Development
Approaches in development stages include the following.
Transthoracic electroporation
Usefully transfecting lung cells in vivo is difficult. Most techniques, such as using viruses or carrier molecules, either fail to transfect the lungs or induce inflammatory responses that compromise our abilities to further study the transfected lungs. Based on methods pioneered by Dr. David Dean (Northwestern U.), we are transfecting mouse lungs using transthoracic electroporation, in which plasmids containing transgenes of interest are instilled into the lungs and cells become transfected after passing electrical pulses across the closed chest. These techniques can be used to study the regulation of transcription factors (e.g., see J. Immunol. 175:7530, 2005), to identify promoter regions functioning during pneumonia, and to determine biological effects of overexpressing genes of interest.
Polyvalent polymers
Polyvalency is biologically relevant and offers numerous advantages over monovalent interactions between receptors and ligands. With other members of the Nanoscale Science and Engineering Center, in particular Dr. George Whitesides, we are using polymers presenting polyvalent ligands to manipulate innate immune responses in the lungs. One example is a strategy for painting multiple diverse bacteria with an antigen of interest, so that antibodies specific for that antigen can then be used to target those bacteria for phagocytic attack by innate immune cells (e.g., see Biomaterials 27:3663, 2006). This approach was featured in a radio broadcast on the Morning Edition show.
Proteomics
There are ~1500 transcription factors encoded in the genomes of humans (or mice). We are developing proteomics techniques to identify transcription factors and other regulatory proteins activated during pneumonia in an unbiased (or at least differently biased) fashion. These studies are in collaboration with Dr. Dieter Wolf and the Harvard NIEHS Proteomics facility, and they have led to the identification of a novel previously uncharacterized protein that has become an ongoing research effort of ours.
Epifluoresecent view of a mouse lung transfected with a gene for enhanced green fluorescent protein using transthoracic electroporation. Transfected cells can be visualized as glowing green through the pleural surface. Untransfected lungs or lungs transfected with other genes show no green fluorescence.
