Article

Xen36, Xen 36, Staphylococcus aureus Xen36, IVIS, Animals; Mice; Mice, Inbred C57BL; Microbial Sensitivity Tests; Nisin/*pharmacology; Peritoneal Cavity/*microbiology; Staphylococcal Infections/*prevention & control; Staphylococcus aureus/*drug effects/growth & development

AIMS: To determine the ability of nisin F to control systematic infection caused by Staphylococcus aureus, using C57BL/6 mice as a model. METHODS AND RESULTS: Twelve mice were intraperitoneally injected with 1 x 10(8) viable cells of Staph. aureus Xen 36 containing the modified Photorhabdus luminescence luxABCDE operon on plasmid pAUL-A Tn4001. After 4 h, six mice were intraperitoneally injected with 640 arbitrary units (AU) nisin F, and six were injected with sterile saline. Six mice, not infected with Staph. aureus, were treated with nisin F, and six not infected were left untreated. The viability of Staph. aureus Xen 36 was monitored over 48 h by recording photon emission levels. Nisin F suppressed Staph. aureus for 15 min in vivo. No abnormalities were recorded in blood analyses and internal organs of mice treated with nisin F. CONCLUSIONS: Nisin F suppressed the growth of Staph. aureus in the peritoneal cavity for at least 15 min. Re-emergence of Staph. aureus bioluminescence over the next 44 h suggests that nisin F was inactivated, most probably by proteolytic enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: A single dosage of nisin F administered in the peritoneal cavity controlled the growth of Staph. aureus for at least 15 min in vivo.

Journal Article

MDA-MB-231-D3H2Ln, IVIS, Bioluminescence, Animals; Antineoplastic Combined Chemotherapy Protocols/*therapeutic use; Cell Hypoxia/physiology; Cell Line, Tumor; Cyclophosphamide/*therapeutic use; Female; Immunohistochemistry; Lung Neoplasms/prevention & control/secondary; Mammary Neoplasms, Experimental/*drug therapy/genetics/pathology; Mice; Mice, Nude; Sirolimus/*therapeutic use; Tumor Burden; Xenograft Model Antitumor Assays

Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has been shown to inhibit the growth of oestrogen positive breast cancer. However, triple-negative (TN) breast cancer is resistant to rapamycin treatment in vitro. We set to test a combination treatment of rapamycin with DNA-damage agent, cyclophosphamide, in a TN breast cancer model. By binding to and disrupting cellular DNA, cyclophosphamide kills cells via interfering with their normal functions. We assessed the responses of nude mice bearing tumour xenografts of TN MDA-MB-231 cells to the combination of rapamycin and cyclophosphamide in both orthotopic mammary and lung-metastasis models. We tracked tumour growth and metastasis by bioluminescent imaging and examined the expression of Ki67, CD34 and HIF-1alpha in tumour tissues by immunohistochemistry and apoptosis index with TUNEL assay, and found that MDA-MB-231 cells are sensitive to rapamycin therapy in orthotopic mammary, but not in lung with metastasis. Rapamycin when combined with cyclophosphamide is found to have a more significant effect in reducing tumour volume and metastasis with a much improved survival rate. Our data also show that the sensitivity of TN tumours to rapamycin is associated with the microenvironment of the tumour cells. The data indicate that in a relatively hypoxic environment HIF-1alpha may play a role in mediating the anti-cancer effect of rapamycin and cyclophosphamide may prevent the feedback activation of Akt by rapamycin. Overall our results show that rapamycin plus cyclophosphamide can achieve an improved efficacy in suppressing tumour growth and metastasis, suggesting that the combination therapy can be a promising treatment option for TN cancer.