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Journal Article

Xen10, Xen 10, Streptococcus pnuemoniae Xen10, IVIS,

Background & objectives: In vivo imaging system has contributed significantly to the understanding of bacterial infection and efficacy of drugs in animal model. We report five rapid, reproducible, and non invasive murine pulmonary infection, skin and soft tissue infection, sepsis, and meningitis models using Xenogen bioluminescent strains and specialized in vivo imaging system (IVIS). Methods: The progression of bacterial infection in different target organs was evaluated by the photon intensity and target organ bacterial counts. Genetically engineered bioluminescent bacterial strains viz. Staphylococcus aureus Xen 8.1, 29 and 31; Streptococcus pneumoniae Xen 9 and 10 and Pseudomonas aeruginosa Xen-5 were used to induce different target organs infection and were validated with commercially available antibiotics. Results: The lower limit of detection of colony forming unit (cfu) was 1.7-log10 whereas the lower limit of detection of relative light unit (RLU) was 4.2-log10 . Recovery of live bacteria from different target organs showed that the bioluminescent signal correlated to the live bacterial count. Interpretation & conclusions: This study demonstrated the real time monitoring and non-invasive analysis of progression of infection and pharmacological efficacy of drugs. These models may be useful for pre-clinical discovery of new antibiotics.

Journal Article

B16-F10-luc2, B16F10-luc2, IVIS

mAb therapy for experimental metastatic melanoma relies on activating receptors for the Fc portion of IgG (FcgammaR). Opposing results on the respective contribution of mouse FcgammaRI, FcgammaRIII, and FcgammaRIV have been reported using the gp75-expressing B16 melanoma and the protective anti-gp75 mAb TA99. We analyzed the contribution of FcgammaRs to this therapy model using bioluminescent measurement of lung metastases loads, novel mouse strains, and anti-FcgammaR blocking mAbs. We found that the TA99 mAb-mediated effects in a combination therapy using cyclophosphamide relied on activating FcgammaRs. The combination therapy, however, was not more efficient than mAb therapy alone. We demonstrate that FcgammaRI and, unexpectedly, FcgammaRIII contributed to TA99 mAb therapeutic effects, whereas FcgammaRIV did not. Therefore, FcgammaRIII and FcgammaRI are, together, responsible for anti-gp75 mAb therapy of B16 lung metastases. Our finding that mouse FcgammaRIII contributes to Ab-induced tumor reduction correlates with clinical data on its human functional equivalent human FcgammaRIIIA (CD16A).

Journal Article

Adjuvants, Immunologic; Allylamine; Amine Oxidase (Copper-Containing); Animals; Antibodies, Blocking; Antibodies, Monoclonal; B16-F10-luc-G5 cells; Bioware; Cell Adhesion Molecules; Cell Line, Tumor; Cell Migration Inhibition; Enzyme Inhibitors; Female; Growth Inhibitors; Lymphoma, T-Cell; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Myeloid Cells; Semicarbazides

Vascular adhesion protein-1 (VAP-1) is an endothelial, cell surface-expressed oxidase involved in leukocyte traffic. The adhesive function of VAP-1 can be blocked by anti-VAP-1 Abs and small-molecule inhibitors. However, the effects of VAP-1 blockade on antitumor immunity and tumor progression are unknown. In this paper, we used anti-VAP-1 mAbs and small-molecule inhibitors of VAP-1 in B16 melanoma and EL-4 lymphoma tumor models in C57BL/6 mice. Leukocyte accumulation into tumors and neoangiogenesis were evaluated by immunohistochemistry, flow cytometry, and intravital videomicroscopy. We found that both anti-VAP-1 Abs and VAP-1 inhibitors reduced the number of leukocytes in the tumors, but they targeted partially different leukocyte subpopulations. Anti-VAP-1 Abs selectively inhibited infiltration of CD8-positive lymphocytes into tumors and had no effect on accumulation of myeloid cells into tumors. In contrast, the VAP-1 inhibitors significantly reduced only the number of proangiogenic Gr-1(+)CD11b(+) myeloid cells in melanomas and lymphomas. Blocking of VAP-1 by either means left tumor homing of regulatory T cells and type 2 immune-suppressing monocytes/macrophages intact. Notably, VAP-1 inhibitors, but not anti-VAP-1 Abs, retarded the growth of melanomas and lymphomas and reduced tumor neoangiogenesis. The VAP-1 inhibitors also reduced the binding of Gr-1(+) myeloid cells to the tumor vasculature. We conclude that tumors use the catalytic activity of VAP-1 to recruit myeloid cells into tumors and to support tumor progression. Small-molecule VAP-1 inhibitors therefore might be a potential new tool for immunotherapy of tumors.

Journal Article

GL261-luc2, IVIS, Glioma, Biolumninescence imaging

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain (1); therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation (1). Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two mul of GL261-luc or GL261-luc2 cells (10(7) cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured. Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150mug luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in >10% loss of weight. Tumors were evident in all of the animals on necropsy.