Research Summaries - Medical & Surgical - Part I (Internal Medicine Dept.)

General Internal Medicine

1. Senior Research Staff

John Popovich, M.D., Chair

Keoki Williams, M.D.

2.  Research Summaries

Principal Investigator:  Williams, Keoki, M.D.
Title:  Mechanism of Endotoxin's Effect on Allergy Risk (NIH R01 AI06177401)

A number of studies suggest that early exposure to endotoxin is associated with a lower risk of asthma and allergies. The overall objective of this research is to understand the mechanism of that effect. This includes 1) clarifying whether endotoxin mediates its effect through the binding and activation of Toll-like receptors (TLRs), and 2) determining endotoxin's role in T-cell differentiation. Toll-like receptor (TLR)-2, TLR-4, and CD 14 are known to bind endotoxin and to mediate its stimulation of the innate immune system. What is not known is whether this pathway also mediates endotoxin's effect on allergy. As type-2 helper T-cells (Th2) appear to be responsible for allergic expression, endotoxin's probable downstream effect is suppression of this T-cell phenotype. Since the TLR family recognizes a broad range of infectious organisms, clarifying this mechanism has obvious implications for long-standing, but unproven, "Hygiene Hypothesis," which posits a protective association between early infections and allergic development. The research proposed here will take advantage of a large, prospective, multi-ethnic, birth cohort which began in the Detroit area in September 2003. To achieve the first objective, endotoxin levels will be measured in the home in the first year of life and related to subsequent serologic and skin prick measures of atopy. Single nucleotide polymorphisms (SNPs) known to cause alterations in either the expression or the function of CD14, TLR-2, and TLR-4 will be studied. If the TLR pathway mediates endotoxin's effect on allergy, children with these polymorphisms should display an altered or diminished response to endotoxin. The second objective will determine whether endotoxin exposure results in skewing of the helper T-cell phenotype (i.e., Thl vs. Th2) or whether it results in T-cell anergy. To achieve this objective, intracellular cytokine production in CD4+ lymphocytes will be prospectively measured. Interleukin (IL)-4, IL-10, and interferon-3, will be measured in cord blood, at 6 months, 1 year, and 2 years of age. As CD4+/CD25+ regulatory T-cells appear to be responsible for anergy, changes in this group of cells over time will be related to the level of endotoxin exposure. It is the hoped that understanding the mechanism of endotoxin's effect will establish new targets for the prevention of allergies.

Principal Investigator: Williams, Keoki, M.D.
Title:  Using Information Technology to Improve Asthma Adherence (R01 HL079055)

It has been estimated that regular use of inhaled corticosteroids (ICS) could reduce asthma-related hospitalization by 80%. Inhaled corticosteroids can significantly improve lung function, as well as prevent asthma exacerbations and death. Yet, ICS use remains low among patients with asthma, and studies measuring ICS adherence suggest that patients take their prescribed dose on only 20% to 73% of days. Together these findings suggest that increase adherence to ICS could result in significant improvements in asthma-related outcomes. Unfortunately, few behavioral or educational interventions have been shown to be very effective at improving adherence and asthma control, despite in some cases considerable time invested with the patient. Given the importance of adherence information to clinical decision making and disease management, it is surprising that not until recently have there been studies looking at the effects of providing this information to physicians. However, even the limited evidence from these small studies suggests that simply providing adherence information to clinicians can have a profound influence on patient adherence. One challenge is how to provide adherence information in the clinic setting where time and resources are often limited. Weighing canisters and electronic recording devices have been used to measure adherence to ICS, but these methods are too time consuming and expensive to be used by most clinicians. Medication adherence can also be estimated using pharmacy claims data. Since claims data are collected for other reasons, these pharmacy-based adherence measures are relatively inexpensive to generate and they are available on a large number of patients. This application seeks to develop and test a tool, which feeds back adherence information to physicians via the electronic medical record. These adherence measures will be generated by linking prescription data to pharmacy claims. This method has the potential benefit of being both easily use in clinical practice and financially sustainable. After a baseline period, primary care physicians will be randomized to receive ICS adherence information on their patients with persistent asthma. The primary outcomes will be the change in adherence from baseline and the difference in adherence between treatment groups. Also assessed will be changes from baseline and differences between the treatment and control groups in patient-clinician communication, patient attitudes toward adherence, outcomes related to disease control, and medical care costs.

1. Senior Research Staff

Michael Eichenhorn, M.D., Division Head
Edward Zoratti, M.D.

2. Research Summaries

Principal Investigator: Zoratti, Edward, M.D.
Etiology of Pediatric Atopy NIH R01 A1051598)

Our objective is to study the relationship of early-life cat and dog exposure to potential persistent effects on T-cell cytokine profiles, biologic markers of atopy, and clinical atopy among a well-characterized birth cohort (n=835), the Detroit area Childhood Allergy Study (CAS), as they reach 18 years of age. Extensive early-life environmental exposure data have been collected on this cohort. Outcomes will include total IgE, allergen-specific IgE and IgG4 levels, intracellular IL-4, IL-10 and interferon (IFN)-γ in mitogen stimulated T-cells and current or history of allergic rhinitis and asthma. Hypotheses: Compared to subjects with low levels of exposure, those with high-level exposure to dogs and cats in the first year of life will exhibit: i) a persistently altered immune response at age 17, typified by a low IL-4/ IFN-γ ratio and elevated IL-10 secretion in response to T-cell mitogenic stimulation; ii) reduced levels of allergen-specific IgE and increased IgG4 to common aeroallergens; and, iii) a persistently reduced risk for allergic rhinitis and asthma. The specific aims include analyzing the number of indoor pets during the first year of life, adjusting for well-established confounders and effect modifiers, for persistent effects on: i) T-cell intracellular cytokine levels of IL-4, IFN-γ and IL-10; ii) serum levels of allergen-specific IgE (cat, dog, dust mite, ragweed, grass and alternaria) and allergen-specific IgG4 (cat, dog, grass and dust mite); iii) current and lifetime history of self-reported and doctor diagnosed allergic rhinitis and asthma. We will also iv) study the relationship between these outcomes and v) evaluate the role of biomarkers and history of atopy in each parent in the above analyses. Methods: The CAS cohort will be followed-up by questionnaire and a home visit at age 18 years. Stimulated T cell cytokine analysis, total IgE and serum allergen-specific IgE and IgG4 levels will be determined and cohort subjects and parents will answer a questionnaire regarding subject and parental history of symptoms and diagnosis of allergic rhinitis and asthma and post early life pet exposure. Blood specimens from parents and subjects will be placed in a biorepository for future molecular epidemiology studies.

1. Senior Research Staff

2. Research Summaries

Principal Investigator: Keteyian, Steven J., Ph.D.
HF-ACTION (Heart Failure - A Controlled Trial Investigating Outcomes of Exercise Training) (NHLBI U01 HL64250)

Despite recent advances and many different therapies to improve longevity and quality of life in patients with heart failure, the prevalence of this disorder continues to rise. One novel strategy now being investigated at Henry Ford Health System is whether regular exercise training improves survival and decreases re-hospitalizations in these patients. To assess the potential benefits of exercise training, HF-ACTION is a controlled trial offered at over 60 centers in North America -- one of which is the Henry Ford Heart & Vascular Institute. In this trial 3,000 patients will be randomly assigned to either, a) usual care involving the medications routinely used in patients with heart failure or, b) usual care plus a program of exercise four to six times per week. The objective of the study is to determine if regular exercise lowers all-cause fatalities and hospitalizations. This study will also assess whether regular exercise impacts on exercise capacity, emergency room visits, quality of life, economic costs and other biological parameters. Recruitment for this study began in April 2003, and Henry Ford was the second site in North America to enroll a patient. After one year of enrollment, 33 patients had been enrolled, which ranked 5^th when compared to all other participating sites.

The scope of work is;

1. Senior Research Staff

Robert Chapman, M.D., Division Head and Director, JFCC
Frederick Valeriote, Ph.D.

2. Research Summaries

Principal Investigator: Chapman, Robert, M.D.
Cancer Prevention and Treatment Demonstration for Ethnic and Racial Minorities (CPTD) (CMS/DHHS IAO 30006810)

This CMS sponsored demonstration project will show the racial disparity in cancer screening, diagnosis, and treatment between African American and Caucasian Medicare recipients in Southeast Michigan can be substantially and significantly reduced.  We are located in a large geographic community with a large African American community (over 80% of the population of Detroit).  The proportion of Detroit residents existing below the poverty line is more than double that in the nation or the state of Michigan.  Corporate data stores indicate that roughly 13,000 African American Medicare recipients receive their care at the Henry Ford Health System (HFHS).  Our Information Technology Systems allow us to readily create a database of those target individuals who elect to enroll in the project.  This database will be enriched by the individuals referred from our partnering organizations (AARP, Adult Well-Being Services, and our Faith Based Initiative).  The data on disparities in stage at diagnosis, along with other data from our Henry Ford linking disparities in stage at diagnosis to disparities in outcomes, strongly suggest the need for additional effort made on enhancing cancer screening rates.  Although some screening tests may not be recorded in the administrative data systems at HFHS, we believe that the cancer screening rates are well below achievable levels in our over-65 population and that there is a clear opportunity for improvement.  The HFHS patient data base indicates very poor utilization of our screening services and presentation of our African American patients with more advanced stages of cancer than their Caucasian counterparts.  Other published studies from HFHS have documented disparities in treatment patters and outcomes for patients with lung cancer or breast cancer.  These disparities have been shown to be related to clinical factors like comorbidities or obesity, pointing the way to identification of patients at higher risk for suboptimal treatment, and suggesting opportunities for provider-directed as well as patient-directed interventions.

Principal Investigator: Valeriote, Frederick, Ph.D.
Discovery of Anticancer Drugs from Cyanobacteria (NIH R01 CA10085)

The overall goal of this project is the discovery and development of new anticancer agents with solid tumor selectivity from leads obtained from marine cyanobacteria. The need for new anticancer drugs is significant given the paucity of agents active against the major solid tumors of man. An underlying hypothesis of our screening strategy is that it will generate drugs active against the major solid tumors (such as lung and colon), which are not effectively treated at present. Marine cyanobacteria are abundant as both free-living and symbiotic tropical organisms, and have a correspondingly rich and diverse secondary metabolism. We propose to produce between 1000 and 1500 extracts per year from field collected and cultured tropical marine microalgae, mainly cyanobacteria, with a focus on those of low natural biomass or found in symbiosis with marine invertebrates, such as sponges and tunicates and to characterize "super-producing" marine cyanobacterial strains. Extracts will also be obtained from collections of tuft-forming marine cyanobacteria and planktonic/thin slime forming marine cyanobacteria for culture as well as cultured cyanobacteria isolated from invertebrate hosts under natural product-eliciting conditions. We will use a unique in vitro disk diffusion assay to both identify solid tumor selectivity in the extracts and to direct the isolation of putative anticancer agent. Drug structure will be determined by using and developing innovative NMR pulse sequences and integrating this with MS and other spectroscopic information. If necessary; we will scale-up the culture or recollect selective species to provide sufficient drug to advance to preclinical studies. The first step requires about 20 mg of drug and incorporates information from in vitro concentration-survival clonogenic studies on a solid tumor with pharmacokinetic information (serum and tumor drug levels). The drug is first formulated for intravenous administration and an HPLC assay is developed to monitor serum and tissue levels. The clonogenic/pharmacokinetic information is analyzed to determine whether the more expensive in vivo therapeutic trial should be undertaken. If positive, then an efficacy trial in tumor-bearing mice will be carried out in at least one xenograft model. Therapeutically active drugs will be pursued outside of this application.

Principal Investigator: Valeriote, Frederick, Ph.D.
Discover & Development of Anticancer Agents from Plants (NIH R01 CA092143)

The goal of this project is the discovery and development of new anticancer agents with solid tumor activity from leads obtained from plants. The need for this is significant given the paucity of anticancer agents active against the major solid tumors of man. During the past 5 years, nearly 4000 crude plant extracts, from the laboratory of Dr. Joseph Hoffmann at the University of Arizona, have been evaluated for solid tumor selectivity by Dr. Fred Valeriote, now at the Josephine Ford Cancer Center. The results of these studies have provided this project with 30 prioritized, primary hits to continue with isolation (through bioassay-directed fractionation) and identification of the active compound(s) from these plants over the next 5 years In order to accomplish the objectives, bulk collections will be made as needed or plants will be grown in experimental field plots to provide sufficient material for expanded testing both in vitro and in vivo. Additional material will be generated through aeroponic/hydroponic systems, especially when root material is the primary source of the active compound(s). Also, 200 new plants will be collected each year with the new and old leads prioritized annually. Six priority plants will be targeted for completion each year to generate at least 6 active compounds for both in vitro and in vivo testing. Greater emphasis will be placed on novel compounds over known compounds with either new or known anticancer activity. The anticancer drug development component will be carried out at the Josephine Ford Cancer Center. The pure compound moves into formulation, pharmacology and therapeutic assessment. There are three essential components to in vivo pharmacology: testing development of analytical methods for chemical analysis of the new compounds, development of a suitable IV formulation through biopharmaceutical measures, and preliminary pharmacology testing for drug clearance and distribution. Doses and target drug concentrations will be established in vitro. Mice will bear bilateral tumors that have grown to 100 to 300 mm3 prior to initiation of dosing. Doses of each agent contained in 0.2 ml volume for parenteral administration will be estimated from traditional dosage demands and dosages adjusted for in vitro pharmacology results. The methodology for the study on therapeutic efficacy to test doses of compound, up to the maximum tolerated dose, in tumor bearing mice (syngeneic with a murine tumor or xenografted wit).

The overall goal of this program is to discover new types of anticancer agents from sponges and sponge-associated fungi that will be effective against solid tumors of man.  The work that will be carried out in the laboratory of Dr. Caleriote is to: 1) test 1000 extracts per year from sponges and sponge-associated fungi obtained from Pacific collections of Dr. Crews using in vitro disk diffusion assay in the laboratory of Dr. Valeriote to first identify extracts with solid tumor selectivity.  Those extracts with solid tumor selectivity (about 2% or 20 per year) will be fractionated. 2) To carry out pharmacokinetic studies in order to assess all compounds.  3) To carry out in vivo therapeutic studies that are integrated with the biological and pharmacological studies.

1. Senior Research Staff

2. Research Summaries

Principal Investigator: Beierwaltes, William, Ph.D.
Renin Regulation by Nitric Oxide. (NHLBI R01 HL076469)

The renin-angiotensin system produces the potent vasoconstrictor angiotensin II, which is involved in regulation of vascular resistance, blood pressure, and virtually every form of hypertension. In the kidney, the vasoconstrictor effects of angiotensin are buffered by the vasodilator endothelium-derived nitric oxide (NO). Renin is the rate-limiting enzymatic step in angiotensin formation. We propose that NO both counteracts angiotensin-induced vasoconstriction and regulates its formation by differentially regulating renin secretion. NO has been reported to both inhibit and stimulate renin secretion, but it is unclear how these different effects could occur. While cAMP is the cyclic nucleotide which stimulates renin secretion, we hypothesize that cGMP, the second messenger of NO, inhibits renin release from JG cells with basal cAMP levels by activating protein kinase GII (PKGII) and increasing intracellular calcium. In contrast, when cAMP levels are elevated by secretagogues such as prostaglandin (PG)E₂, cGMP stimulates renin release indirectly by inhibiting phosphodiesterase (PDE)-3 and exaggerating its effects by reducing cAMP metabolism. In aim 1, we propose that with basal cAMP levels, NO inhibits renin release by stimulating cGMP production and increasing PKGII activity in the JG cells. We will study the effects of NO and cGMP on renin without stimulating cAMP both in vitro (primary cultures of isolated JG cells and isolated glomeruli) and in vivo using eNOS knockout mice. In aim 2, we propose that if cAMP levels are elevated NO enhances renin release by cGMP inhibition of PDE-3 and potentiation of cAMP-mediated renin stimulation. We will study cAMP and macula densa stimulation of renin, manipulating PDEs that break down either cGMP or cAMP, using primary JG cultures, isolated glomeruli (without the macula densa) and renal cortical slices (with the macula densa). We will use nNOS knockout mice to see if NO from the macula densa mediates renin stimulation. In aim 3 we propose that prostanoids, particularly PGE₂ produced by COX-2 in the macula densa, regulate cAMP levels in JG cells. Co-expression of COX-2 and nNOS in the macula densa suggests NO's effect on renin depends on COX-2 expression and activity. We will manipulate COX-2 expression and PG synthesis in the macula densa and study the effects of NO and cGMP, using renal cortical slices and in vivo experiments stimulating macula densa-mediated renin secretion in rats and nNOS knockout mice. These studies should reveal the dual pathways by which NO can either inhibit or stimulate renin secretion.

Principal Investigator: Carretero, Oscar A., M.D.
PPG: Vasoactive Autocoids in Blood Pressure Regulation (NIH P01 HL028982)

This PPG was started in September, 1982. The central theme is "the study of the role of vasoactive systems (autocrine, juxtacrine, paracrine and endocrine) in the regulation of renal function and blood pressure (BP) and mediation of target organ damage". The general hypothesis to be tested is that there is a balance between systems that promote water and sodium retention, hypertension and target organ damage (Ang II, COX-2 products and free radicals), and systems that antagonize these effects (kinins, NO, Ac-SDKP and activation of the Ang II type 2 receptor). Alterations of this balance in favor of the former are responsible for the development of hypertension and target organ damage, while alterations of this balance in favor of the latter have therapeutic effects. We will use molecular, physiological, and pharmacological approaches to study vasoactive systems at the subcellular, cellular, and isolated organ levels and in intact animals in both acute and chronic models, including transgenic mice. In project 1 we will study whether a novel peptide (Ac-SDKP) alters the balance between systems that promote and oppose target organ damage in favor of the latter, thus preventing and regressing this process. In project 2 we will study whether the local effects of Ang II in the heart are antagonized by activation of the AT2 receptor, kinins and NO. In project 3 we will study whether COX-2 via EP1 and EP3 receptors promotes the development of cardiovascular disease and whether this effect is antagonized by the PPAR receptor. In project 4 we will study the regulation of renal microcirculation by the juxtaglomerular apparatus to see whether there is an interplay between vasodepressor autacoids (NO, kinins and vasodilator eicosanoids) and vasopressor systems (Ang II, reactive oxygen species and cP450 vasoconstrictor metabolites). In project 5 we will study whether NO produced by eNOS in the renal tubules alters the water and sodium balance in favor of natriuresis and diuresis, thus opposing hypertensive stimuli. Four core units (Administrative, Analytical and Morphology, Mutant Mouse, Biostatistics) will support and facilitate the scientific efforts of the investigators. Special expertise is centralized in the cores so that resources can be used more efficiently. The Program Project provides integration of our efforts, continuing collaboration and sharing of ideas and expertise; thus it accelerates acquisition of knowledge on the pathogenesis of hypertension and target organ damage.

Principal Investigator: Carretero, Oscar A., M.D.
PPG Project I: Role of Ac-SDKP in Target Organ Damage in Hypertension (NIH P01 HL028982)

Hypertension is a cardiovascular risk factor that often leads to target organ damage. Angiotensin-converting enzyme inhibitors (ACEi) significantly reduce cardiovascular events in high-risk patients. Although there is significant evidence that the effects of ACEi are mediated by inhibition of both the conversion of angiotensin I to II and kinin hydrolysis, we have data showing that in hypertension, another peptide hydrolyzed by ACE, acetyl-seryl-aspertyl-lysyl-proline (Ac-SDKP) prevents and reverses cardiac fibrosis without altering blood pressure or cardiac hypertrophy. In a model of heart failure due to myocardial infarction we found that Ac-SDKP acts as an anti-inflammatory cytokine, preventing and reversing cardiac fibrosis in the non-infarcted area (reactive fibrosis). Following Ac-SDKP treatment, macrophage infiltration was significantly reduced, along with a decrease in the number of TGF-ß-positive cells. We also measured cardiac performance in vivo in adult spontaneously hypertensive rats (SHR) using a pressure-volume conductance catheter system to evaluate systolic and diastolic function at different preloads. We found that at 10 to 11 months of age, before heart failure developed, SHR exhibited increased systolic performance and diastolic stiffness accompanied by delayed relaxation in comparison with Wistar-Kyoto (WKY) controls. When SHR were treated with Ac-SDKP, cardiac fibrosis was normalized but diastolic stiffness was not changed and systolic performance was slightly decreased, suggesting that in order to improve cardiac function it is necessary to coordinate decreased cardiomyocyte size and fibrosis. We have evidence that blocking Ac-SDKP prevents some of the effects of ACEi on cardiac and renal fibrosis. We also studied the effect of a specific aldosterone antagonist, eplerenone, on the progression of heart failure and found that its beneficial effect was equivalent to that of ACEi. In collaboration with the Department of Radiation Oncology, we recently found that ACEi reduce radiation injury to the optic nerve; thus it may be possible to develop a pharmacological approach to reduce injury to normal tissue in patients undergoing radiation therapy. We feel these results are relevant because they give important new information about the mechanism by which target organ damage develops in hypertension, and how drugs used to treat such damage work. In the future, it may be possible to develop nonpeptidic analogues of Ac-SDKP to treat fibrosis in the elderly or in patients with arteriosclerosis or diabetes.

Principal Investigator: Carretero, Oscar A., M.D.
PPG Project IV: Autocrine/Paracrine Regulation of the Renal Microcirculation (NIH P01 HL028982)

In hypertension, regardless of its cause, increased renal vascular resistance shifts the pressure natriuresis curve to the right. The afferent (Af-) and efferent arteriole (Ef-Art) account for most renal vascular resistance, controlling glomerular filtration rate (GFR) and peritubular pressure and consequently renal function. Af/Ef-Art resistance is regulated by a balance between vasopressor and vasodepressor mechanisms and by tubuloglomerular feedback (TGF) via the macula densa, which senses chloride, sodium and/or solute load and sends a paracrine signal that controls Af/Ef-Art resistance. We recently described a new mechanism of Ef-Art control in which the glomeruli release autacoids when stimulated by various hormones, and demonstrated that the glomeruli produce cyclooxygenase (COX) metabolites (prostaglandins) and CP450 metabolites (epoxyeicosatrienoic acids or EETs) as well as nitric oxide (NO) which have a vasodilator effect. However, they also produce a CP450 metabolite with a vasoconstrictor effect, 20-hydroxyeicosatetraenoic acid (20-HETE), so that the circulation downstream from the glomeruli is regulated not only by autacoids produced by the Ef-Art but also by autacoids produced by the glomeruli themselves. We also studied the mechanism by which the macula densa acting via TGF regulates the renal microcirculation. We found that increased intracellular Ca at the macula densa is required for the TGF response, and that in the macula densa the apical Na/H exchanger participates in activation of neuronal NO synthase (nNOS), probably by increasing intracellular p H, and this increase in nNOS activity results in release of NO which in turn inhibits TGF. These studies are important because they may elucidate the feedback mechanism that prevents an extreme TGF response and consequently a decrease in GFR. Understanding the mechanism that regulates the renal microcirculation is important not only for understanding how various autacoids participate in the regulation of renal function but also the development of hypertension.

Principal Investigator: Garvin, Jeffrey L., Ph.D.
PPG Project V: NO Produced by Tubular eNOS Regulates Renal Function (NIH HL 028982 and American Heart Association Fellowship)

We are studying the role of a chemical called nitric oxide in the regulation of salt absorption by the thick ascending limb. Nitric oxide is important in regulating many aspects of blood pressure and is the active molecule in drugs such as nitroglycerin and amyl nitrate. We were the first to show that nitric oxide reduces salt absorption in this segment by inhibiting the activity of a protein that allows salt to enter the cell. This protein is also inhibited by loop diuretics. More recently we have found that nitric oxide not only regulates salt absorption, but is produced in the thick ascending limb by an enzyme called nitric oxide synthase. Thus, this segment is capable of regulating its own salt absorption. A high-salt diet increases the amount of nitric oxide synthase and enhances the ability of nitric oxide to reduce salt absorption. Consequently we hypothesize that a defect in this process may lead to salt-sensitive hypertension. This hypothesis appears to be borne out by a genetic rodent model of salt-sensitive hypertension. Currently we are investigating how the amount and activity of nitric oxide synthase are controlled. In these studies we are using state of the art techniques including confocal microscopy and gene transfer technology that was developed in my laboratory. We have been able to specifically transfer the nitric oxide synthase gene in the thick ascending limb into mice that lack this protein. The development of this technology will allow us to specifically test the role of this protein in the regulation of salt and water excretion by the kidney, and blood pressure.

Principal Investigator: Garvin, Jeffrey L., Ph.D.
Salt-Sensitive Hypertension: Role of Renal Superoxide (NIH/NHLBI 2R01 HL70985-05 and National Kidney Foundation Fellowship)

In our second project we are studying the role of reactive oxygen species or oxidants in the regulation of salt absorption by the thick ascending limb. All cells produce oxidants, but when production is unchecked serious consequences occur. Oxidants have been implicated in many pathological states from atherosclerosis to hypertension. In the kidney, oxidants increase salt and water retention, promoting hypertension. In the thick ascending limb of the kidney, we are investigating how oxidants increase salt absorption. To date we have found that oxidants work in two ways. First, they reduce the amount of nitric oxide. Thus they reduce the levels of a chemical that inhibits salt absorption. Secondly, and perhaps more importantly, they stimulate the proteins involved in salt absorption. Oxidants do not act directly, but rather through a series of other small molecules and proteins that form so-called signaling cascades. In the thick ascending limb, oxidants activate the protein kinase C signaling cascade, named for the final mediator of cellular events in this pathway, protein kinase C. Protein kinase C chemically alters the proteins that allow salt to enter the cell and thereby enhances their activity. Because oxidants increase salt retention by the thick ascending limb, we are now studying whether they also cause hypertension.

Principal Investigator: LaPointe, Margot C., Ph.D.
PPG Project III: Role of COX-2 Products in Cardiac Hypertrophy and Fibrosis (NIH P01 HL028982)

The pro-inflammatory mediator cyclooxygenase-2 (COX-2) is upregulated in the myocardium following ischemia, infarction (MI) and heart failure (HF). Recent studies have implicated COX-2 in the pathophysiology of HF, but the underlying mechanisms are virtually unexplored. In this proposal we are studying the role of COX-2 and its prostanoid product PGE2 in cardiac hypertrophy and fibrosis. To study the role of COX-2 in vivo, we are using a mouse model of myocardial infarction (MI), while our in vitro experiments involve cultured cardiac myocytes and fibroblasts. We are focusing on regulation of COX-2 and PGE2 synthesis, identification of PGE2 receptors, the signaling mechanisms activated by PGE2 in both cardiac myocytes and fibroblasts, and the anti-inflammatory mechanisms by which the nuclear receptor PPAR? inhibits COX-2 and PGE2 synthesis. We found that COX-2 is induced in the heart after MI, resulting in enhanced PGE2 synthesis. Blocking COX-2 with 2 different inhibitors (NS-398 and rofecoxib) improved cardiac function and decreased hypertrophy and fibrosis. The reduction in fibrosis was likely the result of decreased synthesis of a growth factor (TGF-ß) known to regulate extracellular matrix protein synthesis (e.g., collagen synthesis). To understand the mechanisms by which PGE2 regulates hypertrophy, we studied its effects on protein synthesis in cultured cardiac myocytes. We found that PGE2 binds to a receptor that stimulates a signaling cascade causing activation of the regulatory kinase p42/44 MAPK. PGE2-induced protein synthesis was blocked by inhibition of p42/44 MAPK. Finally, we determined that the anti-inflammatory nuclear regulatory protein PPAR? inhibited COX-2 and PGE2 synthesis in cardiac myocytes. Cardiovascular disease is a major health care problem in the USA, and pro-inflammatory mediators are gaining importance as causative factors. Given the controversy over whether COX-2 is protective or deleterious, it is important to study its role in a number of different pathophysiological conditions. Our data so far indicate that COX-2 and its products are deleterious to the heart post-MI. In patients COX-2 inhibition might be helpful in improving function after MI.

Principal Investigator: Liu, Ruisheng, M.D.
Regulation of Superoxide Production by the Macula Densa during TGF

The mechanism of superoxide (O2-) production in the macula densa is not known. We will test our hypotheses with the following specific aims. Aim 1: Hypothesis: The increase in tubular NaCI concentration that initiates tubuloglomerular feedback enhances O2- production primarily from macula densa NAD(P)H oxidase (NOX). We will measure O2- production while increasing luminal NaCI in macula densa. To investigate the Nox isoform expressed at the macula densa, we will isolate macula densa cells using laser capture Microdissection (LCM) and real time PCR. Aim 2: Hypothesis: O2- production is activated by macula densa depolarization. Depolarization of the macula densa activates NAD(P)H oxidase by stimulating the GTP-binding protein Rac. We will measure O2- generation while: a) depolarizing the macula densa; and b) using dominant negative Rac and constitutively active Rac expressed macula densa cells. Aim 3: Hypothesis: O2- production is enhanced by increased macula densa intracellular pH, which process is involved in transporting protons generated during O2- production out of the macula densa by Na/H exchange and/or H/K ATPase. We will measure O2- production while increasing luminal NaCI: a) in the presence and absence of H/K ATPase inhibitors; and b) while clamping intracellular pH. We will measure O2- generation while increasing luminal NaCI in H/K ATPase knockout mice. Aim 4 Hypothesis: Activity of apical Na/H exchangers is higher in SHR than in WKY, resulting in higher macula densa pH, which enhances NAD(P)H oxidase activity and superoxide production and thus augments tubuloglomerular feedback. Inhibition of macula densa apical Na/H exchange tends to normalize tubuloglomerular feedback in SHR. We will study the activity of apical and basolateral Na/H exchangers at the macula densa in SHR and WKY. We will isolate the apical and basolateral membranes of the macula densa using LCM and study the Na/H exchanger expression level using real-time PCR. We will measure tubuloglomerular feedback in SHR while clamping macula densa intracellular pH to the same degree as WKY. In present research, we will study the mechanism and effect of superoxide on renal hemodynamic and blood pressure. We believe the results of the present study will help us better understand the causes of hypertension and might reveal new approaches for treatment.

Principal Investigator: Oritz, Pablo, Ph.D.
Salt Absorption by the THAL: Role of NKCC2 Trafficking (National Heart, Blood, and Lung Institute 5R01 HL080409-02)

The number of Na-transporters in the cell membrane is regulated by dynamic insertion, retrieval and recycling of transporters.  Collectively these processes are known as trafficking.  In some form of human and experimental hypertension, salt retention is caused by abnormally high levels of Na-transporters in the apical membrane of some nephron segments.  The thick ascending limb (THAL) absorbs NaCl and contributes to salt and water homoeostasis, thereby influencing blood pressure.  NaCl absorption by the THAL is primarily regulated by NKCC2, an apical Na/K/2CI cotransporter that mediates Na and Cl entry into the cell.  To date, it is not clear whether trafficking of NKCC2 regulates NaCl absorption.  It is also not clear whether vasoactive hormones that stimulate and autocoids that inhibit NaCl absorption by the THAL regulate NKCC2 levels in the apical membrane by affecting its trafficking.  We hypothesize that NaCl absorption by the THAL is regulated in part by trafficking of NKCC2 into the apical membrane, in a process stimulated by cAMP and inhibited by nitric oxide.  Enhances insertion of NKCC2 into the apical membrane contributes to salt retention in models of salt-sensitive hypertension.  In Aim 1 we will examine whether NKCC2 levels in the apical membrane and NaCl absorption are regulated by insertion and retrieval of NKCC2.  In Aim 2 we will study whether hormones that stimulate NKCC2-dependent NaCl entry into THALs via cAMP increase NKCC2 levels in the apical membrane.  In Aim 3 we will study whether nitric oxide inhibits NKCC2-dependent NaCl entry by decreasing NKCC2 levels in the apical membrane.  Finally we will examine whether basal and hormone-stimulated NKCC2 levels in the apical membrane are enhanced in THALs from salt-sentive animals.  We will use state-of-the-art techniques to measure NKCC2 trafficking and activity in isolated rat THALs.

Data from this proposal will increase our understanding of how salt absorption by the THAL is regulated in normal and salt-sensitive animals, and will identify new targets for the development of diuretics.

NAD(P)H oxidases are broadly activated in cardiovascular diseases, including hypertension, atherosclerosis, and diabetes. Reactive oxygen species (ROS) derived from these oxidases have been implicated in impaired vascular relaxation, medial hypertrophy, and neointimal hyperplasia in various forms of hypertension. NAD(P)H oxidase-derived superoxide anion (O2-) and other ROS are believed to mediate stretch-induced signaling, leading to neointimal hyperplasia. We previously developed a cell-permeant inhibitor of gp91-phox- (nox2-) based oxidase assembly which is capable of abrogating vascular O2- production in response to angiotensin II. The current proposal stems from 3 major findings, demonstrating (a) the importance of the multi-component oxidase assembly in vascular O2- production; (b) the ability of our cell-permeant inhibitor of nox2-based oxidase assembly to inhibit O2- and attenuate neointimal proliferation of the rat carotid artery in response to balloon angioplasty; and (c) the upregulation of novel nox2 homologues, nox1 and nox4, in response to balloon injury. Since nox1 and nox4 appear to be important oxidase homologues involved differentially in vascular O2- production after stretch, we will determine the efficacy of docking sequence mimics (which inhibit nox1and nox4 assembly with other oxidase subunits) to inhibit whole-vessel and endothelial, smooth muscle cell and fibroblast O2- generation and neointimal proliferation. These studies will address the hypothesis that nox1 and nox4 are functionally involved in vascular stretch-induced oxidase assembly and O2- generation, leading to neointimal proliferation. Three specific aims will be tested: (1) to develop specific inhibitors of nox1- and nox4-based oxidases and test them in an in vitro model of hormone-induced vascular NAD(P)H oxidase activation; (2) to investigate the role of docking sequences on individual nox-based oxidases in vascular stretch-induced oxidase activity in vitro; and (3) to determine the role of nox docking sequences in balloon angioplasty-induced neointimal hyperplasia in vivo. Relevance: Therapies aimed at disrupting the various NAD(P)H oxidase systems in blood vessels should substantially improve vascular patency and function following balloon angioplasty. These inhibitors are also expected to provide broad utility in a variety of disease processes involving oxidants, including hypertension, diabetes and atherosclerosis.

Principal Investigator: Rhaleb, Nour-Eddine, Ph.D.
Hypertension and Collagen: Effect of Ac-SDKP

Hypertension is a major risk factor for .cardiovascular and renal diseases. Inflammation and components of the extracellular matrix (EM) have a negative impact on the physiology and function of end target organs such as the arteries, heart and kidneys in hypertension. Blocking angiotensin-converting enzyme (ACE) decreases angiotensin II (Ang II) and increases kinins, leading to decreased cardiovascular inflammation, hypertrophy and collagen. ACE inhibitors (ACEi) increase plasma Ac-SDKP, a negative regulator of cell proliferation present in plasma and tissue. In hypertension and heart failure, Ac-SDKP prevents monocyte/macrophage infiltration and fibrosis in the aorta, kidneys and left ventricle (LV). By virtue of its anti- fibrotic and anti-inflammatory effects, Ac-SDKP was able to improve renal function in hypertension, diabetes and other experimental models of renal diseases. However, the mechanism(s) or receptor(s) involved in Ac- SDKP's cardiovascular and renal effects are not fully understood. We hypothesize that Ac-SDKP exerts its anti-inflammatory and anti-fibrotic effects on the cardiovascular and renal systems in hypertension via specific receptor(s) located on the plasma membrane, contributing to end organ protection. In aim I we will identify and characterize Ac-SDKP receptors using pharmacological tools [ l]Hpp-Aca-SDKP, 5(6)FAM-SDKP and new analogues of Ac-SDKP), proteomic technology, and cloning techniques. In aim II we will 1) perform a more extensive examination of the structural activity of Ac-SDKP in order to a) develop potent antagonists that lack partial agonistic activity and b) improve the affinity of the radio-iodinated peptide; and 2) characterize the Ac-SDKP receptor in fibroblasts and macrophages (rat and human), using [ l]-Hpp- Aca-SDKP and newly developed antagonists; and 3) compare rat cardiac fibroblasts and human cardiac fibroblasts for the inhibitory effect of Ac-SDKP or analogues on collagen synthesis and proliferation. In aim we will study whether Ac-SDKP receptor activity depends on mechanisms closely linked to the regulation of receptor internalization. In aim IV We will determine 1) the effect of Ac-SDKP on the non-receptor tyrosine kinase Src and HB-EGF on Ang II and ET-1-stimulated transactivation of the EGFR; 2) whether Ac-SDKP inhibits the effects of calcium ionophores or EGF on p42/44 MAPK and collagen synthesis; 3) whether PLC, EGFR, cSrc, calmodulin kinase or IP3 inhibitors attenuate MAPK activity and collagen synthesis to the same extent as Ac-SDKP in response to Ang II or ET-1; and 4) whether inhibition of MAP kinase activation by Ac- SDKP is mediated by MAP kinase phosphatase-1, using selective inhibitors of phosphatases and specific SiRNAs. This project will provide important new information on the d the mechanism of action of Ac-SDKP. Consequently, it will identify another component (Ac-SDKP) as part of the multiple mediators participating in the cardioprotective effects of ACEi in hypertension.

Principal Investigator: Yang, Xiao-Ping, M.D.
Gender Influence in Mice with Myocardial Infraction (NHLBI R01 HL07895101)

Cardiovascular disease (CVD) is one of the leading causes of morbidity and mortality in the United States. There is evidence that premenopausal women are much less prone to suffer CVD than males of similar age, and that this advantage disappears after menopause. Using a mouse model of myocardial infarction (MI), we recently found that female mice had a much lower cardiac rupture rate and better preserved left ventricular (LV) function than males after MI. Supplementation of estrogen and/or castration in males reduced the rupture rate and slowed LV remodeling, while supplementation of testosterone in females increased the incidence of rupture and worsened cardiac function and remodeling, indicating a protective role of estrogen and detrimental role of testosterone post-MI. In this proposal, we will further test the hypotheses that estrogen, acting on the a-receptors, protects the heart from early (infarct expansion and cardiac rupture) and late remodeling (cardiac hypertrophy, dilatation and dysfunction) and this effect is partially mediated by facilitating the healing process during acute MI, and by inhibiting the inflammatory response and reducing oxidative stress during the development of heart failure. Conversely, testosterone exacerbates the inflammatory response.

• In Aim 1, we will study the effect of estrogen and testosterone on infarct healing, including inflammatory cell infiltration, collagenase activity, collagen deposition, infarct expansion and neovascularization.
• In Aim 2, we will study whether, during the chronic phase of MI, estrogen decreases nuclear factor-κB and reactive oxygen species, thereby ameliorating the inflammatory response and improving cardiac function in males, whereas testosterone aggravates inflammation and cardiac dysfunction.
• In Aim 3, we will study whether the cardioprotective effect of estrogen is mediated by activation of its a-receptor.
• In Aim 4, we will study whether
  1. estrogen given soon after ovariectomy (mimics early postmenopausal status) will provide better cardiac protection than if it is given a few weeks later (mimics late postmenopausal status) when mice are subjected to MI; and
  2. the cardioprotective effect of estrogen will be attenuated when combined with progestin.

Principal Investigator: Yang, Xiao-Ping, M.D.
PPG Project II: Role of Ang II, Kinins, NO and AT2 Receptors in Target Organ Damage (NIH P01 HL028982)

Heart failure is the leading cause of death in the United States. Activation of the renin-angiotensin system (RAS) plays an important role in this pathological process, since blocking the RAS with ACEi or angiotensin II (Ang) receptor blocker (ARB) improves cardiac function and regresses LV remodeling. We have shown that activation of kinins, nitric oxide (NO) and AT2 receptors contributes to the cardioprotective effects of ACEi and ARB as well. In this project, we propose to study 1) whether Ang II acts directly in the heart via the AT1 receptor independently of its effect on BP, resulting in cardiac remodeling and dysfunction. The cardiac effects of Ang II are mediated by the release of endothelin and reactive oxygen species (ROS), which activate nuclear factor-?B and stimulate inflammatory process; and 2) kinins, NO and AT2 activation antagonize the cardiac effects of Ang II. Using transgenic mice overexpressing Ang II in the heart (Tg-Ang II-cardiac mice, cardiac Ang II 40- to 50-fold higher than wild-type controls), we found that although SBP was similar between strains, Tg-Ang II-cardiac mice had profound cardiac hypertrophy, left ventricular (LV) dilatation and a slight but significant increase in collagen deposition. Thus Ang II may act in an autocrine/paracrine manner, stimulating pro-growth and pro-inflammatory responses and causing cardiac hypertrophy and interstitial fibrosis. We are currently studying whether cardiac remodeling and dysfunction are more advanced when these mice are subjected to myocardial infarction (MI). Using B1 kinin receptor knockout mice (B1-/-), we are testing the hypothesis that kinins acting on B1 receptors play an important role in tissue repair and preservation of cardiac function. We found that B1-/- mice had increased basal LV chamber dimensions and mass and more severe remodeling and dysfunction post-MI compared to B1+/+; blockade of B2 receptors worsened these processes in B1-/- mice, suggesting that kinins acting via both B1 and B2 receptor play an important role in cardiac remodeling post-MI.

Principal Investigator: Zhou, Jia Long, M.D.
Role of Intracrine Angiotensin II in Kidney Cells (R01DK067299)

Angiotensin II (Ang II) plays an essential role in maintaining body sodium and fluid balance and normal blood pressure by regulating proximal tubular sodium reabsorption. Ang II exerts powerful effects on sodium transport and cell growth by activating cell surface receptors on brush borders and basolateral membranes of proximal tubule cells. However, we have new evidence that a) extracellular Ang II is taken up by proximal tubule cells in vivo and in vitro; b) microinjection of Ang II directly into the cells increases intracellular calcium; and c) Ang II induces RNA transcription and expression in isolated nuclei. Our results suggest that internalized Ang II may act as an intracellular hormone to play important physiological and pathological roles in these cells. In this project, we propose to test the general hypothesis that extracellular Ang II is taken up by proximal tubule cells through AT1A receptor-mediated internalization, and that internalized Ang II binds to cytoplasmic and nuclear receptors to induce intracellular responses. To test this hypothesis, we will conduct both in vitro and in vivo studies, using complementary biochemical, morphological, cellular and molecular biology approaches. In Aim I, we will study whether proximal tubular cells take up extracellular Ang II in vitro and in vivo and elucidate the mechanisms by which Ang II receptors and the endocytotic machinery regulate Ang II trafficking. In Aim II, we will use confocal microscopy, state-of-the-art EM autoradiography and immunohistochemistry to trace intracellular trafficking pathways of internalized Ang II to the endosomal compartments and its translocaUon to the nucleus in vitro and in vivo. In Aim III we will study whether microinjection of Ang II increases intracellular calcium through activation of cytoplasmic AT1 receptors and the cellular mechanisms involved. Finally, in Aim IV we will investigate whether internalized Ang II binds to intracellular Ang II receptors to activate transcription factor NFkappaB and its translocation to the nucleus, and whether internalized Ang II stimulates nuclear receptors to increase transcription and expression of the Na+/H+ exchanger NHE3 and pro-inflammatory cytokines. These studies will provide new insights into the important role of internalized Ang II in renal physiology and Ang II-induced hypertensive renal injury.

1. Senior Research Staff

Paul Kvale, M.D.
Michael Simoff, M.D.
Michael Eichenhorn, M.D., Division Head

2. Research Summaries

Principal Investigator: Kvale, Paul, M.D.
Pathology Specimen Collection for the PLCO Project (NIH/NCI N01 CN25512)

The pathology collection for PLCO will collect and analyze pathology tissue samples from the existing cohort of PLCO participants. NCI is funding pathology specimen collection for a period of 12 months with a collection of approximately 300 samples from each of the 10 screening centers for the first year. NCI has developed a strategy to collect as few as 50 and as many as 300 samples per SC for up to an additional 4 years. Tumors of the following tissue types will be collected: colorectal (including adenomas), ovarian, lung, prostate and breast plus additional cancers as determined by NCI. First year collection will begin with colorectal cancer and adenomas.

Pathology tissue from cancer patients provides increasing opportunities for the study of biological questions relevant to tumor etiology. The material obtained from this study is expected to answer crucial questions about cancer etiology and cancer screening tests. Pathology tissue samples will be collected from pathology labs and sent to a central NCI lab and used to generate tissue microarrays.

Based on participant selection criteria developed by NCI, screening centers will receive a list of potential participants for pathology specimen collection. The selection criteria will include participants in both the screening and control arms of PLCO, with a signed Etiologic Studies Consent. Confidentiality will be maintained and protected as stated in the Privacy Act System of Records Notice. Access to study data will be limited to staff working on the study and all staff will sign a confidentiality statement.

Screening centers are responsible for obtaining participant authorization, requesting tissue from pathology departments, shipping and tracking this effort. There will be contact with PLCO participants, medical records departments, pathology laboratories, shipping companies and UCLA all of which will be tracked by each screening center and monitored by the coordinating center.

UCLA is responsible for processing pathology specimens into tissue microarrays for future analysis and for storage of TMA's. In addition UCLA will store any tissue blocks sent for permanent retention. The coordinating center is responsible for coordination, data management, systems support and quality control. PLCO is led by the PLCO Steering Committee, consisting of lead investigators from each screening center, UCLA and the NCI project Officer.

Principal Investigator: Kvale, Paul, M.D.
National Lung Screening Trial (NLST) (NIH/NCI NO1 CN25512)

Approximately 75 per cent of lung cancer patients present with advanced disease, for which there is no effective cure. The best hope of lung cancer survival comes with early stage diagnosis, which generally responds favorably to surgical resection. The National Cancer Institute-sponsored NLST is investigating whether annual screening with low-dose helical computed tomography (spiral CT) can reduce lung cancer mortality, compared to annual chest x-ray, by leading to earlier stage detection. NLST was launched in September 2002 and by February 2004 had completed its nationwide recruitment goal of 50,000 high-risk study subjects. More than 30 study sites nation-wide are participating in this study. With a recruitment goal of 3500, HFHS is one of the largest study centers in the country. Study subjects receive an annual screening, half with spiral CT and half with chest x-ray, for three consecutive years and will be followed up with annual monitoring until 2009.

In 2003, Dr. Paul Kvale took over leadership as principal investigator of NLST at HFHS from Dr. Raymond Demers. In 2003 at HFHS, 2675 individuals were recruited and 2534 individuals received their screening intervention. By the end of 2003, a total of 3395 subjects had been recruited into NLST at HFHS.

Large cohort studies such as NLST frequently generate secondary studies that contribute consequential scientific information far beyond that intended by the primary study hypothesis. In 2003, NLST was at a nascent stage, and not enough data had been generated to initiate many ancillary studies. However, HFHS researchers were developing ideas for a NIH RO1 proposal to investigate the application of artificial intelligence to digital computed tomography images and improve accurate detection of lung cancers. HFHS is making valuable contributions to NLST, which is expected to produce definitive answers to important public health questions within the next 5 years.

1. Senior Research Staff

2. Research Summaries

Principal Investigator: Drake, Chrisopher, Ph.D.
Predisposition Model of Insomnia (NIH/NIMH 5K23MH068372-02)

Candidate's Plans/Training: The candidate's goal is for a career in patient-oriented clinical research in the field of sleep medicine with a focus on insomnia and concomitant psychiatric disease. The training plan will include 11 courses at two local Universities and formal didactic and laboratory training from experts on insomnia pathophysiology and treatment, magnetoencephalography, endocrinology, and post-traumatic stress disorder. Training will be organized in modules each providing specific instruction and consultation regarding topics related to the proposed research plan and the candidate's long-term career goal of becoming an independent investigator. Environment: Henry Ford Sleep Center is a well-established research facility and would be an ideal training site for this award. Proven mentorship, strong within and across departmental collaboration, and an institution with a dedicated research commitment combine to provide a setting well suited for the career development of a young scientist. Research: The prevalence of chronic insomnia has been estimated to be between 10-15 percent of the general population. Insomnia is associated with a two to five-fold greater incidence of depressive disorders across the lifespan, and a significant negative impact on quality of life. Models of primary insomnia generally conceptualize the pathophysiology of this disorder in the context of a precipitating event superimposed upon predisposing and subsequent maintaining factors. However, to date, factors that predispose individuals to acute sleep disturbance and the significance of that predisposition for the development of chronic insomnia has not been investigated. Our model of primary insomnia proposes that hyperarousability (markers: emotional reactivity, beta frequency EEG, and HPA axis activation in response to a "challenge") is associated with vulnerability to acute sleep disruption. It is our view that hyperarousability and its associated vulnerability to acute sleep disruption represents a predisposition to the subsequent development of chronic primary insomnia by sustaining sleep disruption following the removal of a precipitant. Within the framework of this model we propose two experiments to identify and characterize a predisposition to insomnia in which 1) a subset of individuals without insomnia but who have elevated arousal as marked by emotional reactivity (NER) similar to that seen in patients with primary insomnia, have a general vulnerability to sleep disturbance induced by a first night in the laboratory and nocturnal caffeine administration; 2) however, unlike individuals with chronic insomnia, on non-challenge nights, these high NER individuals show normal sleep; 3) high NER individuals have increased physiological arousal similar to chronic insomniacs; 4) finally, predisposed individuals have protracted sleep disturbance following the removal of a sustained sleep disrupting precipitant when the possibility of sleep disruption remains.

Principal Investigator: Roehrs, Timothy, Ph.D.
Abuse Liability Associated with Chronic Hypnotic Use (R01DA01735501A1)

The acknowledged drugs of choice for the pharmacological treatment of insomnia are the benzodiazepine receptor agonist hypnotics (BzRA). Studies show that at therapeutic doses, used over the short-term, the abuse liability of BzRAs is relatively low and their efficacy outweighs their minimal risks. However, an increasing number of patients use BzRAs nightly for longer periods of time than is currently indicated (i.e. 4 weeks) and, minimal data on the long-term abuse liability and efficacy of these drugs exist. This project, using both prospective and retrospective methods, will address questions about the long-term abuse liability and efficacy of the BzRAs. The questions being raised are:

• What is the abuse liability and efficacy of hypnotics currently being used chronically and
• What is the prospective abuse liability and efficacy of hypnotics used chronically?

Zolpidem is the chosen standard for this project as it is the most frequently prescribed BzRA and also arguably the BzRA with the best short-term efficacy and safety profile. The focus of the first question is clinical; it is about the long-term abuse liability and efficacy of BzRAs, specifically zolpidem, as it is currently being prescribed and about the type of patients who receive this drug. The second question addresses the issue of whether a standard BzRA can be prescribed efficaciously and safely for the long-term to patients with primary insomnia. Over the first 4 years of this 5 year project a total 240 insomniacs will be entered into this study to yield 60 insomniacs who have been using zolpidem chronically (>6 months) and 180 who have never used a prescribed hypnotic. The 60 chronic-current zolpidem users will be followed for approximately 1 month. The 180 treatment-naive insomniacs will be randomly assigned to a placebo or zolpidem group and followed for 12 months. In the first month (all groups) and long-term (the two prospective groups), assessments for dependence, including probes for both physical and behavioral dependence, hypnotic efficacy, next-day residual effects, and markers of "hyperarousal' as predictors of dependence risk, will be conducted. Drug self administration will be used to assess behavioral dependence, standard polysomnography (PSG), withdrawal symptom checklists, urinary catecholamine and cortisol levels and Multiple Sleep Latency Testing for physical dependence, and PSG and self-reports for hypnotic efficacy.

1. Senior Research Staff

Abraham Thomas, M.D., Division Head
Fred Whitehouse, M.D.

2. Research Summary

Principal Investigator: Thomas, Abraham, M.D.
Prevention of Cardiovascular Disease in Diabetes Mellitus (NIH subcontract)

The purpose of this program is to conduct a multicenter randomized trial assessing the effect on macrovascular morbidity and mortality in persons with Type 2 diabetes mellitus of the following pharmacologic strategies:

1. Intensive glycemic control using a non-insulin-resistance-lowering drug regimen compared with conventional glycemic control using a non-insulin-resistance-lowering drug regimen
2. Intensive glycemic control using an insulin-resistance-lowering drug regimen compared with conventional glycemic control using a non-insulin-resistance-lowering drug regimen
3. Intensive glycemic control using an insulin-resistance-lowering drug regimen compared with intensive glycemic control using a non-insulin-resistance-lowering drug regimen
4. Intensive compared with conventional levels of lipid and blood pressure control

The Epidemiology of Diabetes Intervention and Complications (EDIC) is primarily an epidemiologic investigation, especially for the study of macro vascular disease in IDDM, and it also takes advantage of intention to treat analyses based on previous involvement of the study population in the DCCT.

1. Senior Research Staff

Norman Markowitz, M.D.
Jose Vazquez, M.D.
Marcus Zervos, M.D., Division Head

2. Research Summaries

This is a 5-year competitive renewal application to continue our studies to identify immune mechanisms of resistance and susceptibility to oropharyngeal candidiasis (OPC). OPC is the most common oral manifestation of HIV infection [1-4]. While OPC can occur during early stages of HIV infection when blood CD4+ cell numbers are still relatively high, the hallmark is the high prevalence during latter stages when CD4+ T cell numbers drop below 200 cells/µl. Despite standard antifungal therapy with azoles as well as highly active antiretroviral therapy (HAART) that has reduced the number of cases of OPC, both through enhanced immunity and direct effects of protease inhibitors [5-9], OPC continues to be the most common oral infection in HIV disease. There can also be significant antifungal resistance that adds yet another level of complexity. The causative agent, Candida albicans, is a commensal organism of the oral, gastrointestinal and vaginal mucosa. This mucosal presence of Candida (asymptomatic) stimulates both Candida-specific humoral and cell-mediated immunity (CMI) detectable in the peripheral blood of the majority of healthy individuals [10-12]. Of these responses, Th1-type CMI by CD4+ T cells is considered the predominant host defense mechanism against C. albicans at mucosal surfaces, including the oral cavity [13-16]. The strong correlate of OPC to reduced CD4+ T cells [1,3,12] supports this. However, OPC may not occur in those with reduced CD4+ T cells, or be recurrent in others, suggestive of a role for additional protective immune mechanisms. We have studied local and systemic immune responsiveness during OPC during the past 4 years. Among several findings, we found that Candida-specific CMI responses in the peripheral circulation was not defective as CD4+ T cells declined [12]. We interpreted these data to imply that CD4+ T cells must be maintained at a certain threshold number to be protective. Below this cellular threshold, protection of the oral cavity against OPC is solely dependent upon local immune mechanisms. Our studies suggest that local mechanisms important for protection against OPC may include Th1-type cytokines in saliva [17], oral epithelial cells that have significant anti-Candida activity in vitro [18], and CD8+ T cells in the oral mucosa. In those with OPC on the other hand, Th2-type cytokines (associated with susceptibility to candidiasis) are present in saliva [17], together with reduced epithelial cell-mediated anti-Candida activity [18], and accumulated CD8+ cells in lesions at a considerable distance from the site of infection at the outer epithelium. We now propose to further investigate these immune mechanisms to better understand their role in protection and any dysfunction that may lead to infection. Our hypothesis is that CD8+ T cells and related cytokines/chemokines together with epithelial cells represent important oral host defense mechanisms when CD4+ T cells drop below protective levels and that dysfunctions in one or more of these defenses contribute to episodes of OPC. To test our hypothesis, we will continue to study individuals with and without OPC in an urban patient population of HIV+ individuals with considerable age, gender, and racial diversity in the greater New Orleans area. Both immunological and molecular biology techniques will be used to (i) characterize the immune status of CD8+ T cells in the lesions through CD8 cell-associated cytokines, chemokines, adhesion molecules, chemokine receptors, and co-stimulatory molecules important for cell trafficking and function, and (ii) identify the effector moiety, role, and mechanism of the oral epithelial cell anti-Candida activity. The long term goals of this project are to identify the specific immune factors associated with the susceptibility to OPC in HIV+ individuals, and to develop immunotherapeutic strategies to enhance local resistance during periods of reduced CD4+ T cells. There are 3 specific aims: 

1. To evaluate the presence and immune status of CD8+ T cells in oral tissue of HIV+ persons with and without OPC. This will be accomplished in biopsies taken cross-sectionally and longitudinally through immunohistochemical staining and RNAse protection assays for various T cell markers and adhesion/activation molecules.
2. To evaluate CD8+ T cell-associated cytokines/chemokines in oral tissues of those with and without OPC. This will be accomplished by analysis of cytokines (IL-2, IL-15, and lymphotoxin-a) and chemokines (RANTES, IP-10, and lymphotactin) in saliva, epithelial cell/Candida cocultures, and biopsy specimens for the cytokines, and the chemokines. Additionally, chemokine receptors will be evaluated on T cells and epithelial cells.
3. To identify the effector moiety, mechanism of action, and role for the oral epithelial cell anti-Candida activity. This will be accomplished by intensified clinical surveys of the epithelial cell growth inhibition activity as well as experimental studies to identify the putative effector moiety and mechanism of action.