The laboratory focuses on signaling in the immune system and study of host processes that HIV-1 exploits. Control of apoptosis, autoimmunity, angiogenesis, retrovirology, and blockade of HIV-1 infection are prominent in our studies.
We use advanced Flow Cytometric analysis (FACS) of phosphoproteins in single cells and dominant effector genetics to achieve many of our goals. For this we have developed a range of FACS assays, cDNA and peptide expression systems using viruses, and single-cell genetic selections, to study pathways of interest to us.
Proteomics: Multiparameter phosphoprotein analysis in single cells by Flow Cytometry and FACS
High complexity retroviral libraries of cDNAs and peptides
HIV-1 Transcriptional Control
Apoptosis: Signaling and Regulation
FLUORETTES, and single cell studies of intracellular signaling
FACS-Gal and associated techniques, published in 1987, are areas in which the laboratory continues an interest for studies of transcriptional events and other events at the single cell level.
New Ways to Detect or Perturb Intracellular Events: The lab is also using genetic selection to develop a new toolset to detect and perturb intracellular events. Kevin Marks and Michael Rozinov have used phage display of peptide libraries to discover short, constrained peptides that bind to fluorescent dyes. Selections of phage-displayed peptide libraries yielded peptides with nanomolar binding to the fluorophore Texas Red These fluorophore-binding peptides (or "fluorettes") were used to target Texas Red inside of mammalian cells. Thus, this short peptide can be used as a tag to monitor proteins in vivo, and it provides proof-of-principle that peptides can be used to target small molecules inside of living cells.
shown that we can target small
molecules to specific proteins
inside of living cells, we are
a creating a generalized system
which uses bifunctional small
molecules: one part of the chemical
binds to the targeting peptide,
and the second part of the small
molecule functions as a sensor
of relevant properties- potentially
ranging from presence of ions
or a specific protein, membrane
localization, pH etc. This modular
system will allow the us to routinely
target a wide variety of chemicals
to any locale or protein within
Autoimmunity: Diabetes and Arthritis
Autoimmunity is a pathological state in which the immune system turns against host tissues. It encompasses diseases such as insulin dependent diabetes mellitus, rheumatoid arthritis, multiple sclerosis and lupus. In collaboration with the laboratories of Garrison Fathman, Ed Engleman and Larry Steinman, we are using our retroviral technologies to deliver dominant negative effectors and cytokines to block autoimmune inflammation.
The T helper response can be considered to have two different arms known as Th1 (pro-inflammatory, promoting cytotoxic T cell activity) and Th2 (non-inflammatory, promoting B cell activity). Although today this is generally considered to be an oversimplistic view, it serves as a framework in which we can consider mechanisms of blocking inflammation. Th1 cytokines (such as interferon (IFN) g and tumour necrosis factor (TNF) a) inhibit Th2 cell activity, whilst Th2 cytokines (such as interleukins (IL) 4, 5 and 10) block Th1 activity.
We are particularly interested in using scFvs against other immune system molecules in an effort to block inappropriate immune responses. In work being carried out by Dr. Richard Smith, targets of current interest include CD3, CD40, CD80 and CD86. We are also starting to use scFv backbones to present intracellular and secreted peptide libraries, in the context of the hypervariable regions, in an effort to identify novel factors involved in antigen presentation, costimulation and T cell activation. Further information relating to our work with scFvs and autoimmunity can be found here.
Gene Transfer/Therapy via Retroviruses: Oncoretrovirus and Lentivirus
The laboratory has developed several rapid, stable high-titre retroviral production systems. Major collaborations include provision of viral systems for IDDM (diabetes) with Dr. Gary Fathman and Edgar Engleman, skin diseases due to genetic deficiency (collaborations with Dr. Paul Khavari), and HIV-1 gene Therapy in a multicenter gene therapy trial involving Stanford, the NIH, University of Michigan, and UCLA.
The cells and vector systems are helper-virus free and useful for production of high-titre retroviruses and lentiviruses. The systems have shown application in delivery of clonal virus and high complexity viral libraries for a variety of therapeutic and research purposes.