Journal Article

B16-F10-luc2, Melanoma, B16F10-luc2, IVIS

In-transit metastasis (ITM) is a unique manifestation of intralymphatic tumor dissemination, characterized by the presence of melanoma cells between the primary lesion and the draining regional lymph node basin that is clinically associated with poor prognosis. In this study, we aimed to establish an experimental animal model of melanoma ITM, as research progress in this field has been hampered by a lack of suitable experimental models. We reproduced melanoma ITM in a mouse hind limb by transplanting melanoma cells into the footpad of a mouse with lymphedema (LE). The tumor cells at the ITM site were highly proliferative, and mice with ITMs were more likely than control mice to develop distant lymph node and lung metastases. Peritumoral lymphatic vessels and tumor-associated blood vessels were increased in the primary tumor site of the LE mice. Our established ITM melanoma mouse model enabled us to clarify the molecular determinants and pathophysiology of ITM. This ITM model is also comparable to the unfavorable clinical behavior of melanoma ITM in humans and, moreover, underlined the importance of lymphangiogenic factors in the tumor dissemination through the lymphatic system.Journal of Investigative Dermatology advance online publication, 6 September 2012; doi:10.1038/jid.2012.274.

Journal Article

Xen31, Xen 31, MRSA, S. aureus, IVIS, Bioluminescence, Animals; Bacterial Infections/*drug therapy; Burns/drug therapy/microbiology; Candida/drug effects; Cations; Gram-Negative Bacteria/drug effects; Gram-Positive Bacteria/drug effects; Male; Methicillin-Resistant Staphylococcus aureus; Mice; Mice, Inbred BALB C; *Photochemotherapy; Photosensitizing Agents/*chemical synthesis/chemistry/pharmacology; Porphyrins/*chemical synthesis/chemistry/pharmacology; Solubility; Staphylococcal Infections/drug therapy/microbiology; Structure-Activity Relationship

Structures of typical photosensitizers used in antimicrobial photodynamic therapy are based on porphyrins, phthalocyanines, and phenothiazinium salts, with cationic charges at physiological pH values. However, derivatives of the porphycene macrocycle (a structural isomer of porphyrin) have barely been investigated as antimicrobial agents. Therefore, we report the synthesis of the first tricationic water-soluble porphycene and its basic photochemical properties. We successfully tested it for in vitro photoinactivation of different Gram-positive and Gram-negative bacteria, as well as a fungal species (Candida) in a drug-dose and light-dose dependent manner. We also used the cationic porphycene in vivo to treat an infection model comprising mouse third degree burns infected with a bioluminescent methicillin-resistant Staphylococcus aureus strain. There was a 2.6-log(10) reduction (p < 0.001) of the bacterial bioluminescence for the PDT-treated group after irradiation with 180 J.cm(-2) of red light.

Journal Article

A549-luc-C8, A549-luc, IVIS, Bioware

Many cancer cell types are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Here, we examined whether HSP70 suppression by small interfering RNA (siRNA) sensitized non-small cell lung cancer (NSCLC) cells to TRAIL-induced apoptosis and the underlying mechanisms. We demonstrated that HSP70 suppression by siRNA sensitized NSCLC cells to TRAIL-induced apoptosis by upregulating the expressions of death receptor 4 (DR4) and death receptor 5 (DR5) through activating NF-kappaB, JNK, and, subsequently, p53, consequently significantly amplifying TRAIL-mediated caspase-8 processing and activity, cytosolic translocation of cytochrome c, and cell death. Consistently, the pro-apoptotic proteins Bad and Bax were upregulated, while the anti-apoptotic protein Bcl-2 was downregulated. The luciferase activity of the DR4 promoter was blocked by a NF-kappaB pathway inhibitor BAY11-7082, suggesting that NF-kappaB activation plays an important role in the transcriptional upregulation of DR4. Additionally, HSP70 suppression inhibited the phosphorylation of ERK, AKT, and PKC, thereby downregulating c-FLIP-L. A549 xenografts in mice receiving HSP70 siRNA showed TRAIL-induced cell death and increased DR4/DR5 levels and reduced tumor growth. The combination of psiHSP70 gene therapy with TRAIL also significantly increased the survival benefits induced by TRAIL therapy alone. Interestingly, HSP27 siRNA and TRAIL together could not suppress tumor growth or prolong the survival of tumor-bearing mice significantly, although the combination could efficiently induce the apoptosis of A549 cells in vitro. Our findings suggest that HSP70 suppression or downregulation might be promising to overcome TRAIL resistance in cancer.

Journal Article

Xen36, Xen 36, Staphylococcus aureus Xen36, IVIS

Wear of metal-on-metal (cobalt-chromium, Co-Cr particles) and metal-on-polyethylene (ultra-high-molecular-weight polyethylene, UHMWPE particles) bearing surfaces in hip prostheses is a major problem in orthopedics. This study aimed to compare the influence of Co-Cr and UHMWPE particles on the persistence of infection. Bioluminescent Staphylococcus aureus Xen36 were injected in air pouches prepared in subcutaneous tissue of immuno-competent BALB/c mice (control), as a model for the joint space, in the absence or presence of Co-Cr or UHMWPE particles. Bioluminescence was monitored longitudinally up to 21 days, corrected for absorption and reflection by the particles and expressed relative to the bioluminescence found in the presence of staphylococci only. After termination, air pouch fluid and air pouch membrane were cultured and histologically analyzed. Bioluminescence was initially lower in mice exposed to UHMWPE particles with staphylococci than in mice injected with staphylococci only, possibly because UHMWPE particles initially stimulated a higher macrophage presence in murine air pouch membranes. For mice exposed to Co-Cr particles with staphylococci, bioluminescence was observed to be higher in two out of six animals compared to the presence of staphylococci alone. In the majority of mice, infection risk in the absence or presence of Co-Cr and UHMWPE particles appeared similar, assuming that the longevity of an elevated bioluminescence is indicative of a higher infection risk. However, the presence of Co-Cr particles yielded a higher bioluminescence in two out of six mice, possibly because the macrophage degradative function was hampered by the presence of Co-Cr particles. (c) 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Journal Article

Xen41, Xen 41, Pseudomonas aeruginosa Xen41, IVIS

BACKGROUND: : Experimental models of intestinal ischemia-reperfusion (IIR) injury are invariably performed in mice harboring their normal commensal flora, even though multiple IIR events occur in humans during prolonged intensive care confinement when they are colonized by a highly pathogenic hospital flora. The aims of this study were to determine whether the presence of the human pathogen Pseudomonas aeruginosa in the distal intestine potentiates the lethality of mice exposed to IIR and to determine what role any in vivo virulence activation plays in the observed mortality. METHODS: : Seven- to 9-week-old C57/BL6 mice were exposed to 15 minutes of superior mesenteric artery occlusion (SMAO) followed by direct intestinal inoculation of 1.0 x 10 colony-forming unit of P. aeruginosa PAO1 into the ileum and observed for mortality. Reiterative studies were performed in separate groups of mice to evaluate both the migration/dissemination pattern and in vivo virulence activation of intestinally inoculated strains using live photon camera imaging of both a constitutive bioluminescent P. aeruginosa PAO1 derivative XEN41 and an inducible reporter derivative of PAO1, the PAO1/lecA:luxCDABE that conditionally expresses the quorum sensing-dependent epithelial disrupting virulence protein PA 1 Lectin (PA-IL). RESULTS: : Mice exposed to 15 minutes of SMAO and reperfusion with intestinal inoculation of P. aeruginosa had a significantly increased mortality rate (p < 0.001) of 100% compared with <10% for sham-operated mice intestinally inoculated with P. aeruginosa without SMAO and IIR alone (<50%). Migration/dissemination patterns of P. aeruginosa in mice subjected to IIR demonstrated proximal migration of distally injected strains and translocation to mesenteric lymph nodes, liver, spleen, lung, and kidney. A key role for in vivo virulence expression of the barrier disrupting adhesin PA-IL during IIR was established since its expression was enhanced during IR and mutant strains lacking PA-IL displayed attenuated mortality. CONCLUSIONS: : The presence of intestinal P. aeruginosa potentiates the lethal effect of IIR in mice in part due to in vivo virulence activation of its epithelial barrier disrupting protein PA-IL.