Search

Resident tissue myeloid cells play a role in many aspects of physiology including development of the vascular systems. In the blood vasculature, myeloid cells use VEGFC to promote angiogenesis and can use Wnt ligands to control vascular branching and to promote vascular regression. Here we show that myeloid cells also regulate development of the dermal lymphatic vasculature using Wnt ligands. Using myeloid-specific deletion of the WNT transporter Wntless we show that myeloid Wnt ligands are active at two distinct stages of development of the dermal lymphatics. As lymphatic progenitors are emigrating from the cardinal vein and intersomitic vessels, myeloid Wnt ligands regulate both their numbers and migration distance. Later in lymphatic development, myeloid Wnt ligands regulate proliferation of lymphatic endothelial cells (LEC) and thus control lymphatic vessel caliber. Myeloid-specific deletion of WNT co-receptor Lrp5 or Wnt5a gain-of-function also produce elevated caliber in dermal lymphatic capillaries. These data thus suggest that myeloid cells produce Wnt ligands to regulate lymphatic development and use Wnt pathway co-receptors to regulate the balance of Wnt ligand activity during the macrophage-LEC interaction.

As the limits of existing treatments for cancer are recognized, clearly novel therapies must be considered for successful treatment; cancer therapy using adenovirus vectors is a promising strategy. However tracking the biodistribution of adenovirus vectors in vivo is limited to invasive procedures such as biopsies, which are error prone, non-quantitative, and do not give a full representation of the pharmacokinetics involved. Current non-invasive imaging strategies using reporter gene expression have been applied to analyze adenoviral vectors. The major drawback to approaches that tag viruses with reporter genes is that these systems require initial viral infection and subsequent cellular expression of a reporter gene to allow non-invasive imaging. As an alternative to conventional vector detection techniques, we developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and human metallothionein. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional metallothionein (MT) as a component of their capsid surface. We demonstrate the feasibility of (99m)Tc binding in vitro to the pIX-MT fusion on the capsid of adenovirus virions using a simple transchelation reaction. SPECT imaging of a mouse after administration of a (99m)Tc-radiolabeled virus showed clear localization of radioactivity to the liver. This result strongly supports imaging using pIX-MT, visualizing the normal biodistribution of Ad primarily to the liver upon injection into mice. The ability we have developed to view real-time biodistribution in their physiological milieu represents a significant tool to study adenovirus biology in vivo.

Tumors deficient in TAP expression cannot efficiently induce T cell immunity, and are resistant to T cell lysis. Here we show that introduction of B7.1 molecule into TAP-negative (TAP-) and TAP1-transfected (TAP1+) CMT.64 tumor cells dramatically augments T cell immunity against TAP- tumors. Differences in the immune responses were observed between TAP- and TAP1+ CMT.64 cells depending on the dose of inoculation. While mice immunized with B7.1 expressing TAP1+ cells rejected TAP- tumors with high and low dose inoculations, only high dose inoculations with B7.1 expressing TAP- cells resulted in tumor rejection. The induced protective immunities were T cell dependent as indicated by depletion of CD8 or CD4 cells in immune competent mice. We also found that B7.1 and TAP1 coexpression decreased tumorigenicity. In addition, when CMT.64 cells infected with vaccinia viruses carrying B7.1 and/or TAP1 were used for immunization, we found that B7.1 and TAP1 coexpression, but not B7.1 expression alone, played a critical role in induction of protective immunity after a high dose inoculation, suggesting that viral antigens significantly affected TAP negative and B7.1 expressing cells for tumor antigen presentation and T cell priming. Our results indicate that B7.1-provoked antitumor immunity against TAP- cancer is facilitated by TAP1 expression and thus, both genes should be considered for cancer therapy in the future.

Two technical hurdles, gene delivery and target specificity, have hindered the development of effective cancer gene therapies. In order to circumvent the problem of tumor specificity, the suicide gene, HSV-1 thymidine kinase (HSV-Tk), was modified with a complex 5' upstream-untranslated region (5'-UTR) that limits efficent translation to cells that express high levels of the translation initiation factor, eIF4E. Previous study data have shown that most tumor cells express elevated levels of eIF4E. Gene delivery was optimized by incorporation of the modified suicide gene (HSV-UTk) into an adenovirus vector (Ad) that infects a broad range of cells. The goal of this project in the development of Ad-HSV-UTk was to target infection of a broad population of cells and yet limit translation of the suicide gene to those cells expressing elevated levels of eIF4E. The efficacy of this novel approach of targeting suicide gene expression and cytotoxicity by means of translational restriction was tested in vitro with the use of the human mammary tumor cells [MCF-7, MDA-MB435, ZR 75-1, MCF-10A and MCF10A cells that over express eIF4E (MCF10A-4E)], as well as primary cultures of human mammary epithelial cells (HMEC) and human mammary stromal cells (HMSC) that express relatively low levels of eIF4E. Real time rt-PCR was used to quantify transcription levels of HSV-Tk for both Ad-HSV-Tk and Ad-HSV-UTk infected cells, as well as the translation initiation factor eIF4E. Translation of HSV-Tk in both Ad-HSV-Tk and Ad-HSV-UTk infected cells was measured by Western blot analysis. In addition, cytotoxicity was determined by assessing cell viability following treatment with the prodrug GCV in Ad-HSV-Tk and Ad-HSV-UTk infected cells with the use of the reductase activity indicator MTT. These data demonstrated enhanced cytotoxicity for the Ad-HSV-UTk /GCV treatment in high eIF4E expressing cells compared to low eIF4E expressing cells, whereas the Ad-HSV-Tk/GCV treatment showed similar cytotoxicity in both high and low eIF4E expressing cells. These results indicate that translational targeting of suicide gene expression in tumor cells in vitro is effective and may provide a platform for enhanced cancer gene therapy specificity in vivo.