Citation Details

Journal Article

AngioSense, Animals; Antigens, CD/metabolism; Antigens, Differentiation, Myelomonocytic/metabolism; Calcium-Binding Proteins/metabolism; Central Nervous System/metabolism; Green Fluorescent Proteins/genetics/*metabolism; Immunohistochemistry/*methods; Lipopolysaccharides/pharmacology; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins/metabolism; Microglia/cytology/drug effects/*metabolism; Microscopy, Confocal/*methods; Receptors, Cytokine/genetics/metabolism; Receptors, HIV/genetics/metabolism; Reproducibility of Results

Microglia are specialized immune cells of the brain. Upon insult, microglia initiate a cascade of cellular responses including a characteristic change in cell morphology. To study the dynamics of microglia immune response in situ, we developed an automated image analysis method that enables the quantitative assessment of microglia activation state within tissue based solely on cell morphology. Per cell morphometric analysis of fluorescently labeled microglia is achieved through local iterative threshold segmentation, which reduces errors caused by signal-to-noise variation across large volumes. We demonstrate, utilizing systemic application of lipopolysaccharide as a model of immune challenge, that several morphological parameters, including cell perimeter length, cell roundness and soma size, quantitatively distinguish resting versus activated populations of microglia within tissue comparable to traditional immunohistochemistry methods. Furthermore, we provide proof-of-concept data that monitoring soma size enables the longitudinal assessment of microglia activation in the mouse neocortex imaged via 2-photon in vivo microscopy. The ability to quantify microglia activation automatically by shape alone allows unbiased and rapid analysis of both fixed and in vivo central nervous system tissue.

Journal Article

IVIS, Xen29, Xen 29, Staphylococcus aureus Xen29, Administration, Inhalation; Aerosols; Animals; Cell Line, Tumor; Cell Proliferation/drug effects; Cell Transformation, Neoplastic; Humans; Lung Neoplasms/drug therapy/genetics/*pathology/*secondary; Mice; Oncogenes/genetics; Plasmids/*administration & dosage/chemistry/*pharmacology; Polyethyleneimine/chemistry

Despite numerous efforts, drug based treatments for patients suffering from lung cancer remains poor. As a promising alternative, we investigated the therapeutic potential of BC-819 for the treatment of lung cancer in mouse tumor models. BC-819 is a novel plasmid DNA which encodes for the A-fragment of Diphtheria toxin and has previously been shown to successfully inhibit tumor growth in human clinical study of bladder carcinoma. In a first set of experiments, we examined in vitro efficacy of BC-819 in human lung cancer cell-lines NCI-H460, NCI-H358 and A549, which revealed >90% reduction of cell growth. In vivo efficacy was examined in an orthotopic mouse xenograft lung cancer model and in a lung metastasis model using luminescent A549-C8-luc adenocarcinoma cells. These cells resulted in peri- and intra-bronchiolar tumors upon intrabronchial application and parenchymal tumors upon intravenous injection, respectively. Mice suffering from these lung tumors were treated with BC-819, complexed to branched polyethylenimine (PEI) and aerosolized to the mice once per week for a period of 10 weeks. Using this regimen, growth of intrabronchially induced lung tumors was significantly inhibited (p = 0.01), whereas no effect could be observed in mice suffering from lung metastasis. In summary, we suggest that aerosolized PEI/BC-819 is capable of reducing growth only in tumors arising from the luminal part of the airways and are therefore directly accessible for inhaled BC-819.

Journal Article

4T1-luc2; Adenocarcinoma; Animals; Bioware; Breast Neoplasms; Cell Line, Tumor; Chemotaxis, Leukocyte; Cytotoxicity, Immunologic; Female; Humans; Immunotherapy, Adoptive; Indium Radioisotopes; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Knockout; Neoplasm Transplantation; Radiopharmaceuticals; Receptors, Antigen, T-Cell, gamma-delta; Spleen; Tissue Distribution; T-Lymphocyte Subsets; Tomography, Emission-Computed, Single-Photon; Transplantation, Heterologous; Transplantation, Isogeneic

In contrast to antigen-specific alphabeta-T cells (adaptive immune system), gammadelta-T cells can recognize and lyse malignantly transformed cells almost immediately upon encounter in a manner that does not require the recognition of tumor-specific antigens (innate immune system). Given the well-documented capacity of gammadelta-T cells to innately kill a variety of malignant cells, efforts are now actively underway to exploit the antitumor properties of gammadelta-T cells for clinical purposes. Here, we present for the first time preclinical in vivo mouse models of gammadelta-T cell-based immunotherapy directed against breast cancer. These studies were explicitly designed to approximate clinical situations in which adoptively transferred gammadelta-T cells would be employed therapeutically against breast cancer. Using radioisotope-labeled gammadelta-T cells, we first show that adoptively transferred gammadelta-T cells localize to breast tumors in a mouse model (4T1 mammary adenocarcinoma) of human breast cancer. Moreover, by using an antibody directed against the gammadelta-T cell receptor (TCR), we determined that localization of adoptively transferred gammadelta-T cells to tumor is a TCR-dependant process. Additionally, biodistribution studies revealed that adoptively transferred gammadelta-T cells traffic differently in tumor-bearing mice compared to healthy mice with fewer gammadelta-T cells localizing into the spleens of tumor-bearing mice. Finally, in both syngeneic (4T1) and xenogeneic (2Lmp) models of breast cancer, we demonstrate that adoptively transferred gammadelta-T cells are both effective against breast cancer and are otherwise well-tolerated by treated animals. These findings provide a strong preclinical rationale for using ex vivo expanded adoptively transferred gammadelta-T cells as a form of cell-based immunotherapy for the treatment of breast cancer. Additionally, these studies establish that clinically applicable methods for radiolabeling gammadelta-T cells allows for the tracking of adoptively transferred gammadelta-T cells in tumor-bearing hosts.

Journal Article

VivoTag, IVIS, Vivotag

Recombinant adenovirus-engineered dendritic cells (Ad.DC) are potent vaccines for induction of anti-viral and anti-cancer T cell immunity. The effectiveness of Ad.DC vaccines may depend on the newly described ability of Ad.DC to crosstalk with natural killer (NK) cells via cell-to-cell contact, and to mediate activation, polarization and bridging of innate and adaptive immunity. For this interaction to occur in vivo, Ad.DC must be able to attract NK cells from surrounding tissues or peripheral blood. We developed a novel live mouse imaging system-based NK-cell migration test, and demonstrated for the first time that human Ad.DC induced directional migration of human NK cells across subcutaneous tissues, indicating that Ad.DC-NK cell contact and interaction could occur in vivo. We examined the mechanism of Ad.DC-induced migration of NK cells in vitro and in vivo. Ad.DC produced multiple chemokines previously reported to recruit NK cells, including immunoregulatory CXCL10/IP-10 and proinflammatory CXCL8/IL-8. In vitro chemotaxis experiments utilizing neutralizing antibodies and recombinant human chemokines showed that CXCL10/IP-10 and CXCL8/IL-8 were critical for Ad.DC-mediated recruitment of CD56(hi)CD16(-) and CD56(lo)CD16(+) NK cells, respectively. The importance of CXCL8/IL-8 was further demonstrated in vivo. Pretreatment of mice with the neutralizing anti-CXCL8/IL-8 antibody led to significant inhibition of Ad.DC-induced migration of NK cells in vivo. These data show that Ad.DC can recruit spatially distant NK cells toward a vaccine site via specific chemokines. Therefore, an Ad.DC vaccine can likely induce interaction with endogenous NK cells via transmembrane mediators, and consequently mediate Th1 polarization and amplification of immune functions in vivo.

Journal Article

ProSense; in vivo imaging; NOD2; mice; inflammatory arthritis; TCR transgenic; knockout

In addition to its role in innate immunity, nucleotide oligomerization domain 2 (NOD2) has been shown to play a suppressive role in models of colitis. Notably, mutations in NOD2 cause the inherited granulomatous disease of the joints called Blau syndrome, thereby linking NOD2 with joint disease as well. However, the role of NOD2 in joint inflammation has not been clarified. We demonstrate here that NOD2 is functional within the mouse joint and promotes inflammation, as locally or systemically administered muramyl dipeptide (MDP; the NOD2 agonist) resulted in significant joint inflammation that was abolished in NOD2-deficient mice. We then sought to investigate the role of NOD2 in a mouse model of inflammatory arthritis dependent on adaptive immunity using TCR-transgenic mice whose T cells recognized the dominant epitope of proteoglycan (PG). Mice immunized with PG in the presence of MDP developed a more severe inflammatory arthritis and histopathology within the joints. Antigen-specific activation of splenocytes was enhanced by MDP with respect to IFN-gamma production, which would be consistent with the Th1-mediated disease in vivo. Intriguingly, NOD2 deficiency did not alter the PG-induced arthritis, indicating that NOD2 does not play an essential role in this model of joint disease when it is not activated by MDP. In conclusion, we demonstrate that in a model of inflammatory arthritis dependent on T and B cell priming, NOD2 activation potentiates disease. However, the absence of NOD2 does not alter the course of inflammatory arthritis, in contrast to models of intestinal inflammation.