Developing a Conceptual Framework for Environmental Health Tracking in Victoria, Australia.

Victoria's (Australia) Environment Protection Authority (EPA), the state's environmental regulator, has recognized the need to develop an Environmental Health Tracking System (EHTS) to better understand environmental health relationships. To facilitate the process of developing an EHTS; a linkage-based conceptual framework was developed to link routinely collected environmental and health data to better understand environmental health relationships. This involved researching and drawing on knowledge from previous similar projects. While several conceptual frameworks have been used to organize data to support the development of an environmental health tracking system, Driving Force-Pressure-State-Exposure-Effect-Action (DPSEEA) was identified as the most broadly applied conceptual framework. Exposure and effects are two important components of DPSEEA, and currently, exposure data are not available for the EHTS. Therefore, DPSEEA was modified to the Driving Force-Pressure-Environmental Condition-Health Impact-Action (DPEHA) conceptual framework for the proposed Victorian EHTS as there is relevant data available for tracking. The potential application of DPEHA for environmental health tracking was demonstrated through case studies. DPEHA will be a useful tool to support the implementation of Victoria's environmental health tracking system for providing timely and scientific evidence for EPA and other decision makers in developing and evaluating policies for protecting public health and the environment in Victoria.

Biodistribution of the RD114/mammalian type D retrovirus receptor, RDR.

BACKGROUND: The limited expression of viral receptors on target cells is a recognized barrier to therapeutic gene transfer. Previous analysis of receptor expression has been performed using indirect methods due to a lack of receptor-specific antibodies. METHODS: In this report we have used anti-RDR antiserum to provide direct histochemical and flow cytometric analysis of the expression of RDR, which is the cognate receptor for RD114-pseudotyped vectors as well as being a neutral amino acid transporter. RESULTS: RDR was present on a range of normal tissues with relevance to gene therapy including: colon, testis, ovary, bone marrow and skeletal muscle. It was also highly expressed on immature cells present in the squamous epithelia of skin, cervix, nasal mucosa, bronchus and tonsil. Of relevance to possible germline gene transfer, we demonstrated a lack of RDR expression on male or female germ cells. RDR expression on mature hemopoietic cell subsets showed up to 5-fold variability between individuals within each lineage-with some individuals expressing low levels of RDR across all blood lineages. Both myeloid and monocytic lineages contained the highest fraction of cells expressing RDR, whereas lymphoid lineages showed the lowest. Coexpression of CD34 and RDR ranged from 2.04 to 0.44% in G-CSF-mobilized peripheral blood samples. CONCLUSIONS: As a means to optimize gene transfer protocols, biodistribution studies such as these are fundamental to enable targeting of the virus receptor most abundantly expressed on relevant populations. The inter-individual variation of receptor expression seen here also raises the possible requirement for tailor-made gene therapy protocols.

Rapid screening for high-titer retroviral packaging cell lines using an in situ fluorescence assay.

The production of high-titer recombinant retrovirus is a major determinant of the efficiency of target cell transduction. Titer assessment for producer clones that contain vectors encoding proteins that can be detected using fluorescence is typically performed by flow cytometry. However, this method is both costly and labor intensive, severely limiting the number of clones that can be screened for each construct. In this report we describe a rapid, high-throughput screening method for viral quantitation of producer clone supernatant on target cells using a 96-well format. Plates were assayed using a multichannel fluorescent reader to determine the percentage of target cells expressing green (EGFP), cyan (ECFP), yellow (EYFP) or red (DsRed) fluorescent reporter genes, or their combinations. The relative fluorescence counts of target cells incubated with viral supernatant from each packaging cell clone correlated with the level of transduction, and hence, viral titer. Correlation of cell fluorescence between the fluorescent plate reader assay and flow cytometric assessment was high (r(2) = 0.96). Independent detection of different fluorescent reporters enabled multiplex assays to be performed. Simultaneous cell density analysis using alamarBlue fluorescence was proportional to cell number per well (r(2) = 1.0). In situ titer assessment of 66 FLYRD packaging cells encoding the EGFP reporter gene identified clones (>10(7) colony forming units per milliliter [CFU/ml]) that provided titers up to sevenfold over the parent population. The application of this rapid, high-throughput screening method overcomes many limitations imposed by the current flow cytometric screening method. This robust assay maximizes the chance of identifying rare high-titer packaging clones and offers a further opportunity to optimize gene transfer protocols.