This application is for a Patient-Oriented Research Career Development Award (K23) for Dr. Judith Tsui, an Assistant Professor in General Internal Medicine at Boston University School of Medicine, who is establishing herself as an investigator in hepatitis C virus (HCV) related outcomes. The research plan both extends her prior work in HCV, and allows her to grow in a new direction as a pain researcher focusing on substance users. The award will provide Dr. Tsui with the support necessary to accomplish the following goals: 1) develop expertise in the etiology of chronic pain in substance users with chronic viral infections like HCV and HIV; 2) publish research that will lead to improved treatments for pain among substance users; and 3) transition into an independently funded researcher by obtaining an R01. The comprehensive training plan includes formal coursework, as well as hands-on training in pain measurement and experimental pain models. Dr. Tsui's primary mentor will be Dr. Jeffrey Samet, an established investigator in the fields of substance use and chronic viral infections. She will receive additional mentoring from Dr. Jianren Mao, a leading translational pain researcher, and Dr. Robert Edwards, an accomplished junior investigator and clinical psychologist. Pain is a major problem among substance users, and a better understanding of its etiology is needed to improve clinical care. There is limited research to determine how viral infections such as HCV and HIV impact risk for developing chronic pain and painful conditions. Dr. Tsui will explore the novel hypotheses that HCV is associated with: 1) hypersensitivity to pain under experimental conditions and 2) clinical pain. To explore these hypotheses, Dr. Tsui will conduct an experimental, cross-sectional study of HCV mono- infected, HIV/HCV co-infected, and uninfected opioid addicts that compares experimental pain responses and self-reported clinical pain. She will assemble a cohort of 120 opioid dependent patients on buprenorphine or methadone: 40 HCV+/HIV-, 40 HCV+/HIV+ and 40 HCV-/HIV-controls. Participants will undergo the following experimental testing: 1) the cold-pressor test to assess cold pain tolerance 2) mechanical stimulus to test mechanical pain thresholds and 3) repetitive pinprick to assess for temporal summation. The study will also collect data on pain and pain-related variables using validated scales such as the Brief Pain Inventory, as well as measurements of pro- and anti-inflammatory cytokines (TNF-1, IL-2, IL-6, IL-2, IL-10 and IL-4) from peripheral blood samples. She will perform appropriate statistical testing to assess whether opioid addicts who are HCV infected (HCV+ alone or HCV+/HIV+) have different experimental pain responses (lower pain tolerance/thresholds and more temporal summation) and a higher prevalence of self-reported chronic pain, and to explore whether inflammatory cytokines mediate found associations between HCV and pain. This research will contribute to our understanding of the causes of pain among substance users, and will shape future studies to test new approaches (possibly including anti-HCV treatment) to prevent and treat pain in this vulnerable patient population. PUBLIC HEALTH RELEVANCE: Pain is a common problem among current and former substance users, many of whom are infected with HIV and HCV. A better understanding of pain etiology is needed in order to effectively prevent and treat chronic pain among patients with substance use problems. This research addresses the simple (yet relatively unexplored) question: how do chronic viral infections contribute to patients' vulnerability to pain? In addition, it begins to explore potential mechanisms for pain hypersensitivity in the setting of HCV by investigating the role of inflammatory cytokines in shaping pain responses. The results of this research may directly benefit patients with a history of substance use and pain by 1) providing an explanation for their pain and 2) leading to improved treatments for pain, including anti-HCV therapy or anti-cytokine medications (such as pentoxyphylline). In addition, it will inform clinicians and policy makers who must consider the long-term consequences of HCV infection when weighing costs/benefits of treatment and interventions to prevent transmission.


We have analyzed the development of B cells in Terminal Deoxynucleotide Transferase (TdT)knockout mice. Two aspects have been studied: 1) the effect on the immune response to phosphocholine (PC) in mice immunized with Proteus morganii; and 2) the effect on B1 B cell development. The immune response of normal mice to P. morganii is dominated by B cells expressing a M603 idiotype. Thus, the V1 H chain of these antibodies has a somatic mutation at the V:D junction in which the germline encoded Asp at position 96 has been changed to an Asn. It has been claimed that anti-PC antibodies of other idiotypes are not capable of binding to the PC-epitope on P. morganii. However, in TdT knockout mice immunized with this bacterial antigen only germline encoded T15-idiotype anti-PC antibodies are produced. This data demonstrates that TdT is responsible for the Asp to Asn change that occurs in the V1 gene of normal mice and it also shows that the response to P. morganii is not restricted to the M603-idiotype. Published work on TdT knockout mice shows that loss of TdT has very little effect on the animals ability to respond to a wide variety of antigens. This was surprising in that TdT isresponsible for generating diversity in the 3rd hypervariable region of the antibody molecule and this region of the antibody often defines the specificity of the antibody molecule. We found that TdT knockout mice have a higher level of B1 B cell development than normal mice. This increase in CD5+ B cells is accompanied by a 3 fold increase in the number of peritoneal B cells that bind to phosphatidyl choline (PtC). In normal mice up to 50% of the PtC binding cells display the VH-12 idiotype. However, in TdT knockout mice the VH-12 PtC-specific B cellsrepresent a much lower fraction of the PtC pool. These data demonstrate that TdT plays a role in controlling the homeostasis of the B1 B cell pool, and it also affects the H-chain makeup andantigenic specificity of this pool of B cells.


Terminally differentiated ventricular muscle cells virtually cease to proliferate, and subsequent cardiac growth occurs almost exclusively by cell enlargement. Hence, failure to increase cell number preludes cardiac regeneration as a means to reconstitute functional mass after infarction or other insult. This irreversible growth arrest poses a therapeutic challenge, especially in the elderly, where the includes of infarction and heart failure is high. Using an array of recombinant viruses to direct the expression of adenoviral EIA proteins, the applicant has recently demonstrated evidence for dual pocket protein-and p300-dependent pathways that govern the cell cycle in cardiac muscle. Drawing on these developments the present application proposes to study in depth the endogenous occupant of the "pocket", the transcription factor known as E2F; modulators of pocket protein function in the heart, known as cyclins and clyclin-dependent protein kinases (Cdks); and the molecular relationship between the pocket protein-and p300-dependent mechanisms. Specific Aims of the present project are: (1) to establish molecular mechanisms for the phenotype effects of E2F ( activation of the cell cycle, and repression of tissue-specific genes) in cardiac muscle cells; (2) to clarify what role is played by endogenous regulators of free E2F (G1 cyclins, Cdks, and Cdk inhibitors) in cardiac muscle cells; (3) To determine the epistatic relationship between E2F and the p300 pathway in cardiac muscle cells; and (4) To investigate the function of E2F in adult cardiac muscle in vivo, by direct injection of e2F virus and by cardiac-restricted homologous recombination of the pocket protein gene, Rb.


This proposal is directed toward the synthesis of cembranoid natural products, a class of compounds whose chemistry and biological activity have been little studied. The specific objectives, isolobophytolide, anisomelic acid, asperdiol and peunicin are cytotoxic and may exhibit antimicrobial, antiprotazoal, and cardiosvascular activity. A major objective is the development of reliable synthetic routes to this widespread class of macro carbocyclic natural products. Of particular interest is the assemblage of multifunctional chiral intermediates suitable for cyclization to the characteristic 14-membered cembranoid macrocycle. Biological testing of synthetic intermediates will be arranged with the appropriate N.I.H. division.


Neurological complications occur commonly in HIV infection, with over 40% of individuals being at risk for developing HIV-related neurocognitive impairment (NCI), with the numbers increasing in the era of effective antiretroviral therapy (1). Factors that underlie the development of NCI include neuronal dysfunction due to enhanced production of proinflammatory cytokines (e.g., TNF-? and IL-1?) and other inflammatory mediators that are secreted by HIV-infected cells in the brain (2,3). Contributing to such responses is impairment metabolism mediated by nicotinamide adenine dinucleotide (NAD), which is essential for normal mitochondrial function and the production of energy substrates in cells. In this grant, we will analyze potential effects of exposure to cigarette smoke (CS) and nicotine as co-factors for increasing the risk of NCI in HIV infected individuals. Cigarette smoking has been shown to alter a number of innate and adaptive immune mechanisms and can elicit cellular oxidative stress responses that can promote injury. Most marketed brands of cigarettes contain varying amounts of nicotine. In smokers, CS causes a leukocytosis and, in HIV-infected individuals, smoking is associated with higher plasma HIV viral loads and an increased mortality. It has been estimated that over 40% of HIV+ individuals are cigarette smokers (6), a number that is twice the estimated prevalence of smoking among adults in the general population (7). Nicotine, via activation of nicotinic acetylcholine receptors (nAChR), promotes cigarette smoking through its addictive properties. By activating these receptors, nicotine has been also shown to suppress immune activation and proinflammatory responses through direct effects on immune cells as well as through pathways that are mediated by activation of nicotine receptors (8-10). However, due to effects of HIV protein and with chronic nicotine exposure and upregulation of nAChR, proinflammatory responses have been observed to increase, thereby potentially contributing to CNS damage that occurs in the context of HIV infection. In previous studies in adult Lewis rats, we demonstrated that CS exposure can result in marked inflammatory and oxidative stress responses in the brains of the exposed animals (11). We have subsequently demonstrated that such effects can be also observed in HIV-1 transgenic rats, with more prominent responses developing than what are observed in corresponding wild-type animals. We also provide evidence that such responses can be driven by nicotine that is present in the CS. Activation of nAChR can also regulate cellular levels of NAD and activation of associated metabolic pathways. The potential effects of such activation in HIV infection and the generation of chronic inflammation in brain has not been previously studied. The proposed studies will be performed in vivo utilizing the transgenic rat model as well as in vitro utilizing primary astrocyte cultures that express the same HIV genes as the transgenic rat in vivo.


DESCRIPTION: What is proposed is to continue and expand a collaboration between an outstanding peptide and organic synthesis laboratory in Latvia and an outstanding cytoskeleton laboratory in Boston. Potential actin or polyphosphoinositide lipid (PPI)-binding peptides will be synthesized based on the sequences of various proteins identified by genetic or biochemical data. The structures of some peptides will be calculated by molecular modeling in Riga and their biochemical effects on lipids, actin binding proteins, and intact cells will be studied in Boston. These studies will be expanded to include fluorescent peptides shown to retain the PPI-binding properties of unlabeled analogs. One of these peptides exhibits the unusual property of crossing the plasma membrane of living cells. This peptide has potential applications as a research tool to identify or disrupt PPI localization in vivo or clinically as a carrier of pharmaceutical agents into specific cell types. These possibilities will be tested using derivatives synthesized in Riga and shown in Boston to form gels at very low concentration by assembling into rodlike micelles. These peptide gels are ideal materials to be studied by rheologic methods and have potential for application in drug delivery or antigen presentation.


In rural South Africa, only two-thirds of HIV+ pregnant women seeking antenatal care at community health centers took full advantage of available prevention of mother-to-child transmission (PMTCT) services in 2010 (SADOH). While engagement of male partners has been encouraged as a potential means of increasing PMTCT uptake, men have been reluctant to accompany their wives/partners to antenatal care. Recent studies generally support male involvement in promoting PMTCT, but the nature and impact of that involvement is unclear and untested. It is also clear that factors such as stigma, disclosure and intimate partner violence pose significant barriers to PMTCT uptake and retention in care, suggesting that male involvement may be necessary, but not sufficient to accomplish the WHO goal of <5% infant HIV incidence. Additional measures may be needed to increase participation by HIV positive pregnant women in PMTCT. In 2011, Mpumalanga Province had the highest rates of HIV in the country (36.7%) and rates of infant HIV incidence in rural clinics ranged up to 50%. Rates of PMTCT uptake in the Province have been among the lowest in South Africa (69%). This application proposes to expand on a successful PEPFAR- supported, PMTCT couples intervention pilot study conducted in Mpumalanga Province, (Vikela Umndeni: Protect Your Family) to include a more representative population of HIV positive pregnant women and their partners, the primary objective being to determine whether male partner involvement plus a behavioral intervention would significantly reduce infant HIV incidence by increasing levels of adherence to ARV/ PMTCT protocols, including breastfeeding and family planning, during the antenatal and post-natal periods. The proposed study will enroll two cohorts of HIV positive pregnant women recruited from 12 randomly assigned Community Health Centers (6 experimental, 6 control): a) Women attending without their male partners (n = 720), followed by b) Women attending with their male partners (n = 720 couples), to determine whether the influence of male participation itself or combined with a behavioral PMTCT intervention can significantly reduce infant HIV infection ante-, peri- and post-natally. It is our intention to significantly increase PMTCT participation from current levels (69%) in Mpumalanga Province to 90-95% through engaging women and couples in a unique, controlled, six session ante- and post-natal risk-reducing/PMTCT promotion intervention addressing the barriers to PMTCT (e.g., stigma, disclosure, intimate partner violence, communication, infant feeding practices, safer conception) that prevent women and men from taking full advantage of the treatment opportunities available to them and their infants. Based upon the encouraging preliminary results from our pilot study, successful CHC adoption of the Vikela Umndeni: Protect Your Family program could have major public health policy implications for containing the epidemic among the most vulnerable populations in rural South Africa: HIV+ pregnant women and their infants.


Mammographic screening leads to many women being diagnosed with ductal carcinoma in situ [DCIS], yet we cannot accurately predict which lesions will undergo malignant progression to invasive ductal carcinomas [IDC] or effectively block this transition. Studies of human breast biopsies have implicated in this process cysteine cathepsins V/L2 and B in tumor cells and macrophages and cathepsins F, K and L in myoepithelial cells/[myo]fibroblasts. Aberrant signal transduction, for example through p21-activated kinase 1 [PAK1], may contribute to increased pericellular proteolysis. Our working hypothesis is that the transition from pre-invasive DCIS to invasive carcinomas and the rapid progression of some DCIS lesions are mediated through alterations in proteolytic pathways in DCIS cells and DCIS-associated cells, and that dysregulated PAK1 contributes to the induction of these aberrant proteolytic pathways. To test this hypothesis, we will recapitulate the transition from pre-invasive DCIS to invasive carcinoma using in vitro and in vivo progression models that we have designated MAME for mammary architecture microenvironment engineering. In these models, we will use isogenic MCF10 cell lines [AT1, DCIS1 and CA1d] and two human DCIS cell lines [SUM-102 and SUM-225]. Our specific aims are to: 1. Modulate expression and activity of cysteine cathepsin V or B in the isogenic and SUM DCIS cell lines, both by direct targeting and through intervention at the level of PAK1, and determine using the in vitro MAME model whether the invasive phenotype is altered; 2. Determine using the in vitro MAME model whether the invasive phenotype can be altered by co-culturing modified cells from Aim 1 with myoepithelial cells, [myo]fibroblasts or both cell types, using wild-type cells and ones in which expression and activity of cysteine cathepsin F, K or L have been modulated; 3. Determine using the in vivo MAME model whether the malignant phenotype of xenografts of modified cells from Aim 1 can be altered by simultaneous implantation of myoepithelial cells, [myo]fibroblasts or both cell types, using wild-type cells and ones in which expression and activity of cysteine cathepsin F, K or L have been modulated; and 4. Screen [via our Hu/Mu ProtIn chip] the in vivo MAME model for proteolytic pathways that may contribute to the transition from DCIS to IDC and use the in vitro MAME model to define functional changes with libraries of reagents from the Center on Proteolytic Pathways. Validating, in the context both of the tumor and its microenvironment, proteases key to progression of DCIS to IDC, and kinase pathways that regulate them, should identify potential targets for therapeutic intervention as well as biomarkers to distinguish DCIS lesions that will rapidly progress to IDC. PUBLIC HEALTH RELEVANCE: Proteases and kinases are the subject of intensive efforts by the pharmaceutical industry to develop new treatment strategies for human diseases, including cancer. Our proposed studies will discover and validate protease pathways that are active in the tumor microenvironment and that mediate the transition to a full-blown malignancy, and kinase pathways that regulate these protease pathways. We anticipate that our studies will identify biomarkers to distinguish premalignant lesions that will progress to invasive cancers and define targets that will abrogate that progression. [unreadable] [unreadable] [unreadable]


The immune system plays a critical role in the pathogenesis of pain triggered by injury or illness. A precise understanding of the mechanisms through which particular immune mediators contribute to sensitization of nociceptive neuronal pathways will be essential for the development of more efficacious treatment strategies. The complement system is a principal component of innate immunity. This proposal focuses on two highly active split products of the complement system, C3a and C5a. Increased production of C3a and C5a has been reported in various pathological states associated with pain, including arthritis, pancreatitis, inflammatory bowel disease, burns and surgical trauma. Blocking the synthesis of C3a and C5a or antagonizing their receptors produces analgesic effects in animal models of inflammatory, neuropathic and postoperative pain. Moreover, direct administration of the complement fragments increases nociceptive sensitivity to heat and mechanical stimuli in animal models. In spite of this strong evidence implicating the complement system in the development of pain hypersensitivity, the underlying mechanisms are not understood. The goal of this project is to investigate mechanisms that are responsible for the enhanced pain sensitivity produced by the generation of C3a and C5a in the affected tissues. We hypothesize that C3a and C5a receptors are expressed and functionally coupled with TRPV1 in a subset of primary nociceptors; activation of these receptors sensitize nociceptors via protein kinase C-dependent modulation of the TRPV1 channel, which is known to function as a molecular integrator of pain producing stimuli. We will use a multidisciplinary approach involving immunohistochemistry, single-cell RT-PCR, [Ca2+]i imaging, patch-clamp analysis, single-fiber recordings and measurement of nociceptive behavior to test our central hypothesis. In Aim 1, we will characterize the expression and subcellular distribution of C3a and C5a receptors (C3aR and C5aR, respectively) and TRPV1 in various classes of sensory neurons identified by specific molecular markers. We will also use Ca2+ imaging and patch-clamp recordings to test functional coupling of TRPV1 with C3aR and C5aR. In Aim 2, we will investigate intracellular signaling cascades that link the activation of C3aR and C5aR with TRPV1 sensitization. In Aim 3, we will use single-fiber recordings and behavioral studies to examine the role of PKC- dependent modulation of TRPV1 downstream of C3aR and C5aR activation in regulating nociceptor excitability as well as heat and chemical sensitization of nociceptors. The proposed studies will help to characterize the novel roles of C3a and C5a receptors in the regulation of nociceptor function, and may lead to the development of new therapeutic strategies targeting the complement system to alleviate pain. PUBLIC HEALTH RELEVANCE: Pain management remains one of the most serious public health problems. The proposed studies will help to better understand how the complement system contributes to the pathogenesis of pain caused by surgical trauma or illnesses such as arthritis, pancreatitis and inflammatory bowel syndrome, and may lead to the development of new therapeutic strategies targeting the complement system to alleviate pain.


Focal neuropathies of the hand are among the most common diseases treated by neurologists. The two most common are median neuropathy at the wrist (carpal tunnel syndrome) and ulnar neuropathy at the elbow. The current electrophysiological methods of assessment of focal neuropathies can be painful, and include nerve conduction studies and electromyography. Electrical impedance myography (EIM) is a new technique that can be used to assess neuromuscular disease. We have had success in using EIM in small muscles in animals, and limited experience using EIM in small muscles in human studies. We plan to refine these techniques in this proposal, while studying focal neuropathies of the hand. The long-term goal of this proposal is to make EIM a valuable tool for the assessment of neuromuscular disease. Establishing standards for use in small muscles will complement the known utility of EIM in larger muscles and help make it such a tool. We have three specific aims for this study. First, we aim to establish a range of normality for EIM for the three hand muscles that are tested most commonly in standard neurophysiologic assessment: the abductor digiti minimi, first dorsal interosseous, and abductor pollicis brevis. Second, we will evaluate EIM parameters in a group of patients with median or ulnar neuropathy, predicting that EIM parameters recorded from affected muscles in patients with median and ulnar neuropathy will differ from normal subjects due to the underlying architectural alterations in the muscle caused by neurogenic injury. Third, in order to determine the ability of EIM to judge lesion severity, we seek to determine the correlation between EIM parameters with standard electromyography (EMG) parameters in patients with focal neuropathy. Relevance to public health: Focal neuropathies of the hand, which include median neuropathy at the wrist (carpal tunnel syndrome) and ulnar neuropathy at the elbow, occur in approximately 10% of people. The standard neurophysiologic assessment of these problems includes electromyography and nerve conduction studies, both of which may be poorly tolerated. We are developing electrical impedance myography as a painless, noninvasive, quantifiable technique to assess focal neuropathies of the hand, and neuromuscular diseases in general. [unreadable] [unreadable] [unreadable]


Type I collagen is a fibrous protein which is commonly used in the human body for structural and functional integrity. Mutations in the alpha-1 and alpha-2 chains of this triple-helical protein induce the disease Osteogenesis Imperfecta (OI). OI is expressed in people by a number of phenotypes ranging from a disease process that is lethal in utero to one that is only detected through gene analysis. We are using the techniques of molecular dynamics and free energy perturbation to predict the phenotypic severity that result from single point mutations of type I collagen. In particular, we are focusing on the following three issues in our investigation: 1) validation of the AMBER molecular mechanics force field for studying fibrous proteins; 2) determination of protein and water-protein structural changes induced by single point mutations of type I collagen; 3) determination of changes in stability between native collagen fragments and collagen fragments in which a single point mutation has occurred. We have completed the validation of the AMBER molecular mechanics force field for both molecular dynamics and free energy perturbations of collagen-like peptides. We have determined that solvent provides an overall network of stability to the collagen molecule, but does not provide the inherent stability to the triple helix. We have begun a collaboration with Professor Ron Raines at the University of Wisconsin to determine what molecular interactions/effects (e.g., hydrogen bonding, induction effects) contribute to the stability of the triple helix.


Project 4: Abstract Spliced X box-binding protein-1 (sXBP1) is prominent endoplasmic reticulum (ER) stress response factor of the Unfolded Protein Response (UPR) of the ER. The highly developed ER and protein processing and trafficking organelles of the pancreatic acinar cell are consistent with its specialized role as the site of synthesis and trafficking of large quantities of digestive enzymes. XBP1 is specifically necessary for development of exocrine pancreatic tissue and we have published and present further preliminary data here that inhibition of its expression promotes organellar disorders and cellular failure of the pancreatic acinar cell; and that increases in its expression are associated with protection from organellar disorders and cellular failure with stressors that cause pancreatitis. We find that XBP1s inhibition correlates with activation of additional ER- originating signals associated with inflammation and cell death, i.e., the PKR-like endoplasmic reticulum kinase (PERK) and C/EBP homologous protein (CHOP), i.e., pathways that promote the latter responses. Little is known about the regulation of XBP1s in exocrine pancreas. Our preliminary data suggest roles for calcium metabolism, ATP and a functionally pleiotropic ER transmembrane protein, Bax inhibitor-1 (BI-1) in regulating sXBP1. On the basis of these observations, we propose a series of hypotheses: that pancreatic acinar cell sXBP1 expression is subject to regulation by multiple factors including cellular Ca2+ responses; BI-1 expression; and mitochondrial ATP production. In response to stress, sufficient sXBP1 responses are required to feed back to restore control over many of these same factors to maintain cellular homeostasis. In contrast, sXBP1 deficiency exacerbates ER stress and leads to over-activation of the pathologic arm of the UPR (PERK/eIF2 /CHOP), disturbances of protein trafficking, and pancreatitis. We will pursue 3 aims in this project. Aim 1 will investigate the regulation of sXBP1 expression; Aim 2 is to determine the effects of sXBP1 expression insufficiency on UPR (PERK/eIF2 /CHOP), disturbances of protein trafficking, and pancreatitis; and the final aim is devoted to demonstrating that increasing sXBP1 expression enhances protective responses in the exocrine pancreas. Because of our initial findings that sXBP1 has significant effects on the endo-lysosomal/trafficking systems investigated in other projects in this program, it is highly integrated into the Program Project. Because we will show that sXBP1 can attenuate trafficking disorders and protect against pancreatitis, the project is highly translational.


Defects in human left right (LR) axis formation cause birth defects of the heart, vasculature, lungs, and gastrointestinal tract that are life-threatening and costly to the healthcare system. To understand how laterality defects develop, it is essential to understand the molecular mechanisms by which LR asymmetry is established. My long-term goal is to understand the mechanism of how LR asymmetry is established during early embryogenesis. My previous work including collaboration with my colleagues in this field has revealed important steps in this process in the mouse model. However, there are still gaps in our understanding of the entire process, such as the mechanisms by which the node structure and rotating node cilia develop, and how LR polarity in the node is transferred to the left LPM. Here I will address these questions by investigating roles of definitive endoderm in LR determination. To date, no report has been focused on roles of endoderm in LR asymmetry. But several series of evidence suggest importance of endoderm in this process. Our hypothesis is that endoderm cells regulate LR determination in two ways: regulating node formation and/or function, and transferring LR signal from the node to the LPM. This hypothesis will be addressed by analyzing Sox17 mutants because Sox17 is the only gene that specifically expressed in the endoderm and primary defects of the mutants are specific to the definitive endoderm. Aim 1 will study the nature of the initial LR determination defects occurring in Sox17 mutants by characterizing the node and cilia formation, in addition to the development of nodal flow. Aim 2 will examine the possible defects occurring in the process of transferring LR signals from the node to the LPM regarding the gap junctional transport and intracellular Ca2+ signaling. In addition to chemical blockers, the roles of gap junctions will be tested by a sophisticated transgenic approach using a dominant negative form of connexin that can be temporally and/or spatially controlled. Conditional mutants of Sox17 will be studied to identify the importance of endoderm cells in the signal transfer from the node to the LPM, being separated from the initial abnormalities in the node. We have found that the Nodal gene continues to be expressed in migrating endoderm in the Sox17 mutants, which could be the cause of endoderm defects in Sox17 mutants that affect LR determination. In Aim 3, I will test this possibility by transgenic techniques. I will rescue Sox17 mutants by expressing Nodal antagonists in endoderm and the Sox17 mutant phenotype will be reproduced by ectopically expressing the Nodal gene in the endoderm of wild type embryos. The 20 yrs of experience that the PI possesses in this model system has enabled us to design and optimize novel tools and techniques that are ideal for addressing the questions I have put forth in this proposal. The proposed experiments will reveal novel roles of endoderm cells in establishment of LR asymmetry in mammals. This work may ultimately provide insight into the mechanisms underlying human birth defects due to problems in laterality. PUBLIC HEALTH RELEVANCE: The proposed experiments will reveal novel roles of endoderm cells in establishment of LR asymmetry in mammals. This work may ultimately provide insight into the mechanisms underlying human birth defects due to problems in laterality.


Prediction of RNA secondary structure is important in many areas of molecular biology. For example, in phylogenetic studies, prediction of the secondary structure of "16S-like" rRNA is an important piece of information used in determining the correct alignment of sequences. The "well-defined" regions of the multiply aligned sequences are then used to make phylogenetic inferences. Secondary structures can be used in part to explain translational controls in mRNA, and replication controls in single-stranded RNA viruses. RNA structure also plays an important role in the regulation of retroviruses and cellular messenger RNAs. Secondary structure modeling can be used as a first step to the more intricate process of three-dimensional modeling. This could include the modeling of ribosomal RNA or catalytic RNA's such as group I introns. The folding patterns of individual RNA molecules are most often predicted using energy minimization approaches based on dynamic programming algo rithms. The optimization of the most modern versions of these algorithms on highly parallel computers will allow the application of these methods to biological systems previously too large for these calculations. Although the RNA folding problem is ill-conditioned, this drawback can be mitigated by the use of special "energy dot plots" that show the superposition of all possible foldings in the vicinity of a global energy minimum. Multiple predicted foldings indicate either the inability of the present modeling procedure to yield an unambiguous answer, or the actual presence of multiple structures (or both). We are now focusing on the MFOLD program, a more recent version of the program that incorporates these advances. Several cycles of optimization have been performed on MFOLD. First, the existing SGI version was simply ported to the Paragon. As expected this version was not particularly efficient since it required large global data arrays and frequent communication. In a second step, it was determined that the computationally intensive part of the code lies in the calculation of multiply branched loops. After optimizing this part of the computation the code performed relatively well, but was limited to relatively small molecules because of its replication of the stored energy values on each processor. Finally, a distributed memory version has been implemented that allows molecules of at least 20,000 bases to be analyzed. This appears to be both the fastest implementation, as well as the most memory efficient, currently available. In the current year, we proceeded with the porting of the parallel code from the paragon to the CRAY T3E. This meant a translation of the parallel communication calls from the nx library to the MPI library. Moreover, this port involved many declaration changes, and other formatting modifications needed to compile the code on the T3E. At this point in time, we have completed nearly 95% of the port. There remains a small error in the traceback routine. In the near future, we will complete the testing of the code, and add in a couple of communications optimizations that should improve performance. In the next year we will make the MFOLD code publicly available through the NBCR as an additional transparent supercomputing service.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objective: To convert human pluripotent stem and somatic cells into hematopoietic stem cells (HSCs) and self renewing cardiac progenitor cells. The Midwest Progenitor Cell Consortium is a collaboration between the UW-Madison and the University of Minnesota, which will serve as a research hub within the NHLBI's Progenitor Cell Biology Consortium. The goal is to develop new strategies to convert human pluripotent stem and somatic cells into hematopoietic stem cells (HSCs) and self renewing cardiac progenitor cells through identification of genetic program leading to formation of pre-HSCs and cardiac progenitors and activation of self-renewal program in lineage restricted cells. This project uses WNPRC Stem Cell Resources.


Pulmonary fibrosis is the leading cause of death in systemic sclerosis (SSc). The NIH/NHLBI-sponsored Scleroderma Lung Study (SLS) was a 13-center, double-blind, randomized controlled trial designed to evaluate the effectiveness and safety of oral cyclophosphamide (CYC;less than or equal to 2 mg/kg/d) versus placebo as a 1- year treatment for patients with active, symptomatic scleroderma-related interstitial lung disease (SSc-ILD). The goal of this hyper-accelerated award application is to evaluate stored biologic samples from the SLS in order to define the pathophysiologic mechanisms that underlie SSc-ILD, to predict the presence and activity of SSc-ILD, to predict patient subsets that will benefit the most from cytotoxic therapy, and to aid in the design of future clinical studies in which other biologic agents, with other mechanisms of action, can be evaluated for their ability to improve clinical responses beyond those observed with CYC alone. Analysis of the BAL fluid, cell pellet mRNA, and plasma samples will be carried out with a focus on candidate biomarkers representative of the inflammatory, proliferative and obliterative vascular, and fibrotic/tissue matrix components of SSc-ILD. The unique strengths of this proposal are the near complete set of biologic samples which include material from the plasma as well as the primary target organ (lung);the rich data set from the SLS in which there are both extensive baseline features that define the presence and extent of disease and multiple positive outcomes in response to treatment;the identification of distinct pathogenic components upon which to focus our biologic analysis. By the completion of this study, we hope to have gained important new insight into the biology of SSc-ILD, to be able to use plasma and/or BAL samples to identify patients with potentially responsive lung and skin disease, and to provide important insight to the SLS Investigators as they plan future therapeutic clinical trials. Furthermore, we will garner new information as to the biology and pathogenesis of SSc-ILD. Relevance to Public Health: Successful completion of this study will further our understanding of scleroderma lung disease. It will also allow us to develop biologic markers and predictors of disease progression and to determine which patients would benefit most from treatment with cytotoxic therapy.


A study of the macroscopic properties of thermo-sensitive poly(DIOMMA) revealed a reversed temperature dependence in water versus alcohols. Gels made of the cross-linked analog also showed the same reversed trend between both solvents. We have studied the role of solvent in molecular processes of the polymer aggregations (PA) and the volume phase transitions (VPT) of gels in water or alcohols. We have completed extensive temperature dependence studies of 1H NMR spectra and some relaxation times for poly(DIOMMA) and its cross-linked analog (systems A and B) at both 360 and 500 MHz. Based on these molecular level data, we can deduce the relative strength and nature of the polymer-polymer, polymer-solvent and solvent-solvent interactions. Volumes of gel bead systems (these were created by cross-linking poly(NIPA), poly)NNPA), poly(NIPMA) and poly(NNPMA) with water) shrink at temperatures above the phase transition temperature (ph. tr.T). Spectral changes in 1H NMR (360 MHz) have recently been analyzed from 3 to 65 *C. These data have revealed details of differences in the molecular processes of the VPT among [unreadable]these gel systems. Spectra at temperatures below ph. tr. T (swollen state) consist of several peaks representing various chemical groups of the polymer. The linewidths of all peaks in the spectra suddenly broaden just above the ph. tr. T. These broadenings increase with rising temperature and, at the same time, a broad peak emerges near the HOD peak. Eventually all peaks coalesce to this new broad peak, except in the poly(NNPMA) system where the linewidths of all peaks become rather narrow again at high temperatures. Determination of the ph. tr. T. is usually defined as the onset of volume contractionexpansion measured by certain macroscopic methods. However, the IH NMR spectra indicate that significant spectral changes of the polymer component certainly begin within a narrow temperature range around the ph. tr. T., though gradual changes still continue for 10 to 30* C above the ph. tr. T. These results suggest that molecular processes in the VPT occur rather gradually by manifesting various conformational changes in the polymer chain with changing temperature. Our recent measurements of the temperature dependence of density of various gels demonstrated that onset of volume contractionexpansion corresponds to losing or gaining solvent molecules to or from the bulk liquid phase. Therefore, broadening of NMR linewidths in the collapsed state can be attributed to close proximity of various chemical groups caused by the loss of water and thus increasing the nuclear magnetic dipolar interactions between protons.


Structural abnormalities of the amygdala/hippocampal complex are a consistent finding in schizophrenia. In animal models, neonatal damage to the amygdala/hippocampal complex results in adult onset dopamine (DA) dysregulation, another key feature of this illness. Maintaining the integrity of amygdala/hippocampal circuits therefore appears critical to later DA function. We have previously shown that the extended amygdala, a major output region of the amygala and other temporal lobe structures, has broad inputs to the dopamine neurons. This pathway is thus a potential route by which amygdala-hippocampal abnormalities may eventually lead to DA dysregulation. The proposed studies will examine how the amygdala and hippocampus can influence the midbrain DA system through the extended amygdala. The fact that temporal lobe injury results in DA dysregulation only later in development suggests that plastic changes eventually influence DA output. Our preliminary results show that B lymphocyte 2 protein (bcl-2), which protects cells from excitotoxic damage and also has neurotrophic effects, is highly concentrated in specific subregions of the adult primate temporal lobe. Our preliminary results show high concentrations of Bcl-2 positive cells in the extended amygdala, and in subregions of the amygdala and hippocampus associated with schizophrenia. The presence of bcl-2 in specific circuits may help to identify excitatory pathways most susceptible to plastic changes and/or excitotoxic stress in adult animals. The proposed studies will identify temporal lobe circuits that influence DA through the extended amygdala. Specifically we will: 1) identify direct amygdaloid and hippocampal inputs to the extended amygdala-DA pathway, 2) identify indirect hippocampal pathways through the amygdala that influence the extended amygdala, 3) determine whether specific amygdaloid and hippocampal input/output paths contain Bcl-2 immunoreactive cells, 4) determine the extent to which hippocampal inputs overlap amygdala subregions that project to the extended amygdala, and the extent to which this input overlaps inhibitory interneurons and bcl-2-containing cells.


Cultured human melanocytes provide an excellent model for the study of neural crest development. Discrete growth and differentiation signals can be delivered to homogeneous cell populations and the effects of these signals monitored by changes in morphology, growth rate, differentiated functions and gene expression. In this way, an in vitro model of melanocyte differentiation can be defined and ultimately correlated with characteristic developmental abnormalities of the pigmentary system. The goal of these studies is to critically examine the effect of local tissue environment and exogenous factors suspected to impact on human melanocyte differentiation during development utilizing a sophisticated tissue culture system. We will use known and potential modulators of melanocyte growth and differentiation to examine the effect of these factors on the expression of specific genes and protein products potentially involved in the differentiated function of melanocytes. The effect of nerve growth factor, laminin, fibronectin and vitamins A and D, and medium conditioned by cultured keratinocytes on melanocyte morphology (dendricity), proliferative capacity, melanin synthesis and the expression of the receptor for nerve growth factor will be determined using the appropriate techniques (immunofluoresence, immunobloting, northern blotting). We will subsequently use phorbol esters, known to affect melanocyte morphology, to examine the induction of genes potentially involved in melanocyte growth and differentiation. We will ultimately attempt to transfect the human nerve growth receptor gene into cultured human melanocytes in an effort to explore the tissue specific expression of this gene. In this way, we hope to construct a model of normal human melanocyte differentiation in vitro.


The initial aim of this research is to determine whether persistently elevated luteinizing hormone (LH) in circulation is causally related to excessive follicular development and superovulation in the cyclic hamster. The ultimate aim is to determine the usefulness of the elevated LH system as an animal model for the study of cystic follicles. Elevation of the circulating LH levels will be accomplished by sc insertion of Alza mini-pumps containing ovine LH S 22 (less than 0.5% contamination with FSH). Preliminary studies have shown that elevation of serum LH concentrations (using 400 ug LH minipumps) beingnning on day 1 of the cycle (estrus) culminates in superovulation 4 days later. Further elevation of the circulating LH (using 1000 ug LH minipumps) blocks ovulation and cystic follicles persist in the ovary. Perhaps, the elevated serum LH levels and cystic follicles in the hamster may indeed mimic a condition found in some women who have elevated serum LH and polycystic ovaries. In order to substantiate the hypothesis that elevated LH (in the presence of normal serum concentrations of FSH) induces follicular development and to 'rule out' the possibility that FSH contamination of the LH is the active component in superovulation, several preparations of highly pure LH (less than 0.05 contamination with FSH) will be tested for the ability to induce follicular development. To provide some universality to the discovery that elevated LH enhances follicular development, I will attempt to induce superovulation in the rat, mouse and hamster. Since the recruitment of follicles by the persistently elevated LH (0.5% contamination with FSH) may be due to FSH contamination, several experiments are designed using hypophysectomized hamsters implanted with 400 and 1000 ug LH minipumps to assess whether LH (and less thna 0.5% FSH) can stimulate follicular development. In addition, hypophysectomized and cyclic hamsters will be implanted with 2 and 5 ug ovine FSH minipumps (the doses of FSH contamination in 400 and 1000 ug LH minipumps, respectively) in order to ascertain any effect of these small doses of FSH on follicular dynamics. The result should provide some insight into whether the LH-minipump system is a useful model for the study of the effects of persistently elevated circulating LH on follicular development and cystic follicles.


The Cold Spring Harbor Laboratory proposes to continue its highly successful summer course in Advanced Bacterial Genetics. This course has its roots in the original Cold Spring Harbor Bacteriophage Course initiated by Dr. Max Delbruck in 1945. The course will be offered for three weeks in June from 1993-1997. This course prepares the participant to enter directly into research that makes use of advanced and/or specialized techniques and concepts in molecular genetics. Participants gain extensive first-hand experience with materials and systems that have led to recent advances in prokaryotic molecular genetics. A solid grounding in classical genetic analysis supports training in contemporary uses of transposon mutagenesis, operon and gene fusion technology, molecular cloning, site-specific mutagenesis and polymerase chain reaction as applied to diverse problems in bacterial and bacteriophage genetics. This course, which is unique in providing instruction in both genetics and molecular biology, gives participants the wherewithal required to perform analogous experiments in their home laboratories. The course instructors are chosen on the basis of their contributions to and knowledge of the field of bacterial genetics. Daily discussion periods cover the basis of the methodology used and fine points of experimental design and analysis. The course instructors invite guest lecturers to give up-to-the-minute reports on current research. The lecturers have all made significant contributions to their fields, so that direct contact with the lecturers is a valuable experience, especially for the younger course participants. Because the Laboratory is open at all hours, and because of the informal atmosphere, spontaneous discussions between participants, instructors, lecturers and laboratory staff are constant occurrences. Course participants range from graduate students to senior investigators. They are chosen by the course instructors from a pool of highly-qualified and deserving applicants. Because of the short duration of the course, senior scientists can easily attend to experience a period of intense training in an environment that is remote from other demands on their time and attention.


Continuing a systematic study of the inotropic effect of changes in extracellular potassium concentration, including: clear demonstration of voltage-independence of these inotropic effects over as large a range of potassium concentrations as will allow adequate voltage control during voltage clamp; quantitative analyses of the effect of increased and decreased extracellular potassium on the voltage-tension relationship, examination of potassium's inotropic effects under experimental conditions, including potassium-excess and sodium lack contractures, as well as activation induced by voltage clamp depolarizations. Continuing mathematical modelling of activation-related calcium transport, with particular regard to its interraction with potassium.


Acne is a multifactorial disease affecting ~50 million people in the US. Three main factors contribute to the pathogenesis of acne;1) P. acnes growth 2) P. acnes-induced inflammation and 3) overproduction of sebum, all targets for therapeutic intervention. Treatments, such as antibiotics and benzylperoxide (BPO) are directed at the bacterium P. acnes and retinoids target sebum control. Several antimicrobial and retinoids also possess anti-inflammatory activity. However, to date a treatment directed at all three components of acne has not been identified. Signum's isoprenylcysteine (IPC) analogs represent a novel class of topical therapeutics that potentially targets all three contributors of acne pathology. Our preliminary results demonstrate IPC analogs inhibit P. acnes growth. In addition, when topically applied, using an in vivo irritant-dermatitis inflammatory model, our results demonstrate IPC analogs have strong anti-inflammatory activity inhibiting neutrophil infiltration, edema and erythema of the skin. Preliminary results support a mechanism involving the reduction in keratinocyte inflammatory mediator production. We have extended these observations to in vitro and in vivo P. acnes-induced inflammation models, demonstrating IPC inhibition of several pro-inflammatory cytokines. Lastly, IPC analogs have also been shown to modulate GPCR signaling, suggesting a potential role in regulating sebum control through inhibition of the GPCR melanocortin 5 receptor (MC5R) (critical for sebaceous gland lipogenesis). Based on our previous work, we hypothesize by screening our library of IPC compounds we will be able to identify IPC chemotypes with plieotropic effects to be developed as a novel therapeutic for acne. Structure-activity relationship of these results will lead to proof of principle by identifying, designing and testing candidate compound(s) having the highest combined potency towards each pathological factor, which will be subsequently developed in a Phase II application. PUBLIC HEALTH RELEVANCE: Acne is the most common disorder of the human skin and affects up to 80% of individuals in their lives. Three main factors contributing to the pathogenesis of acne are 1) P. acnes growth 2) P. acnes-induced inflammation and 3) overproduction of sebum all of which are potential targets for therapeutic intervention. To date a treatment directed at all three components of acne has not been identified. Successful development of this novel class of topical therapeutics will be the first that potentially target all three contributors of acne pathology.


The long term objective of this proposal is to develop and identify compounds which are effective in treating AIDS and other HIV- related diseases. The studies described in this proposal will aid in the collaborative efforts of this drug discovery group to develop and identify compounds which specifically inhibit HIV enzymes, either reverse transcriptase (RT), endonuclease, or a protease which is thought to be virus specified. The specific aims of this laboratory will be to: 1) collaborate to better characterize biochemical events following HIV infection in cultured cells, both T-lymphocytes and monocytes/macrophages; 2) furnish immunoaffinity purified RT to the group; 3) immunoaffinity purify the HIV endonuclease, characterize its substrate and develop an endonuclease assay; 4) evaluate anti RT, endonuclease, and protease compounds for cytotoxicity and anti-HIV activity in cultured cells, both T-lymphocytes and monocytes/macrophage; 5) using HIV infected cultured cells, collaborate with other investigators to determine the mode of action of anti-HIV compounds; 6) study selection of virus resistant to anti-HIV compounds; and 7) study the interaction of new compounds with other current or new compounds in inhibiting HIV in cultured cells.


Plasmacytoid dendritic cells (pDC), a distinct type of bone marrow-derived cell, play an important role in linking innate and adaptive immune responses. pDC are pivotal in the first line of defense against viral infections through recognitio of viruses by toll-like receptors as well as their ability to produce large amounts of type I interferons (IFN). In preliminary studies, we have discovered a novel population of resident pDC in the cornea. In vivo pDC depletion in our preliminary studies demonstrate that pDC are the major source of IFN-a in the cornea and play a protective role in the host defense in herpes simplex keratitis (HSK). Thus our results suggest that pDC are key participants in fighting viral keratitis, while preserving vision. Identifying specific functions of pDC in HSK and understanding the critical pathways of pDC and T cell migration in the cornea may provide new molecular targets for pharmacological intervention through immunotherapy. However, defining organ- and cell-specific molecular migratory mechanisms is critical, in order to inhibit cell subsets driving disease, without affecting leukocytes required for protective immunity. To address these questions, we have developed a new multiphoton intravital microscopy (MP-IVM) model to study pDC in intact corneas of living mice. This imaging approach uses transgenic mice, with fluorescent pDC, and fluorescent viruses, and will visualize pDC and viruses at subcellular resolution in living animals, allowing us to study their interaction with surrounding cells. Based on preliminary work and that up-regulation of organ-specific combination of vascular adhesion molecules and chemokines regulate pDC and T cell recruitment to the cornea and subsequent migration of pDC from the cornea in inflammation and infection. We further hypothesize that pDC are protective to the cornea in HSK through local corneal IFN-? and TNF-? production and migrate to distinct areas of the dLN after activation in HSK, where they mediate differentiation pathways of CD4+ T cells to T regulatory or T helper (Th)17 cells through IFN-? and IL-6 production. pDC will be studied to investigate the traffic signals that guide them to normal and inflamed corneas and will be used to address the following two specific aims: 1.) To characterize corneal pDC and dissect the molecular mechanisms that mediate their recruitment and egress during inflammation; 2.) To characterize corneal pDC and dissect the molecular mechanisms that mediate their recruitment and egress during inflammation. Identification of these critical pathways of cell migration to and from the cornea will provide new and highly specific molecular targets for pharmacological intervention in inflammatory, infectious, alloimmune and autoimmune diseases. Few effective anti-inflammatory drugs have emerged over the last decades in the ophthalmic field and an urgent need for new drugs exists. HSK is a leading cause of blindness. Effective therapy for HSK would significantly reduce visual impairment, increase productivity, and reduce the burden of treating HSK.


A combination of various pancreatic, morphologic and functional assessments, along with gut hormone release, serves more accurately to gauge severity of disease, and these criteria may then be used to trace, in a longitudinal fashion, the course of the disease and to assess the efficacy of various treatment options. In addition, on the basis of the observation that chronic pancreatitis is biochemically similar to Type I diabetes mellitus, the investigators will compare the complications of these two diseases.


Over 1,000,000 Americans and 15 million individuals worldwide are infected with HlV. These numbers have been projected to be in the range of 40 - 100 million by the year 2000. Chemotherapeutic suppression of viral replication provides the greatest likelihood of beneficial intervention in the natural history of disease progression, at least for the near future. Three nucleoside analogs (zidovudine [AZT], dideoxyinosine [ddI], and dideoxycytosine [ddC]) have been approved for use in the United States. In certain patient populations these can prolong life and reduce morbidity; nevertheless, much room for improvement remains. In addition, herpesviruses (HSV, VZV, and CMV) represent the most prevalent and significant opportunistic viral complications of HIV disease. The identification of effective drug regimens for both HIV and the herpesviruses must contend with a number of hurdles. One of the most important of these is drug resistance. The objectives of this proposal are to characterize the expression, the genetic basis, the mechanisms, and approaches to treat antiviral drug resistance. Specific aims include 1) the investigation of the nature and significance of nucleoside drug resistance, 2) the confirmation or denial of the hypothesis that higher plasma levels of non-nucleoside reverse transcriptase inhibitors can effectively suppress drug resistant virus, 3) the determination whether the clinical failure of the tat antagonist Ro 24-7429 is attributable to TNFalpha and whether inhibitors of TNFalpha can synergize tat antagonist activity, 4) to examine drug resistance with protease inhibitors, 5) to determine whether HIV gene therapies, like antisense oligonucleotides and ribozymes, can select for escape mutants (resistance), and 6) to develop compounds that effectively bypass drug resistance caused by acquired or innate kinase deficiencies of herpesviruses like HSV and CMV. These studies involving both specimens from clinical investigations and more basic molecular biologic and biochemical approaches are intended to elucidate new approaches to treat HIV and opportunistic herpesvirus infections more effectively.


Characterization of complications arising from cardiovascular invasive treatment procedures were analyzed using CDMAS.


Hepatitis C virus (HCV) infection is a major public health problem, putting infected individuals (~180 million worldwide) at risk of developing cirrhosis, hepatocellular carcinoma, and liver failure. Chronic hepatitis C is the leading cause for liver transplantation. Current standard interferon-based therapy, a costly and time-consuming process, has only a ~50% cure rate, and no anti-HCV drugs have yet been approved for hepatitis C therapy. Despite the promise shown by HCV-specific proteases and polymerases as drug targets, the rapid emergence of viral resistance indicates that additional targets and combinations of antivirals will be necessary for effective treatment. We propose to isolate genetic suppressor elements (GSEs) from a library comprising a fragmented HCV genome which could be used both as potent anti-HCV therapeutic agents and as probes to identify and validate new targets for further drug screening. GSEs are nucleic acid or protein/peptide molecules derived from a gene or genome that act as transdominant inhibitors of a particular biological function through a variety of mechanisms, which includes binding to and blocking essential interaction surfaces for protein activity. In order to identify GSEs from within the HCV genome that exert inhibitory activity against HCV, a novel function-based selection system will be developed and implemented. Briefly, a fragmented HCV genome will be delivered to a hepatoma derivative cell line that is sensitive to a cytopathic effect exerted by HCV. The cells containing the HCV-derived genetic fragments will be subjected to a cytopathic challenge by exogenously administered cell culture-derived HCV (HCVcc) infectious particles, and cells surviving this HCV challenge will be enriched in GSEs that exert an inhibitory effect against HCV. Iterative application of this selection procedure will result in the identification of highly potent GSEs that protect cells against HCV infection and cytotoxicity. Preliminary studies will evaluate the degree of the protective effect conferred by the identified GSEs against HCV-mediated cytotoxicity, and the stage in the HCV life cycle at which the anti-HCV effect of the GSEs is exerted. Future studies originating from the identified GSEs are expected to reveal the molecular basis of the anti-HCV GSE activity, thus providing new leads for anti-HCV drug development. It is expected that the GSEs identified from the proposed research, and molecular mimetics derived therefrom, will inhibit HCV infection/propagation through diverse mechanisms, including the inhibition of virus-host cell interactions, thus contributing to the urgent quest for new and effective HCV antivirals on multiple fronts. PUBLIC HEALTH RELEVANCE: Infection by hepatitis C virus is a serious global health problem that causes numerous debilitating liver conditions. The current treatment regime for hepatitis C is time-consuming, expensive, and often ineffective, creating an urgent need for new and effective drugs. Novel anti-hepatitis C genetic suppressor elements isolated from this research will serve both as hepatitis C drugs, and as keys to open doors to new avenues of research in hepatitis C antiviral development.


Investigate activation and deactivation of insulin action on hepatic glucose production and peripheral glucose utilization. There are two separate mechanisms which may account for changes in activation kinetics, namely, changes in regional blood flow in response to insulin and transit of insulin from the circulation to the intersttial space.


Summary The cilium is a widely distributed cell surface organelle that functions as a signaling hub for the vertebrate cell. Ciliary defects leads to a wide range of human diseases collectively referred to as ciliopathies. Multiple ciliopathies, including nephronophthisis, show renal fibrosis. However, the role of cilia in renal fibrosis has not been studied extensively. In this project, we use Arl13b, a gene essential for cilia biogenesis, as an entry point to dissect the role of cilia in renal fibrosis. We generated conditional Arl13b knockout mice. Our results show that deletion of Arl13b in renal epithelial cells leads to renal fibrosis and cysts. Moreover, multiple signaling pathways, including the Wnt, HH and Hippo pathway, are mis-regulated in the mutant kidney. This project focuses on dissecting tissue specific functions of cilia and communications between different cell types in renal fibrosis through both candidate and unbiased approaches. Aim 1 is focused on signaling in renal epithelial cells regulated by ciliary defects and the relationship between different pathways. Aim 2 studies targets in interstitial cells that are affected by ciliary defects and the functional significance of interstitial cilia. Through this study, we will elucidate how ciliary defects in epithelial cells triggers a signaling cascade that eventually lead to renal fibrosis and whether and how cilia in interstitial cells modify this response. These results not only will provide insight to the mechanism of renal fibrosis in ciliopathies, but will also be informative to fibrosis in general.


A recently discovered human epilepsy gene, LGI1 (leucine-rich glioma-inactivated; mutated to cause human autosomal dominant lateral temporal lobe epilepsy or ADLTE) encodes a protein secreted at glutamate synapses during postnatal glutamate synapse development. Consequently, we hypothesize that LGI1 might promote epilepsy through a novel mechanism, by regulating postnatal glutamate synapse maturation. We propose ADLTE mutant LGI1 acts as a dominant negative to inhibit native LGI1 function and arrest maturation. To directly address our hypothesis and contrast the functional effects of epilepsy-associated mutant LGI1 with those of excess wild-type LGI1 on native neural circuits, we created transgenic mice using bacterial artificial chromosomes (BAC) carrying a large 226 kb fragment of mouse genomic DNA encoding the full-length LGI1 gene. The ADLTE 835delC mutation introduced a premature translational stop codon to generate a truncated LGI1 protein. The full-length gene BAC transgenic approach is important to maintain native patterns of gene expression and transcript splicing in order to assess the genes effects on the glutamate synapse development process in vivo. LGI1 is heavily expressed presynaptically at medial entorhinal cortex perforant pathway glutamate synapses innervating dentate granule neurons (MPP- dentate) and also separately in a synapse targeting thalamus. Our overall goal is to define the cellular basis for human ADLT epilepsy. We specifically test whether excess LGI1 magnifies and mutant LGI1 blocks maturation of the following glutamate synapse properties during the postnatal periods when it becomes expressed and throughout adulthood in dentate gyrus and thalamus. We examine: 1) presynaptic glutamate release down-regulation and role of Kv1.1 K+ channel activity; 2) postsynaptic NR2B NMDA receptor current and synaptic plasticity down-regulation, PSD95-Src complex formation, and the role of Src kinase activity; and 3) dendrite branch (e.g., dentate) and axon input (e.g., thalamus) pruning. This murine model of human ADLT epilepsy could link a novel human epilepsy gene to the arrest of glutamate synapse maturation and potentially defining a cellular basis for human seizure disorders beyond ADLTE. PUBLIC HEALTH RELEVANCE: This project seeks to identify the cellular basis of the inherited human epilepsy disorder caused by mutations in LGI1, a secreted synaptic protein. The results should provide new insights into the cellular and molecular basis of human epilepsy, and help identify new potential therapeutic targets to treat this common brain disorder. [unreadable] [unreadable] [unreadable] [unreadable]


Continuous wave EPR spectroscopy is being used to characterize the iron-binding domain of a synthetic model (PMA) of the antitumor antibiotic, bleomycin. The EPR spectra of two very similar low-spin ferric complexes have been differentiated and assigned as a methoxide and hydroperoxide form of Fe(III) PMA.


The U.S. appears to have worse health than people in a number of European countries. Because health reflects an accumulation of processes over a lifetime, we will study how stressful events during the working years may affect pre-retirement health and ultimately longevity. The broad aims of this application are to find whether in panel data the health gap between the U.S. and Europe increases during the working years, but not during the post-retirement years, and to explain these broad facts by differences in the stress induced in the labor market and by differences in the ameliorating effects of economic and social policy (including health insurance). Our analysis will use data on 15 countries, which provides a wealth of variation in institutions in time as well as in space. The principal outcomes will be fourfold. First, this study will clarify at what point in the life-cycle health differences emerge and chart their path thereafter. Second, this study will propose a formal economic and biological model linking indicators of physiological functioning to other health and economic outcomes clarifying some of the concepts measured in the literature and guide empirical work on the topic. Third, this study will provide micro as well as cross-country empirical evidence on the relationship between economic stressful events and physiological dysregulation using biomarkers but also reported health measures. Fourth, because of its cross-country design, this study will exploit differences in social policy across countries and over time to see estimate how these policies contribute to the international differences in health through the effect of physiological dysregulation on health. PUBLIC HEALTH RELEVANCE: The U.S. has fallen behind in terms of life expectancy in the pre-retirement years and large differences in health have emerged between the U.S. and Europe. This project aims to test the hypothesis that differences in physiological dysregulation due to a more stressful working life in the U.S. are responsible for those differences. This project will use a large array of longitudinal datasets in 15 countries to link health outcomes in the pre-retirement years to life histories and exploit differences in social policy across countries.


Squamous cell bronchogenic carcinomas continue to be chemically induced at preselected endobronchial sites in dogs. These preparations allow serial testing during the evolution (transitional continuum) of lung cancers like those which occur in humans. Material from patients and a hamster model will be utilized in adjunctive fashion. We aim to: 1) Make the existing cancer models more cost effective; 2) Delineate relationships between polyploidy and stages of respiratory epithelial carcinogenesis; 3) Develop methods for in vivo preparation and propagation of pre-neoplastic and cancer cells; 4) Determine the role of DNA methylation during lung carcinogenesis; 5) Explore factors of genetic control upon pulmonary carcinogenesis. Toward cost effectiveness we shall use endobronchial sustained release implants (SRI) of carcinogens and heterotopical autogenous segmental tracheo-bronchial transplants treated with carcinogen. Using image analysis and FACS separation techniques, we shall study ploidy serially during carcinogenesis and work toward improved cell culture methodology. Transfer of tumor cells to nude mice shall be used for cancer cell propagation; importantly adjunctive, in vivo autogenous tracheal segment grafts treated with carcinogen shall be used to propagate autogenous bronchial cancer cells. Pre-neoplastic cell populations shall be harvested from autologous tracheo-bronchial grafts and endobronchial sites undergoing bronchial carcinogenesis. Lung cancer specimens from humans, dogs, and hamsters shall be used to test the hypothesis that undermethylation is a key factor in bronchial carcinogenesis. The effect of the methylation inhibitor 5-azacytidine upon the continuum of bronchiocarcinogenesis will be tested in dogs and hamsters. Using karyotyping in correlation with assessment of total DNA and DNA methylation measurements, the biologic and genetic differences between more and less carcinogen susceptible hosts will be explored. This project involves collaboration between surgeons, cytogeneticists and molecular biologists seeking better to understand lung cancer. It utilized specimens from three species - man, dog, hamsters and proposed specifically to use the canine model for studies not possible with humans or hamsters.


This proposal concerns the roles of Axin and the related gene Axin2 in regulating the canonical Wnt signal transduction pathway during mammalian embryogenesis. The Wnts are a family of secreted factors that play important roles in cell proliferation, patterning and differentiation during development. Axin is a critical component of a protein complex that controls intracellular signaling downstream of Wnts, by regulating the levels of [_-catenin, a transcriptional co-activator and a key effector in the pathway. Axin2 is believed to have a similar function, although it is not as well characterized. Mutations in both genes in humans have shown that they are tumor suppressors, consistent with their roles in negatively regulating the Wnt pathway. The analysis of mice with mutations in Axin and Axin2 have revealed that these genes are important for early axial patterning, as well as for craniofacial and brain development. Craniofacial development is a complex process that involves interactions between the surface ectoderm, endoderrn, mesoderm and the neural crest, and it is highly susceptible to genetic and environmental perturbations, as craniofacial abnormalities are among the most common birth defects in humans. The Wnt signaling pathway has been implicated in eraniofacial development through several mouse mutations. In this proposal, the hypothesis will be tested that Axin and Axin2 are required to negatively regulate the response to certain Wnts by the cranial neural crest cells that form much of the face, and that mutations in Axin and Axin2 lead to inappropriate activation of the Wnt pathway. This will be addressed by analysis of mutant mice, as well as through the conditional manipulation of the Wnt pathway in the cranial neural crest, using transgenic approaches. To understand how Axin and Axin 2 cooperate during development, it is important to define the extent to which they are functionally redundant, and this issue will be investigated through a gene replacement strategy in mice. Finally, to test in vivo the importance of specific domains of Axin that are believed to mediate its functional interactions with other components of the pathway, several novel mutant alleles of Axin will be generated, and examined for their effects on mouse embryogenesis.


Critically ill patients with acute lung injury and acute respiratory distress syndrome (ALI/ARDS) who receive mechanical ventilation can suffer from severe sleep disruption despite continuous sedative infusions. Sleep disruption, in turn, may activate the sympathetic nervous system and cause elevation of circulating inflammatory cytokines, which, in turn, may play a causative role in delirium and post-traumatic stress disorder through consolidation of unpleasant memories during awakenings from sleep. Currently, there is very little understanding of the inter-relationship between critical illness, sleep, and neuropsychological well-being, due to the lack of intervention-based trials that improve sleep during critical illness. The central purpose of this application is to study the short-term effects of sedation with sympatholysis (central 12 adrenergic agent) on sleep and inflammation in critically ill patients with ALI/ARDS. Sedation with sympatholysis will be achieved by a novel sleep-promoting agent with central 12 adrenergic properties. We will undertake sleep studies and measure circulating inflammatory cytokines that modulate sleep in patients with ALI/ARDS randomized to receive two different sedation strategies: central 12 adrenergic sedative-analgesic (dexmedetomidine) versus a conventional sedation strategy (midazolam and fentanyl) in a randomized, double blind, cross-over study. Specific Aim 1: To assess the short-term effect of an 12 adrenergic agent on sleep quality in critically ill patients with ALI/ARDS. Specific Aim 2: To assess the short-term effect of an 12 adrenergic agent on sleep-modulating inflammatory cytokines in critically ill patients with ALI/ARDS. Specific aim 3: To determine the effect of 12 adrenergic agent on the in-vitro production of sleep-modulating inflammatory cytokines by peripheral blood mononuclear cells of patients with ALI/ARDS. Collectively, our study will identify the best sleep assessment tool in patients with ALI/ARDS and whether sleep disruption in such patients can be minimized. In the long-term, this program of research will identify sedation practices that are least associated with adverse short- and long-term consequences of critical illness, and thereby ultimately help improve quality of life of patients surviving critical illness. PUBLIC HEALTH RELEVANCE: During life support for respiratory failure, patients frequently awaken from sleep despite sedatives, and upon surviving may suffer from flashback of traumatic experiences that were memorized during awakenings from sleep (called 'PTSD'). There is little understanding of how such patients' sleep should be measured or improved. We will find the best sleep measurement tool and administer a new medication that improves sleep by soothing inflammation which may, in the future, improve the quality of life of patients.


Early onset generalized dystonia is a common but poorly understood movement disorder, affecting young people, with little meaningful intervention. Clinical evidence has strongly implicated abnormal dopaminergic transmission in the basal ganglia as a major etiology of dystonia. Two recent findings by our colleagues now allow the investigation of the molecular and cellular basis of early onset dystonia: 1) a strong candidate gene DYTl has been identified on human chromosome 9, and 2) expression of the DYTl message in the brain is restricted neuroanatomically to the substantia nigra, cerebellum and hippocampus. The DYTl gene product, torsinA, is a member of a novel family of human genes (torsin-related proteins) of unknown function. TorsinA has sequence homology with functional domains of the large Clp ATPase/HSP100 gene family. Prototype members of this family are characterized by ATP-binding and formation of functional oligomeric complexes. This project is designed to use an efficient expression system to test hypothesis of torsinA function modeled from its homology to the Clp/Hsp family, including protein stability in stress conditions. The regulation of torsinA gene expression, subcellular distribution of the protein will be determined in neuronal cell lines and primary neuron cultures. The role of mutant torsinA as a dominant negative will be measured directly in functional studies of neuronal function, such as stress response, oxidative phosphorylation and dopamine release. The expression system will be designed to differentiate different torsin members, and mutant from wild type torsinA and allow efficient delivery into neurons in different experimental systems. This expression system is designed as a 'bridge' technology, to link the new discoveries of the genetics of the torsins to the cell biology and function of these proteins in the normal and diseased state.


Project Summary/Abstract The emergence and reemergence of pathogenic viruses represent continuous infectious disease threats to public health. Among these, the paramyxoviruses, which include many important human and animal pathogens, also include two excellent examples of emerged, zoonotic viral pathogens of importance: the henipaviruses; Nipah virus (NiV) and Hendra virus (HeV). NiV and HeV have a uniquely broad host tropism capable of infecting at least 18 animal species across 6 orders of mammals. NiV and HeV can also cause a systemic and often fatal respiratory and/or neurological disease in 11 mammalian species including humans. These henipaviruses remain significant biothreats to humans and economically important livestock in Australia and throughout South East Asia. In addition, there are no vaccines or therapeutics approved for human use. The henipaviruses are single-stranded, negative sense, enveloped RNA viruses and possess two membrane anchored glycoproteins involved in virus entry, one mediates host cell receptor attachment (G glycoprotein) and the other is a Class I fusion (F) glycoprotein which facilitates virion and host cell membrane fusion. The viral G and F glycoproteins are the major antigenic targets of neutralizing antibodies and are the main focus of active vaccine strategies. We have been characterizing the non-pathogenic species of henipavirus, Cedar virus (CedPV). Using recombinant viral glycoprotein mediated cell fusion assays, we have determined that CedPV- mediated fusion tropism is similar to NiV and HeV, however, the ephrin receptor tropism of CedPV is remarkably broad and fusion is triggered by ephrin-B1 and -B2 as well as the glycophosphatidylinositol- anchored A subtype ephrins-A1, -A2, and -A5. The fusion tropism activity by each of these ephrins is also correlated with CedPV G binding, and using a recently developed reverse genetics system for preparing recombinant CedPV (rCedPV) we have confirmed the fusion tropism findings with rCedPV infection studies. The rCedPV platform now offers a new virological system that can be used in a variety of applications to study henipaviruses safely under BSL-2 containment which will be broadly used by the CETR Research Projects and Cores. But of additional importance, it is an authentic henipavirus system that is attenuated in comparison to NiV and HeV, and can be genetically manipulated to serve as a live-attenuated universal henipavirus vaccine platform, and one likely capable of inducting a long-lasting and balanced immune response. Using this new system our objectives here will be to develop rCedPV chimeric viruses encoding the F and G glycoproteins of NiV or HeV and characterize and explore their potential a novel henipavirus vaccine. Specifically, we will: 1) Construct and rescue rCedPV-NiV/HeV viruses; 2) Characterize the chimeric viruses in cell-based infection and tropism studies; 3) Analyze the immune responses and outcomes in both immunocompetent and immunodeficient mice following infection with rCedPVs; 4) Test the protective abilities of rCedPVs by vaccination in animal subjects, in response to NiV and HeV challenge.


[unreadable] The Center for Clinical Epidemiology and Biostatistics (CCEB), the Gastroenterology Division (GD), both of the University of Pennsylvania (Penn) School of Medicine (SOM), and Penn's Leonard Davis Institute (LDI), in collaboration with the Division of Gastroenterology, Hepatology, and Nutrition (DGHN) within the SOM's Department of Pediatrics, seek the continuation of a highly successful research training program for clinically oriented investigators in Gastrointestinal Clinical Epidemiology. The overarching objective of this program is to train individuals who have completed their clinical training to be rigorous and independent academic clinical investigators in the broad field of gastroenterology. [unreadable] This training program consists of (1) a core curriculum of required courses in clinical epidemiology and health services research methodology and biostatistics; (2) a series of seminars in gastroenterology combining faculty expertise from the CCEB, LDI, and GD; (3) extensive independent readings and individualized tutorials; (4) a required course in gastroenterology epidemiology; (5) elective courses in advanced methods for epidemiology, health services research, and biostatistics; (6) a series of seminars and workshops designed to improve trainees' skills in presentations and manuscript/grant writing; (7) instruction in the responsible conduct of research and regulatory affairs; and (8) the development/completion of a clinical research project in gastrointestinal diseases, under supervision of a mentoring team comprised of at least one methodological, one clinical, and one biostatistical preceptor. The program is designed to achieve: (1) indepth knowledge of the research techniques appropriate for clinical epidemiology and clinically-oriented health services research investigation; (2) intensive, supervised research experiences; and (3) integration and cohesiveness among faculty and fellows through participation in CCEB, GD, and LDI lectures, seminars, and journal clubs. Fellows are candidates for a Master of Science in Clinical Epidemiology (MSCE) degree. When feasible, candidates are encouraged to seek a Ph.D. degree. [unreadable] The strengths of the ongoing Gastroenterology Clinical Epidemiology Training Program are a long history of successful NIH T32 research training programs in the CCEB, GD, and LDI; comprehensive and rigorous courses, seminars, and research programs for trainees; and a rich history of collaborative links among the programs. In addition, there are excellent basic science laboratories in the GD (supported by R01 grant funded investigators and an NIH/NIDDK Center for Digestive and Liver Diseases) as well as throughout the SOM, several large databases and tissue banks, and a deep commitment of all SOM Chairpersons and Program Directors to the successful implementation of this training program. The existence of a K30 grant (Clinical Research Curriculum Award), diverse faculety expertise in the CCEB, GD, DGHN, and LDI, and recent successes of trainees provide a strong rationale for the continuation of this successful program [unreadable] [unreadable]


We are concerned with the intracellular expression of the murine thymus-leukemia antigens in normal cells and tumor cells together with changes that follow modulation of surface antigen by specific antibody. The work focuses on mitochondria because they are relatively easy to isolate and have a characteristic morphology. TL antigens are demonstrated by immunoelectron microscopy. We are also characterizing plasma membrane differences on subsets of murine T cells by electrophoretic methods after iodination of intact cells.


In 2004, the Centers for Disease Control reported that AIDS was the leading cause of death for African- American women between the ages of 25 to 34 and African-American men between the ages of 35 to 44 in the United States. Researchers have explained these ethnic/racial disparities in terms of delays in accessing care and difficulties in adhering to medication regimens. These findings suggest that culturally appropriate interventions developed to reduce HIV/AIDS stigma could help improve treatment adherence and in turn, improve health outcomes for African-Americans living with HIV/AIDS. It is the career aspiration of Dr. Rao, applicant for this Patient Oriented Research Career Development Award (K23), to help lessen the burden of stigma associated with HIV/AIDS in vulnerable populations. In this proposal, Dr. Rao outlines a career development plan that will lead to a line of work aimed at making available an effective, culturally- appropriate intervention to reduce self stigma for African-Americans living with HIV/AIDS. Through coursework and mentorship, Dr. Rao will train in culturally-specific models of health beliefs and advanced qualitative and quantitative research methodologies. She will implement a research project that will adapt a self stigma reduction intervention for African-Americans living with HIV. In addition, she will adapt and validate an outcome measure of self stigma for use in a future clinical trial. These adaptations will lead to a pilot test and then directly to a full-scale clinical trial of the intervention. Drs. David Cella and Patrick Corrigan, senior investigators with extensive experience in behavioral interventions and patient reported outcomes measurement, will serve as co-sponsors for this project. The clinical site for this project, the outpatient HIV Clinic at Northwestern Memorial Hospital, serves a patient population with much ethnic/racial and gender diversity, and its patients enthusiastically participate in clinical research. During the award period, Dr. Rao will be based at Evanston Northwestern Healthcare and have faculty appointments at Northwestern University. Her location at these institutions, with their outstanding reputation in research and education, will provide her with an outstanding environment, rich with the research resources she will need to become an independent investigator. Dr. Rao will be uniquely positioned to carry out this work aimed at reducing HIV stigma, and subsequently improving health outcomes, for African-Americans living with HIV.


We face an epidemic of hospitalizations in heart failure (HF), which are often unrelated to HF. We hypothesize that extensive efforts have not successfully reduced hospitalizations in HF because management standards rely on disease-centric clinical guidelines, applied within provider-centric systems of care. Care models often overlook that the HF syndrome arises within a complex and poorly understood multi-morbidity context and how comorbid conditions present according to the type of HF (preserved vs. reduced EF) is not known. Further, as a chronic disease that coexists with multiple other conditions, HF causes self-management difficulties in elderly patients who may lack the needed support. Understanding the epidemiology of coexisting conditions and the determinants of successful self-management is fundamental to design new practice models and reduce hospitalizations in HF. The central goal of our revised application is to respond to the urgent need for new approaches by fulfilling 3 objectives: firstly, understand the complex epidemiology of coexisting conditions in HF, secondly, identify patient-centric attributes conducive to successful self-management by drawing upon the Chronic Care Model, thirdly, develop models that integrate cardiovascular characteristics, coexisting conditions and determinants of self-care to be used at the point of care to engage community support for at risk patients. Our 3 Specific Aims, designed to address these objectives, align with the priorities of the Department of Health and Human Services, outlined in their report Multiple Chronic Conditions: A Strategic Framework. Aim 1 will study the epidemiology of coexisting conditions in HF in a community cohort of persons with HF within the Rochester Epidemiology Project. We will assess the prevalence of coexisting conditions (comorbid diseases, geriatric syndromes) and determine which clusters of conditions most impact hospitalizations. We will assess the emergence and progression of coexisting conditions after HF diagnosis and their association with hospitalizations, according to the type of HF (preserved vs. reduced EF). Aim 2 will evaluate the role of patient-centric factors (social support and self-management) on hospitalizations in HF guided by the Chronic Care Model in a prospective cohort of patients living with HF. Aim 3 will develop and evaluate prediction models to identify patients with HF at high risk for hospitalizations. In doing so, we will leverage the unique Health Information Exchange (HIE) capabilities and community partnerships established by the Office of the National Coordinator for Health Information Technology-Funded Southeast Minnesota Beacon Program and assess the feasibility of adapting an existing HIE architecture to implement such models at the point of care, identify patients at high risk for hospitalizations and alert community care coordinators when their patients have been hospitalized to enable proactive community support. By executing these aims, we will understand how the epidemiology of coexisting conditions and how patient-centric factors contribute to the burden of hospitalizations in HF. We will develop risk prediction models, which integrate cardiovascular characteristics, coexisting conditions and patient-centric factors to optimize tools for interventions to reduce hospitalizations in HF.


The introduction of the PCR technique has provided a powerful tool to analyze the quality of complete responses in low grade follicular lymphomas. Complete remissions confirmed by PCR ("molecular complete remissions") rarely occur with traditional chemotherapy regimens for low grade lymphomas. Preliminary studies conducted in our Institution have shown that a new strategy consisting of 3 alternating non-cross resistant chemotherapy combinations, is capable of inducing a high rate of molecular complete remissions in these disorders. The primary objective of project #1 is to use the molecular response as a surrogate endpoint to compare this new strategy against less intensive strategies. All low grade follicular lymphoma tissues will be phenotyped and genotyped. The peripheral blood and bone marrows will be examined by the PCR technique to detect bcl-2 or JH rearranged sequences before and' after therapy. Patients with Ann Arbor stage I-III will then be randomized to either the novel intensive alternating therapy or to standard management with total nodal radiation. Those with stage IV presentations will be randomized to either the same intensive alternating therapy or a new and effective but less toxic regimen consisting of Fludarabine, Mitoxantrone and Dexamethasone. Patients will be followed periodically with the standard clinical restaging techniques as well as with peripheral blood and bone marrow PCR. The primary endpoint of these trials will be the percentage of cases who achieve a molecular complete response at the end of 18 months. A secondary goal will be to more firmly establish the value of the PCR as a means of assessing response by correlating it with the clinical outcome. Rigorous evaluation of these therapeutic alternatives demands a large patient population with close and uniform disease monitoring as well as a cohesive group of investigators.


The proposed research will continue investigations on: Alterations in the metabolic pool at the site of both aqueous humor and cerebrospinal fluid production in groups of unoperated animals and in animals in which bilateral superior cervical sympathetic ganglionectomy will be performed. In addition, a third group of sham-operated animals will be used. That supersensitivity to catecholamines takes place in sympathectomized animals has been shown previously in this and other laboratories. Ocular fluid dynamics in all animals will be determined by repeated weekly testing of ocular pressure (IOP) (with the Mackay-Marg tonometer), as well as measurement of pupil size. A base-line of measurements will have been made for a period of about two weeks prior to the performance of any surgery. Again, prior to surgery, this base-line will be followed by a period of testing of ocular responses to phenoxybenzamine (PBA), norepinephrine (NE) and prostaglandins (PGs). At the termination of a study, the animals will be anesthetized, the eyes cannulated and outflow resistance and IOP measured. The animals will then be sacrificed and tissues and fluids removed. The metabolism and/or concentrations of cyclic adenylic acid (cyclic-AMP) and the PGs will be of particular interest. We shall, in other words, look closely at the metabolic and physiological nature of ocular and non-ocular tissues in animals which would be made catecholamine-supersensitive (via the above surgical procedure) at sites of sympathetic innervation as compared with sham-operated and unoperated animals. BIBLIOGRAPHIC REFERNCES: Waitzman, M.B. and Law, M.L., "Changes in Prostaglandin Concentration in Blood Subjected to Repetitive Freezing and Thawing", Prostaglandins 10:949-962, 1975. Jackson R.T., Waitzman, M.B., Pickford, L. and Nathanson, S.E., "Prostaglandins in Human Middle Ear Effusions", Prostaglandins 10:365-371, 1975.