IC's goal is to develop innovative diagnostic assays to help doctors effectively identify and treat disease.

Diagnosing Graves' Disease

IC is a leader in creating functional bioassays to identify Graves' disease (Primary Hyperthyroidism), an autoimmune thyroid disease.

Most recently, IC worked with Diagnostic Hybrids, Inc. (DHI) to develop the Thyretain TSI Reporter BioAssay. The Thyretain Assay is the first functional bioassay approved by the FDA that specifically identifies thyroid stimulating immunoglobulins (TSI) directed at the thyrotropin (TSH) receptor, which are able to pathologically stimulate the hyperthyroidism of Graves'. Graves' patients have a multiplicity of autoantibodies to the TSH receptor. Many bind but do not stimulate and cause hyperthyroidism. Instead, they can bind and actually inhibit TSI action. Termed TSI Blocking Antibodies (TSBAb), they are implicated in causing hypothyroidism in neonates, Hashimoto's patients, or patients with idiopathic myxedema (primary Hypothyroidism). Another group of TSH receptor autoantibodies bind and inhibit TSH activity, thyrotropin binding Inhibitor immunoglobulins (TBII); they have no known function but can cause hypothyroidism during treatment of Graves' patients if the inhibit normal TSH functional action. The Thyretain assay is unique because it measures only the stimulating antibodies causing the expression of Graves' disease. The Thyretain Assay works by using a chimeric TSH receptor that largely eliminates the functional site for TSBAb and thus can, for the first time, measure stimulating autoantibodies only. Combined with a luciferase reporter gene linked to a luciferase response element in a genetically engineered cell, it can detect cAMP and non cAMP activation signals by non radioactive means in the presence of assay components patented by DHI. The Thyretain assay is a highly sensitive measure of TSI signaling; it is the first assay to offer prognostic significance, to be useful in assessing exophthalmos, and to be useful to follow therapeutic course in patients with oral immunosuppressive drugs. Moreover, it is more convenient to perform than binding assays and is more accurate. The Thyretain Assay in kit form is sold by DHI a division of Quidel. To learn more about or purchase the Thyretain Assay click here.

The second generation of TSI assays developed by IC used genetically engineered cells transfected with the entire human TSHR (hTSHR) plus the luciferase reporter gene. The first generation assay used the FRTL-5 cell line developed by a consortium of researchers making up the Interthyr Research Foundation (IRF). Both had the defect of measuring the sum of TSI and TSBAb activity, thus lowering sensitivity and specificity. Both continue to be useful; the FRTL-5 cell continues, in fact, to be widely used by research laboratories throughout the world since it is a thyroid cell in continuous culture that expresses most thyroid functions. Their use in a broad array of projects is exemplified below:

1) Italy (University of Pisa, Department of Endocrinology and University of Milan, Dept. of Biomedical Sciences) - biochemical response to electromagnetic fields and protein kinase A, respectively.

2) France (Institute of Biology and Technology, Gif sur Yvette) - iodide efflux.

3) Japan (Nagasaki University, Atomic Bomb Disease Institute, School of Biomedical Sciences) thyroid tumorigenesis.

4) Belgium (University Libre de Bruxelles, Department of Interdisciplinary Research) - thyroid cell carcinogenesis.

5) Germany (Berlin, St. Hedwig Hospital and Humboldt University, Division of Endocrinology) - stem cell markers.

6) United States (Albert Einstein College of Medicine, Department of Endocrinology) - intracellular trafficking of thyroid specific genes

7) England (University of Birmingham, Division of Medical Sciences) Viruses, FGF, and goitrogenesis.

A Marker of Vulnerable Plaques in Atherosclerosis

With Drs Douglas Goetz, Mitchell Silver, and Sudhir Deosarkar, work is proceeding on a diagnostic to uncover vulnerable plaques in atherosclerotic arteries.

In spite of the vast research in atherosclerosis, cardiovascular disease (CVD) remains the major cause of death worldwide. The timely diagnosis and therapy of CVD is limited due to the complex nature of CVD and atherosclerosis. Nevertheless, one of the Autoimmune-inflammatory diseases that IC is aggressively pursuing in its long term mission is atherosclerosis. Arguably, the most vexing problems in caring for atherosclerotic patients is determining which patients harbor a vulnerable plaque, i.e. a plaque that at any moment may become compromised, and thus precipitate a clinical event. Atherosclerosis is asymptomatic over an extended period of a patient's lifespan and in most cases it is only manifest through a severe clinical event such as a heart attack or a stroke. In 2006, in the United States alone, CVD accounted for 34.3% of total deaths. Total estimated cost (including direct and indirect costs) of CVD and stroke in the United States was $475.3 billion and $503.2 billion for the years 2009 and 2010 respectively. The product to be developed is an assay that allows the identification of such patients.

Assay 1: In this project, we will develop a quantitative real-time polymerase chain reaction (qPCR) based diagnostic assay to detect inflamed/activated endothelial cells in patients with atherosclerosis.

We hypothesize that the pathophysiological insult in atherosclerosis that causes vascular damage imparts a distinct inflammatory signature to the circulating endothelial cells present in atherosclerotic patient’s blood. We have identified inflamed endothelial cells as indicators of the severity of atherosclerosis and its severe clinical manifestations. If successful, the diagnostic assay developed here will be able to assess the number and the extent of activation of circulating endothelial cells (CECs) in blood samples; depending on the extent of the departure of the number and the activation state of the endothelial cells from the normal, the diagnostic assay may be used to identify high-risk individuals and hence aid in the avoidance of the adverse clinical events. The qPCR assay proposed here, if successful, will improve or aid the current diagnostic methods in identifying high risk atherosclerotic individuals and will improve the likelihood of saving lives through proper therapeutic intervention.

Assay 2: The assay to be developed in this project will use Wnt5a as a biomarker to define and classify the degree of plaque vulnerability

Atherosclerosis is a chronic inflammatory disease that develops in the wall of large and medium arteries. It is characterized by the accumulation of macrophages in the intima followed by plaque development which makes the wall thicker, reduces its lumen and finally triggers adverse vascular or thrombotic events through multiple mechanisms. We hypothesized that Wnt5a could play an important role in the diagnostic identification of vulnerable plaques. And have demonstrated that human atherosclerotic plaques have a different expression pattern of Wnt5a at both protein and mRNA levels in different areas of the arterial wall. We characterized Wnt5a expression in murine and human atherosclerotic lesions. Tissue sections derived from the aortic sinus to the aortic arch of apolipoprotein E deficient mice and sections derived from the carotid arteries of patients undergoing endarterectomy were subjected to immunohistochemical analysis. Wnt5a was detected in macrophage rich regions of both human and murine atherosclerotic plaques Wnt5a protein and mRNA expression were detected in the plaque sections of each subject, however both Wnt5a protein and mRNA were found at very low or non detectable levels in areas of the artery where the lesions look less vulnerable, i.e. arteries with (i) non active inflammation; (ii) thick cap and small lipid core; (iii) no fissured or injured plaque; (iv) non stenotic, non calcified nodules, (v) non hemorrhagic; and (vi) less remodeling. The converse was true where lesions looked more vulnerable. We concluded that the expression of Wnt5a at protein and mRNA levels is higher in areas of the artery where the lesions show advanced stages. These observations suggest that Wnt5a could works as a biomarker to define and classify the degree of plaque vulnerability.

The commercial potential for such a product is enormous. Additionally, such an assay would likely be used multiple times on any given patient thus further magnifying the commercial impact of such an assay. We suggest the market opportunity for the proposed assay exceeds 25-50 million dollars/year. The primary diagnostic tools used in the clinic to determine a patient’s risk of developing CVD include measurement of blood cholesterol levels, blood pressure, and heart rate. Although these tests give an indication of whether the patient may be predisposed to the development of atherosclerosis and possible cardiovascular complications, they are not accurate in determining the state of vascular health (e.g. a vulnerable plaque) and the severe clinical events that follow. Most recently, an enzymelinked immunosorbent assay (ELISA) based test has been developed that measures C-reactive protein (CRP) in patient's serum samples; however its clinical usefulness in detecting vulnerable plaques is untested.