Novel Methodologies Year 3 Awardees

Mark Sullivan PhD and Iñaki Sanz MD

Project: Novel Platform for Biomarker Discovery in Autoimmune Disease

Investigators:

Brief Description of Project: At the end of this project, we expect that we will have demonstrated the ability to detect disease-specific immune responses associated with rheumatoid arthritis. A rapid, convenient immunoassay for specific auto-antibodies should facilitate identification of optimum treatment options for patients. This novel methodology can be applied to essentially any disease where there is an immune response to disease-related structures (proteins, aberrant degradation products, cell structures, unusual modifications, etc.) in addition to the autoimmune diseases we are targeting for initial validation. Potential applications include neurodegenerative diseases (Parkinson’s and Alzheimer’s), cancer, pre-eclampsia, and type I diabetes, where anti-GAD65 antibodies and an anti-idiotypic antibody network appears important in disease progression.

David Herrmann MD

Project: In-vivo Reflectance Confocal Microscopy as a Biomarker for Peripheral Neuropathy Clinical Trials

Investigators: J. Forrester M.S, Director IT, Dept. of Neurology, and Dr. J. Huang, Dept. of Neurosurgery, U of R.

Brief Description of Project: Reflectance confocal microscopy (RCM) imaging of Meissner corpuscles would have wide application as a rapid, painless, non-invasive, reiterative outcome measure/biomarker for peripheral neuropathy trials of various types that could obviate the need for biopsy in some circumstances. As noted there are existing research programs at U of R, involving clinical trials in diabetic, HIV and hereditary neuropathies that would be advanced by development of new biomarkers. Utility would also extend to clinical and pre-clinical investigators in Neurosurgery and Orthopedics who require objective, reiterative biomarkers for sensory nerve regeneration and cutaneous reinnervation to advance research aimed at improving surgical and other neuroregenerative therapies for focal nerve injuries. MCs are of interest to investigators studying sensory input and gait and balance disorders (including related to aging), and to investigators in occupational medicine where use of vibrating power tools/platforms is hypothesized to injure cutaneous sensory receptors including the MC.

Lianping Xing PhD and Ronald Wood PhD

Project: Development of in vivo Near Infrared Imaging Technology for Longitudinal Assessment of Lymphatic Draining Function

Although we will use Indocyanine green (ICG) near-infrared (NIR) imaging to examine lymphatic function in TNF-Tg arthritic mice, successful assessment of lymphatic transport will certainly have many other applications due to the great importance of lymphatic function in many human diseases. Several immediate benefits include 1) The PI (Dr. Xing) will use this method to generate preliminary data for her R01 application to study the role of lymphatics in pathogenesis of RA next year; 2) Dr. Schwarz will use this method to study the role of LN blockage on arthritis flare and will resubmit his A1-R01 grant; 3) Dr. Jorge Yao is interested in enhancing the detection of LNs from clinical specimens by injecting ICG near tumor sites; and 4) Dr. Ritchlin (MD, Rheumatology) is interested in investigating if impaired lymphatic flow is associated with the size of draining LNs and with arthritic flare in RA patients and if anti-TNF therapy improves lymphatic flow and LN sizes. ICG-NIR imaging can be used to assess lymphatic flow in RA patients along with MRI and Doppler ultrasound to measure LN sizes. Apart from these immediate applications, ICG-NIR lymphatic imaging can be used in other clinical settings: 1) to visualize LNs with cancer cell metastasis in animal models and cancer patients, for which there is a growing medical literature and intellectual property protection efforts; 2) to assess lymphatic drainage in disorders of lymph transport, such as surgery induced lymphedema in breast cancer patients. Another advantage of ICG-NIR is that intravenous ICG has been administered routinely to patients for several decades [5], thus safety will not be an issue inhibiting translation of these methods from the bench to the clinic.

Novel Methodologies Year 2 Awardees

Robert Block MD, MPH and Steve Georas MD

Project: Potent Lipid Mediator Measurement Methodology
Investigators: Robert C. Block, MD, MPH, Steve N. Georas, MD, Alan Friedman, PhD, (University of Rochester) and J. Thomas Brenna, PhD (Cornell University)
Brief Description of Project: This project will develop new assays for a variety of potent lipid mediators in a variety of human diseases, most notably (at first) heart disease and asthma. The accurate measurement and analysis of complex lipids is emerging as an important field in biomedical science. Although still in its infancy compared to other analytical disciplines, "lipidomics" is gaining momentum. For example, the NIH funded Lipid Metabolites And Pathways Strategy (LIPID MAPS) is providing an unprecedented picture of cellular lipid metabolism, cataloging several thousand structures (www.lipidmaps.org). Lipids are increasingly recognized for their role as potent mediators in the resolution of inflammation, cell growth, signal transduction, tissue protection and homeostasis.

Two examples at SMD: (1) Dr. Block is investigating the role of the "resolvin and protectin" family of lipids in patients with heart disease, testing the hypothesis that commonly used medications in patients with coronary artery disease, such as statins and aspirin, exert beneficial effects by generating the production of these lipid mediators; (2) Dr. Georas is exploring the role of the lipid mediator lysophosphatidic acid and its receptors in asthma. This compound is detectable in bronchoalveolar lavage fluids and may contribute to the resolution of allergic inflammation. Although other "-omic" technologies are well established at SMD, there is an absence of local expertise in measuring complex lipids. The goal of this proposal is to help establish this technology on-site.

Frances Eun-Hyung Lee MD and Marc Hellerstein MD, PhD

Project: Using Heavy Water in vivo Labeling to Measure Human Proliferation and Die-Away Kinetics-Development of the Core Facility at the University of Rochester
Investigators: Frances Eun-Hyung Lee, MD, Marc Hellerstein, PhD, Hulin Wu, PhD, Ignacio Sanz,MD, Tim Mosmann, PhD
Brief Description of Project: This project will establish a "human in vivo labeling" core facility at SMD that will be accessed by investigators in a diversity of disciplines such as Cancer biology, Microbiology, Immunology, Medicine, and Biodefense. Heavy water (2H20) or glucose (2H-glucose) labeling of DNA is the only safe and direct human measure of cell kinetics in vivo. Kinetics of rare immune populations has been difficult to study, however, due to extremely low frequencies. In this proposal, protocols will be developed for in vivo labeling, focusing particularly on techniques for studying extremely low frequency immune cell populations (approximately 50,000-100,000 cells). As an initial example, the immune kinetics of VH4-34 B cells, a marker of B cell tolerance, will be measured in vivo in patients with Systemic Lupus Erythematosis (SLE) and in healthy control subjects. This study will be the first to measure in vivo proliferation and die-away rates of these low frequency immune cells as a biomarker of autoimmune disease. This core facility will be instrumental in securing future NIH funding for many UR investigators.

Novel Metholdologies Year 1 Awardees

Manish Kohli MD and Benjamin Miller PhD

Project: Arrayed Imaging Reflectometry-Based Detection of Circulatory Peptides in Prostate Cancer
Investigators: Manish Kohli, MD; Benjamin L. Miller, PhD.
Brief Description of Project: This proposal represents a collaborative effort to develop a novel diagnostic system fordetecting and quantitatingcandidate pathway-specific biomarker proteins in prostate cancer. Drs. Kohli and Miller built on two unique resources developed in their laboratories: (1) a new chip-based diagnostic platform (Arrayed ImagingReflectometry, or AIR) that can quantitatively detect concentrations of proteins with a sensitivity comparable to ELISA, but with significantly greater speed and lower cost; and (2) a bank of high quality blood and urine specimens collected from prostate cancer patients for the specific purpose of biomarker development. At presentthis on-going repository has collected over 350 samples ofplasma and urine specimens on 200 unique subjects in four groups of prostate disease that are readily available for biomarker testing. The four research groups include (i) a control group of subjects with elevated PSA and without cancer; (ii) subjects diagnosed with local stage disease; (iii) subjects with rising PSA after primary prostatic therapy and (iv) patients with progressive disease during androgen deprivation therapy. Serial sampling is performed once every three to four months in all four groups, and serum/plasma from blood along with urine is stored at -80 °C.

AIR is a biosensor platform that enables the detection of any probe/target system by optically sensing molecular binding on a silicon chip. As a label-free technique, the assay procedure can be completed in less than 30 minutes, with nearly instantaneous readout. Comparable labeled techniques such as ELISA requires processing time of more than 2 hours. The AIR method is also amenable to multiplexing, potentially allowing 100s of different probes to be arrayed on the chip surface for simultaneous detection. In this first phase of biomarker development, Drs. Kohli and Miller will focus on the production of a chip capable of detecting several candidate angiogenesis and bone biochemistry biomarker proteins in patients with prostate cancer and controls, using specimens from the clinical groupings described above.