Transgenic mice engineered to target Cre/loxP-mediated DNA recombination into catecholaminergic neurons

To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We crossbred TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of "floxed" genes in catecholaminergic neurons.     

Role of Fyn in the rearrangement of tubulin cytoskeleton induced through TCR

The translocation of the microtubule-organizing center (MTOC), its associated signaling complex, and the secretory apparatus is the most characteristic early event that involves the tubulin cytoskeleton of T or NK cells after their interaction with APC or target cells. Our results show that Fyn kinase activity is essential for MTOC reorientation in an Ag-dependent system. Moreover, T cells from Fyn-deficient mice are unable to rearrange their tubulin cytoskeleton in response to anti-CD3-coated beads. Analysis of conjugates of T cells from transgenic OT-I mice with dendritic cells revealed that an antagonist peptide induces translocation of the MTOC, and that this process is impaired in T cells from Fyn(-/-) OT-I mice. In addition, Fyn deficiency significantly affects the MTOC relocation mediated by agonist peptide stimulation. These results reveal Fyn to be a key regulator of tubulin cytoskeleton reorganization in T cells.     

Permanent Occlusion of Feeding Arteries and Draining Veins in Solid Mouse Tumors by Vascular Targeted Photodynamic Therapy (VTP) with Tookad

Background

Antiangiogenic and anti-vascular therapies present intriguing alternatives to cancer therapy. However, despite promising preclinical results and significant delays in tumor progression, none have demonstrated long-term curative features to date. Here, we show that a single treatment session of Tookad-based vascular targeted photodynamic therapy (VTP) promotes permanent arrest of tumor blood supply by rapid occlusion of the tumor feeding arteries (FA) and draining veins (DV), leading to tumor necrosis and eradication within 24–48 h.

Methodology/Principal Findings

A mouse earlobe MADB106 tumor model was subjected to Tookad-VTP and monitored by three complementary, non-invasive online imaging techniques: Fluorescent intravital microscopy, Dynamic Light Scattering Imaging and photosensitized MRI. Tookad-VTP led to prompt tumor FA vasodilatation (a mean volume increase of 70%) with a transient increase (60%) in blood-flow rate. Rapid vasoconstriction, simultaneous blood clotting, vessel permeabilization and a sharp decline in the flow rates then followed, culminating in FA occlusion at 63.2 sec±1.5SEM. This blockage was deemed irreversible after 10 minutes of VTP treatment. A decrease in DV blood flow was demonstrated, with a slight lag from FA response, accompanied by frequent changes in flow direction before reaching a complete standstill. In contrast, neighboring, healthy tissue vessels of similar sizes remained intact and functional after Tookad-VTP.

Conclusion/Significance

Tookad-VTP selectively targets the tumor feeding and draining vessels. To the best of our knowledge, this is the first mono-therapeutic modality that primarily aims at the larger tumor vessels and leads to high cure rates, both in the preclinical and clinical arenas.