ABSTRACT Alzheimer?s disease (AD) is the most common form of neurodegenerative disorder. Inflammatory changes in the brain are thought to represent key processes in the onset and progression of AD, but it remains unclear whether neuroinflammation confers neuroprotection, accelerated degeneration, or possibly both. Such an understanding in living humans is critical if we are to begin clinical trials using the array of FDA-approved immunomodulatory drugs in the future. We propose that complement- mediated neuroinflammation is protective in the early AD stages, while suppression of complement activities is accompanied by the development of greater cognitive deficits and faster cognitive decline. Our preliminary data from multiple cohorts support this hypothesis by showing 1) reduced levels of cerebrospinal fluid (CSF) complement-related markers occur in the dementia stage but not mild cognitive impairment (MCI) stage of AD; 2) reduced CSF complement-related markers and elevated CSF interleukin-10 (IL-10) levels are associated with faster decline in AD; and 3) CSF inflammatory protein alterations reveal networks of cellular and protein regulations. In the In the current application, we will build on the association between complement related proteins and rates of cognitive decline in AD to identify associated changes in soluble CSF cytokines and chemokines, differential inflammatory cell type regulation, and imaging correlates of neuroinflammation. This application takes advantage of our group?s strengths in performing CSF cytokine measurements, CSF immunophenotyping, molecular imaging of neuroinflammation through positron emission tomography (PET) and iron-enhanced MRI, and network analysis through a novel biochemical-bioinformatics pipeline. We will directly identify individual and networks of soluble CSF cytokines that accompany the transition from the MCI to the dementia stage of AD, correlate the complement and other altered pathways with microglial activation through two modern PET tracers (11C-PBR28 and 18F-FEPPA), and measure changes in individual T helper cell (type 1, 2, 17) and non-T cell populations. This application represents the first attempt to correlate, at the individual level and at the group level, CSF and imaging measures of neuroinflammation. If successful, this application will advance the understanding of neuroinflammation in AD through parallel approaches, form the basis of a new biomarker panel (and algorithm) to diagnose AD through a combination of degenerative and inflammatory markers, and accelerate the target identification of future therapeutics aimed at modulating the immune system in AD.


The purpose of this protocol is to develop novel methods of performing magnetic resonance imaging (MRI) evaluations so that these methods can be transferred to the clinical environment. Normal volunteers are recruited to optimize imaging techniques and the protocol has been very successful in recruiting normal volunteers. Among the accomplishments of this protocol over the last year include optimizing contrast administration rates during magnetic resonance angiograms, automatic table motion techniques for peripheral run-off magnetic resonance angiography (MRAs), phase contrast angiography, motion tracking for knee and patella movement, functional MRI of the brain, gated MRI to image the soft palate, and stroke protocols. We have made substantial gains in technical development in all of these areas and there have been no complications. We have developed protocols to be used in conjunction with Suburban Hospital in MRA, stroke, and cardiac imaging, and will continue this study.


The long-term goals of this project are to understand how cell-to-cell signaling systems interface with regionally expressed transcription factors during development to generate correctly proportioned and patterned body parts. The experimental system that will be used is the fruit fly, Drosophila melanogaster. The starting point for these experiments are two genes, homothorax and extradenticle, that are important for specifying the identity of the proximal portion of the appendages in flies. These genes appear to limit or modify the activities of signaling pathways known to operate during appendage development. Both extradenticle and homothorax encode homeodomain-containing transcription factors. However, Extradenticle protein is only nuclear in the presence of Homothorax. In several tissues, Homothorax and Extradenticle are co-expressed with a Zn-finger-containing transcription factor encoded by the teashirt gene and, in the eye, these three proteins are co-expressed with another homeodomain protein encoded by the eyeless gene.The specific aims for this funding period are to: (1) determine the mechanism by which Extradenticle's nuclear localization is controlled by Homothorax, (2) characterize the roles of homothorax and teashirt in wing and eye development, (3) characterize the genes involved in repressing hth expression during leg development, and (4) characterize the role of novel genes, identified by a forward genetic screen, in proximo-distal patterning in the fly appendages. homothorax and extradenticle both have vertebrate homologs that, when misexpressed, can contribute to leukemias, suggesting that they also play a role in regulating growth and development in vertebrates. In addition, the transcriptional and post-transcriptional regulation of these factors appears similar in vertebrate and fly limb development, suggesting that many of their functions may also be conserved. An understanding of their basic functions in fly development should therefore generate information that is relevant to leukemia, as well as to defects that can occur in vertebrate limb development.


Fungal infections are becoming an increasing public health concern, particularly among immune compromised patients or those undergoing chemo- and/or radiotherapy. Two fungi of particular concern are Aspergillus niger and Candida albicans. We have obtained crystals of the essential DNA repair enzyme dUTP pyrophosphatase (dUTPase) from C. albicans. Determination of the structure of the fungal dUTPase, and comparison with the structure of the human enzyme, recently determined in our laboratory, may allow design of inhibitors specific for the C. albicans enzyme for use as anti-fungal agents.


The object of the proposed research is to further understanding about the mechanisms that all vertebrates have evolved to survive the thermal stresses of the environment. Four major areas will be examined with this objective: (1) Thermal acclimation in teleost fish. The importance of oxygen transport and acid-base imbalance will be evaluated by allowing cold acclimated fish to return to the final thermal preferendum while dissolved oxygen and carbon dioxide concentrations are varied. Appropriate measurements of blood gases will also be made; (2) The mechanism of torpor in teleost fish. Fish will be acclimated to a large number of temperatures. Metabolic rate and spontaneous activity will be monitored at each temperature. ECG, brain electrical activity, oxygen uptake, and ventilatory frequency will be monitored as fish are slowly cooled. The importance of anterior brainstem temperature in the induction of torpor will be evaluated. (3) Thermal inputs to respiratory-cardiovascular regulation in teleost fish. Dorsal aorta temperature, blood flow, and PO2, and ventilation frequency or ventilatory minute volume will be monitored during rapid thermal shifts. Effects of changes in the anterior brainstem temperature on the above parameters will be evaluated. (4) Phylogenetic development of CNS integrated responses to thermal stresses. Certain of the above experiments will be performed on lampreys. It is felt that answers provided by this research will aid in unifying -- for all vertebrates --the common mechanisms for sensing and integrating important thermal variables as well as serving to better understand the development of specialized vertebrate adaptations such as endothermy and hibernation.


Early knowledge of HIV status is critical to prevent transmission to others, to link those who are HIV positive to medical care and services that can reduce morbidity and mortality and improve quality of life, and to reduce health care expenditure. Consistently, the National HIV/AIDS strategy has established a goal of increasing the awareness of HIV status in Americans from 79% to 90% in the next four years. The proposed research is in direct response to PA-11-119, HIV/AIDS Testing and Follow-up Among the Underserved in the United States, and proposes to investigate the acceptability, feasibility, and initial efficacy of a rapid HIV testing and counseling protocol in residents of domestic violence shelters. Intimate partner violence (IPV) is a pervasive public health problem, and a growing body of research highlights the association between IPV and HIV/STI risk (e.g., increased unprotected sexual occasions, sex with risky partners, trading sex, multiple partners). Further, IPV is associated with many barriers to receiving adequate health care (e.g., transport difficulties, child care needs, safety issues, lack of health insurance). Domestic shelters present an opportune setting for providing health care services for women, providing a safe and supportive environment for testing, while addressing many of the barriers to testing and linkage to care faced by shelter residents. RESPECT-2 is an evidence- based CDC Diffusion of Effective Behavioral Interventions (DEBI), utilizing a client-focused, interactive HIV risk reduction counseling model delivered in conjunction with rapid testing. Residents of domestic shelters, however, face distinct HIV risk factors, such as difficulty in negotiating condom use out of fear of retaliation from their abuser, and thus needs to be tailored to meet the specific needs of shelter residents. Thus, in the proposed exploratory/developmental research plan, we aim to expand RESPECT-2 for our target population (i.e., RESPECT-IPV), and to collect preliminary data on RESPECT-IPV + rapid testing's feasibility, acceptability, and initial efficacy in a sample of 100 high-risk shelter women. Predictors of acceptability (e.g., PTSD symptoms, substance use, IPV severity, prior testing history, HIV knowledge, HIV risk behavior, HIV anxiety, stage of change) will also be explored. Our primary hypotheses are that RESPECT-IPV will be associated with reduced unprotected vaginal or anal sex occasions at 1-week and 3-months post-shelter and fewer cases of vaginal trichomoniasis 3-months after leaving shelter.


A workshop convened in 2013 by National Institute of Aging and the National Institute of Neurological Diseases and Stroke prioritized research on cognitive impairment of multiple etiologies, contributions of vascular brain injury (VBI) to neurodegeneration and Alzheimer's Disease (AD), and disparities involving race, ethnicity, socioeconomics, and rural residence. VBI is a leading cause of accelerated brain aging and a major risk factor for stroke, cognitive decline, depression, and probable Alzheimer's disease (AD). However, few studies have evaluated VBI, cognitive impairment, and AD in American Indians (AIs), who bear a heavy burden of risk factors for these conditions. This exclusion is unfortunate, since the distinctive history, risk profiles, environmental stressors, social environments, and healthcare systems of AIs likely result in patterns of disease that differ substantially from other populations. Our research team has conducted the only cohort study to date of covert VBI in AIs. In 2010-2013, Cerebrovascular Disease and its Consequences in American Indians study completed standardized clinical examinations, neuropsychological testing, and cranial MRI on more than 1,000 AIs aged 64-95 years from 10 tribes in 3 states. In 2016, we began re-examination of surviving participants using the same protocols, augmented by assessment for probable AD. However, the particular grant mechanism from the National Institute on Aging that provides funding for this follow-up examination is limited to data collection, with no resources allocated for analysis. In this proposal, we will capitalize on the accumulated longitudinal data to apply sophisticated imaging and analysis methods to quantify and evaluate associations for incident VBI, neurodegeneration, and changes in cognitive status in this elderly minority population. Our Specific Aims are to: (1) establish normative and diagnostic standards for mild cognitive impairment and dementia in elderly AIs, and evaluate associations and determinants for incident VBI, cerebral atrophy, cognitive decline, and probable AD; (2) conduct cluster analyses to identify novel neuroimaging profiles that predict subgroups of elderly AIs at highest risk for cerebral atrophy, cognitive decline, and probable AD; and (3) complete a rigorous career development plan that will position the applicant as an independent investigator and leader in innovative neuroepidemiology research with minority populations. This proposal leverages a timely opportunity to generate the first population-based estimates of and address novel scientific questions on VBI and cognitive impairment in AIs, a minority population with pervasive health disparities.


Practice-based research networks (PBRNs) are organizations of clinicians working in practices that engage in research. PBRNs have emerged as a vital lynchpin in efforts to translate research into practice by translating practice into research. However, the development and maintenance and the continued engagement of busy community practices in cancer research requires specialized expertise and ongoing support. Therefore, the mission of the PBRN Core Facility is: [unreadable] To develop and sustain practice-based research networks that engage in cancer research; [unreadable] To advise and assist Case Comprehensive Cancer Center (Case CCC) investigators in developing community-based cancer research studies for implementation in PBRNs; [unreadable] To provide infrastructure that supports the implementation of Case CCC investigators' research studies in PBRNs, and that supports network practice-initiated research. The Practice-Based Research Network Core Facility supports the engagement of patients, clinicians, practices, and health systems in cancer research by: 1. Starting new PBRNs; 2. Developing the infrastructure of existing PBRNs; 3. Providing PBRN methods consultations to Cancer Center investigators and participating practices; 4. Advising and assisting investigators on research project implementation in PBRNs. The PBRN Core has started and sustained 8 successful PBRNs and is developing 5 new PBRNs. The established PBRNs represent major healthcare systems and include family medicine practices, federallyqualified health centers, community oncology practices, pediatrics practices, and general dentistry practices. The PBRN Core has facilitated the development and implementation of 23 funded research projects within the PBRNs that it has developed and sustained. Fourteen of these projects have been funded by NCI, and 6 supported by ACS. Grant funding for these projects totals over $15 million. The demand for the PBRN Core's services is strong and growing, and the potential for continued and expanded use of the facility is excellent. At present, 19 members of the Case CCC are pursuing 15 different lines of inquiry likely to result in funded studies to be implemented in Core-supported PBRNs.


Electrical activity in circadian clock neurons plays an important role in cellular oscillation. However, the particular voltage-gated ionic conductances necessary for circadian function and the mechanisms by which electrochemical signals interact with transcriptional events in the nucleus of clock neurons remain unknown. To identify the particular ionic conductances necessary for cellular oscillation, ion channel subtype-specific membrane-tethered toxins are transgenically expressed in clock neurons to interfere with voltage-gated conductances. Preliminary studies reveal substantial effects of in vivo expression of Ca2+ and K+ channel tethered toxin blockers on free-running behavioral rhythms. The proposed aims are (1) to test dose- dependence of these behavioral effects, (2) to examine effects of tethered toxins on cellular oscillation, and (3) to determine the molecular identities of the blocked ion channels. The long-term goal of this proposal is to improve understanding of circadian clock function, an issue of substantial health relatedness, given the great costs to society of genetic and environmental disruption of human sleep/wake cycles. [unreadable] [unreadable] [unreadable]


Transcription from Recombinationally Activated Immunoglobulin Genes. Specialized nucleic acid elements are involved in the expression of immunoglobulin genes. These elements are 'enhancers' which act positively to promote transcription. The DNA rearrangements that occur during the construction of an antibody gene bring the enhancer near an otherwise weak promoter, thus activating the promoter. Particularly significant is the fact that unlike other enhancers, which are carried on viruses, the immunoglobulin enhancers are tissue specific--they only function in B lymphocytes. This suggests that B cells contain, as a result of their differentiation, proteins which are designed to function as specific transcriptional factors at this site. This application proposes two general lines of experimentation aimed at identifying and isolating the transcriptional factors involved in the expression of immunoglobulin genes. The first proposed approach is biochemical and involves trying to isolate the enhancer recognition protein directly, for example by assuming that it will specifically bind to enhancer DNA. In addition, an in vitro transcription system will be developed in which messenger RNA synthesis from DNA fragments--containing immunoglobulin genes with or without the enhancer--is tested for stimulation by protein fractions derived from B lymphocytes. The second general strategy is to seek mutants in the gene which codes for the enhancer recognition protein, and then use the mutants to isolate the gene itself. The fact that the immunoglobulin enhancer works only in B cells has allowed the design of three specific experimental strategies. Each is based on the idea of placing a drug resistance gene (neo) under immunoglobulin enhancer control and introducing the construction into various cells. For example, in B cells which are also caused to contain extra copies of the cloned enhancer DNA sequence, only a mutation to overproduction of the enhancer recognition protein will allow the neo gene to be expressed. This mutation would aid the biochemical studies described above, and potentially also allow the regulatory gene which produces the enhancer recognition protein to be cloned. With both the protein and nucleic acid components involved in immunoglobulin gene expression in hand, it should be possible to study this example of a regulated eukaryotic gene to the same depth that has been possible with, for example, the lactose and tryptophan operons in bacteria. Ultimately, purification of the enhancer recognition protein and analysis of mutations in the enhancer site that affect the proteins' ability to bind to the DNA will begin to allow a basic molecular description of immunoglobulin regulation and may provide key insights into the processes of development and differentiation in general. Malaria, DNA Rearrangements and Burkitt Lymphoma. Another experiment related to the recombinational activation of antibody genes attempts to probe the relationship between malaria infection and Burkitt lymphoma, which show a geographic coincidence. By co-cultivation of malaria-infected erythrocytes in vitro with tester mammalian cells, we will test the idea that infected erythrocytes release a clastagenic (chromosome-breaking) compound which could trigger the chromosome translocation associated with Burkitt lymphoma.


(Revised) DESCRIPTION (provided by applicant): Adolescents are at great risk for infectious diseases including the human immunodeficiency virus (HIV). Though the CDC reports that overall AIDS incidence is on the decline, there has been no comparable decline in the number of newly diagnosed HIV cases among young people aged 13-19, and young people of color are particularly at risk. Compared to the general adolescent population, adolescents involved with the criminal justice system are at higher risk for unintended pregnancy and infections. Alcohol use is commonly cited as a reason for risky behavior among high-risk adolescents such as those involved in the criminal justice system (e.g., Morris et al., 1998) and recent data from our research suggests that it is heavy alcohol use that is most strongly related to risky behavior (Bryan, Rocheleau, & Robbins, 2002a). The goal of this research is to design, implement, and test a successful HIV/Alcohol risk reduction intervention that is theory-based, empirically targeted to adolescents, and articulated to a criminal justice setting. The study compares a risk reduction intervention that incorporates an alcohol risk reduction component to a standard risk reduction intervention and a no treatment control condition. We hope to show that: 1) A combined risk reduction intervention will result in larger decreases in risky behavior, 2) The intervention will exert effects through reductions in alcohol use and changes in other mediators derived from a theoretically-based model of intentions and behaviors, and 3) A risk reduction intervention including an alcohol component will be especially effective for those adolescents with higher levels of existing alcohol problems. Finally, if the hypotheses are supported, the long-term objective will be to disseminate the intervention curricula and materials for use in adolescent detention facilities throughout the state.


The low rate of cure of certain cancers such as lung cancer, the most common causes of cancer death, is an important health problem. Early detection appears currently to be the only way of improving the high mortality rate, but is quite difficult because of the lack of symptoms in early disease. Moreover, lung cancer is mimicked in its in vivo image appearance by benign lesions and processes that lower the specificity of detection. Current imaging methods include chest x-ray, CT, and MRI. While these current methods are able to identify curable lung cancer they also result in many false positives. They are also limited in the size of the lung nodules they can detect. Using available criteria, sensitivity for lung cancer detection is high, but specificity and positive predictive value are only moderate. Thus there is a need for enhanced sensitivity and specificity for cancer cells. Our Anti-transferrin Receptor scFv-antibody fragment (TfRscFv) immunoliposome complex (scL) is a nanoconstruct (~100 nm) for delivery of gene therapy to tumors. It has been shown to target various types of human tumor cells in vivo when implanted as xenografts in mice and is now in Phase I clinical trials for delivery of wtp53. What we are proposing in this application is a quantum jump in diagnostic accuracy, an approach specific to cancer and best for small cancers such as lung cancer. The method we are developing is a nano- sized immunoliposome complex delivering superparamagnetic iron oxide particles (SPIO). Iron Oxide particles are both paramagnetic and super-paramagnetic, giving a biphasic response with both T1 and T2* features. This complex targets cancer cells with high selectivity. Thus the efficient delivery of SPIO directly into the tumor cells by the scL-SPIO complex of this application can increase the conspicuity of the lung tumor cells. Moreover, based on previous studies, the nanocomplex delivered contrast agent which is the focus of this application should accumulate within the cancer cells themselves remaining for an extended period (hours) allowing the contrast in non-cancer areas to wash out, further enhancing cancer conspicuity. In preliminary studies using an as yet unoptimized scL-SPIO complex, we demonstrated tumor cell specificity as compared to free SPIO, and enhanced image intensity. More importantly, in earlier studies with scL complexed with another imaging agent, gadopentate dimeglumine (gad-d), in a lung tumor model, the scL-gad-d (and not free gad-d) was able to enhance and identify lung tumors as small as 1-4 pixels (0.1-0.4mm), a size smaller than possible with current technology. No toxicity was found with this complex. In this application we will optimize the scL- SPIO complex and fully characterize its capabilities for use in early detection of lung cancer in a mouse model of human lung cancer and extend our studies to a mouse model of primary lung cancer. In collaboration with investigators at NIST and NCI we will also asses the magnetic properties of the complex and determine it sub cellular localization and trafficking through the cell. Our goal is to perform the majority of the studies necessary for filing an IND as we aim to move rapidly towards clinical trials. If cancer is detected early (e.g. Stage I), it can in many instances be cured (lower mortality). The challenge is to be able to find and positively identify the cancer at this early stage, particularly lung cancer. While the current methods of detection are good, they can only detect tumors of a certain size. Moreover, lung cancer is often mimicked during imaging by non-cancerous lesions, resulting in uncertainty and many false positives, which are often resolved only by following growth of the tumor. Thus there is a pressing need for imaging agents that increase sensitivity and specificity. Our tumor-specific nano complex delivery of an MR imaging agent, e.g. gad-d and iron oxide, has shown great promise in our preliminary studies in this regard, demonstrating that its high affinity for cancer cells in the lung can result in improved sensitivity in detecting tumors and in overall specificity. The development of an imaging agent that can lead to earlier detection is a high priority in the war on cancer and could lead to increased survival.


In order to protect against the four epidemiologically important rotavirus serotypes, we developed a rotavirus vaccine that contains four distinct strains: rhesus rotavirus (RRV) representing serotype 3 (the Jennerian approach), and three human RV-RRV reassortants, each possessing ten RRV genes and a single human RV gene that encodes VP7 (a major outer shell protein) that is responsible for serotype 1, 2, or 4 specificity (the modified Jennerian approach). Efficacy trials of this orally delivered vaccine have been completed in over 10,000 infants and young children with single or multiple vaccine components with successful results in most but not all studies. In five of these trials which included over 7000 infants and young children, the quadrivalent formulation, given orally in three doses, has been shown to be effective: protective efficacy against severe rotavirus diarrhea ranged from 69% to 91% in various locations. The vaccine was 75% to 100% effective in preventing dehydrating diarrheal illnesses. Because of the demonstrated safety (transient fever) and efficacy of the quadrivalent vaccine, on January 31, 1997 Wyeth Laboratories, the licensee, submitted a Product License Application to the Food and Drug Administration (FDA) and on December 12, 1997, the FDA Advisory Committee concluded that the clinical data presented supported the safety and efficacy of the quadrivalent rotavirus vaccine. On February 11, 1998, and again on June 25, 1998, the Advisory Committee on Immunization Practices recommended its use for infants at ages 2, 4, and 6 months of age. Subsequently, on August 31, 1998 the FDA granted a Biologics License to Wyeth Laboratories. and on November 4, 1998, the American Academy of Pediatrics recommended the rotavirus (Rv) vaccine. In addition, on May 7, 1999, the European Commission issued marketing authorization for the Rotavirus Vaccine with this approval covering all 15 member countries of the European Community.In related continuing rotavirus vaccine studies, we are evaluating collaboratively the feasibility of administering the licensed vaccine to neonates to determine its reactogenicity and immunogenicity with different administration schedules. This study will not only provide information on the immunogenicity of the vaccine when the initial dose is given during the neonatal period, but is also designed to compare the immunogenicity of a two-dose vs a three-dose schedule administered at 2 and 4 months and at 2, 4, and 6 months, respectively, the latter the currently approved schedule. We are also evaluating collaboratively several second generation vaccines. One of these, is a quadrivalent reassortant rotavirus vaccine that possesses a single VP7 gene with serotype 1, 2, 3, or 4 specificity and the remaining ten genes from bovine RV (UK). - Human Subjects


The proposed study is a third follow-up of 581 male narcotic addicts admitted to the California Civil Addict program (CAP) during 1962-1964. The subjects were interviewed once in 1974/75 (DA01146), and again ten years later in 1985/86 (DA03425). The combined prospective and retrospective follow-up interviews provide a unique natural history database that can address issues specifically related to long-term narcotics addiction, patterns and consequence of use. Similar interview instruments will be used to collect self-reported measures about narcotics use, legal supervision status, criminal involvement, drug trafficking, employment, marital status, and treatment episodes over the entire addiction career. Corroborative data will be obtained from official arrest records for each interviewed subject, from a voluntarily provided urine specimen to be taken at interview, and from official death certificates for identified decedents. Specific aims of the study are: (1) to provide an additional ten years of data to allow a detailed natural history description over an almost 40-year addiction career of a sample of narcotics addicts; (2) to assess addiction patterns over time identifying factors which influence the divergence of outcomes, in particular, relapse, cessation of use, the process of "maturing-out ", and mortality; (3) to analyze and describe morbidity and death among this aging addict sample; (4) to evaluate the extensiveness of criminal activity and to identify specific criminal career patterns in relation to narcotics use; (5) to conduct a treatment intervention history analysis, examining successive and cumulative treatment effects and the extent to which these individual and cumulative treatment episodes have reduced narcotics use; and (6) to assess long-term costs and consequences of prolonged careers of addiction. Augmentation of the existing addict career database provides a unique opportunity for investigation into the current status of addicts now between 47 and 73 years old.


The object of these investigations is to elucidate the biochemical mechanisms responsible for the reduced cardiac function and mortality that may follow open heart surgery. The availability of additional sufficiently refined methods of studying the cellular chemistry and ultrastructure of relatively intact human myocardium obtained by drill biopsy at the time of cardiac surgery has created a need for this supplementary application to support this work. This will be in addition to the histochemical and biophysical studies already being carried out on biopsy specimens at different stages of an operation. It is hoped that once the abnormalities are fully identified, it may be possible to begin to discover what is required to restore the function of such dysfunctional myocardium (or possibly what modification of the surgical procedure will be necessary to avoid further damage to a diseased heart).


Immune dysregulation may contribute to the pathophysiologic findings seen in SCLS. A monoclonal gammopathy of unknown significance (MGUS, a premalignant precursor to multiple myeloma (MM), in which a clonal plasma cell population secretes monoclonal immunoglobulin (Ig, also referred to as a paraprotein) detectable in patient sera, is present in a majority of SCLS cases. Several patients with SCLS in whom MGUS evolved into myeloma or plasma cell leukemia experienced fewer capillary leak episodes after chemotherapy for their hematopoietic disorder. These findings suggest that the monoclonal paraprotein from the dysregulated plasma cell population may be the direct or indirect source of the pathophysiologic findings observed. We are characterizing the transcriptome of blood cell RNA and the proteome of SCLS serum/plasma, both pre- and post-attack, to determine whether specific biomarkers of acute symptoms and/or etiological factors can be identified. We have now evaluated 58 patients with a confirmed diagnosis of SCLS under this protocol in the last 7 years. We are the primary referral center in the U.S. for SCLS. The transient episodes of hypotensive shock and anasarca in SCLS are thought to arise from reversible microvascular barrier dysfunction. Application of episodic but not convalescent SCLS sera to human microvascular endothelial cells (ECs) caused vascular endothelial cadherin internalization, disruption of interendothelial junctions, actin stress fiber formation, and increased permeability in complementary functional assays. EC contraction and temporary attenuation of adherens junctions may thus permit leakage of solutes and proteins into the extravascular space during acute episodes. Circulating permeability factors, vascular endothelial growth factor (VEGF), angiopoietin 2 (Angpt-2), CXCL10, CCL2, and IL-6, were elevated in episodic SCLS sera compared to remission sera. Thus, angiogenic proteins and proinflammatory cytokines that induce EC hyper-permeability may contribute to transient EC barrier dysfunction around SCLS flares. To test the possible contribution of the Angpt2 pathway and/or other permeability inducers to SCLS, we have expanded blood-outgrowth ECs (BOECs) from circulating precursors obtained by venipuncture. These cells possess structural and molecular characteristics of mature ECs. Preliminary results suggest abnormal gene expression patterns in SCLS BOECs compared to those from healthy controls. Current studies are aimed at examined responses of ECs derived from subjects with SCLS and controls to various mediators of permeability to test the hypothesis that the SCLS endothelium is prone to exaggerated responses to otherwise mundane inflammatory stressors. The role of specific gene defects in SCLS, if any, is also unknown; e.g. whether the endothelium is genetically programmed for hyper-responsiveness to routine stimuli. There are no consistent familial aggregations in SCLS. Using Affymetrix Single Nucleotide Polymorphism (SNP) microarrays, we performed the first genome-wide SNP analysis of SCLS in a cohort of 12 disease subjects and 18 controls. From unbiased high-density mapping of single-nucleotide polymorphisms (SNPs), a small genetic interval, 3p25.3, was identified as the highest-ranking candidate susceptibility locus (p 10-6) with an odds ratio of 41. Odds ratios (7-41) and p values (10-4 and 10-6) for the top SCLS-associated variants were outsized for such a small sample size. These results imply high penetrance for a rare disease allele that remains to be identified. An inbred mouse strain was identified (SJL/J), whose phenotype bears similarity to SCLS, including spontaneous vascular hypersensitivity to a variety of permeability factors including histamine (HA), serotonin, and bradykinin, important mediators of endothelial hyperpermeability in humans, and B cell neoplasms characterized by the presence of serum paraproteins. A recessive locus in SJL mice controlling systemic vascular hypersensitivity to HA (Shs) was mapped to an interval on mouse chromosome 6 syntenic with human 3p25.3, which harbors the primary genetic association with SCLS in humans. This interval contains several gene candidates, which will be analyzed in the SCLS cohort and various mouse strains.


PROJECT SUMMARY Organophosphates (OPs) pose a constant threat to human health due to their widespread use as pesticides and their potential employment in terrorist attacks. The acute toxicity of OPs has been extensively studied; however, the consequences of prolonged or repeated exposure to levels of OPs that produce no overt signs of acute toxicity are poorly understood. Further, there is clinical evidence that such low-level exposures to OPs leads to prolonged deficits in cognition, although the mechanism for this effect is unknown. One long- term goal of our laboratories is to elucidate the mechanisms responsible for the prolonged neurobehavioral deficits associated with chronic low-level OP exposures such that more effective therapeutic strategies can be developed. The results of our experiments conducted during the initial funding period established that low- level exposures to the commercial pesticide, chlorpyrifos, resulted in protracted deficits in prepulse inhibition (a model of pre-attentive processing) and spatial learning without significantly affecting locomotor function. Further, chlorpyrifos was associated with decreases in neurotrophin receptors and cholinergic proteins in brain regions that are important to cognitive function. These deficits were accompanied by decreases in axonal transport measured in sciatic nerves ex vivo. However, the molecular mechanisms for the deficits in axonal transport and the extent to which such effects on axonal transport occur in the brain are unclear. The objective of this application is to identify the mechanisms responsible for alterations in axonal transport as well as to further define the long-term effects of low-level OP exposure on cognitive function. Our central hypothesis is that OPs covalently modify key proteins that are involved in axonal transport and that such modifications compromise the function of neuronal pathways that support cognitive function. To achieve our objective, we propose three specific aims: 1) Determine the consequences of chronic low-level exposure to representative OPs on attention and cognitive flexibility, 2) Determine the consequences of chronic low-level exposure to representative OPs on axonal transport in the brain, and 3) Identify the molecular mechanisms responsible for OP-induced deficits in axonal transport. To address these aims, we will use a five choice serial reaction time task to assess sustained attention, a water maze task to measure extinction (a form of cognitive flexibility) and stereotaxic injections of traceable dextrans, immunohistochemistry, and mass spectrometry to determine OP effects on axonal transport in the brain and the consequences of its impairment. The significance of this project and its relevance to public health is that by mechanistically defining OPs based on their long-term effects on essential components of information processing in animals, we will have addressed a fundamental gap in our knowledge of how OPs likely affect humans over time. The experiments will contribute to a better understanding of the toxicity associated with a class of chemicals that continues to pose a significant environmental risk to millions of people worldwide.


Hepatitis C may in time result in progression damage to the liver and lead to cirrhosis or liver cancer. Interferon alone or in combination with Ribavarin are the only FDA-approved therapies for Hepatits C infection. Treatment in the early stages may prevent progression to cirrhosis. Interferon has several undesireable side effects, therefore, investigators are looking for new drugs that may be useful in the treatment of Hepatitis C. Amantadine is a drug that was developed 35 years ago for the treatment of the flu. Previous research has shown that amantadine may be useful for patients with Hepatitis C and results in a 30% response rate when given at a dosage of 200mg daily. The purpose of this study is to evaluate the efficacy of amantadine in hepatitis C with higher doses of the drug.


Sickle cell disease (SCD) is a systemic ilness effecting over 50,000 patients in the U.S. Transfusions are an important component in the the treatment program for many of the serious complications of SCD. Although a life saving therapy, transfusions also present complications and the persistence of donor white blood cells following transfusion may underlie the immunomodulatory phenomena. This study will examine, prospectively, the natural history of transfused white blood cells and correlate the observations with clinical and laboratory features considered etiologically related.


Project Summary. Estrogen receptor positive (ER+) breast cancers comprise the majority (~70-80%) of breast cancers, the majority of late recurrences emanating from indolent or dormant breast cancer, and the majority of breast cancer deaths resulting from metastatic disease. Anti-endocrine therapy with tamoxifen remains the cornerstone of adjuvant therapy for ER+ breast cancers, particularly in premenopausal women, but also following treatment with aromatase inhibitors in the post-menopausal setting. Nevertheless, many women do not respond to tamoxifen in initial therapy, and of those that do, one third will relapse with resistant and metastatic disease within 15 years. Endocrine resistant ER+ breast cancers therefore remain one of the major causes of breast cancer metastasis and mortality. In fact, initial or acquired resistance to tamoxifen is involved in more than half of all ER+ breast cancer deaths. Reversing resistance to tamoxifen therapy is a crucial overarching breast cancer challenge. We provide preclinical research demonstrating the discovery of a crucial axis that provides tamoxifen resistance to ER+ breast cancer cells mediated by the epidermal growth factor receptor (EGFR)/estrogen receptor ? (ER?) pathways, and their impact on selective mRNA translation through the kinase mTOR. Moreover, we demonstrate that there are several experimental drugs developed for other indications that can be repurposed to target this axis for the treatment of tamoxifen resistant ER+ breast cancers. Tamoxifen is an estrogen receptor antagonizing small molecule used for treatment for ER+ breast cancer worldwide. Resistance is well established to commonly involve overexpression of EGFRs on breast cancer cells, and hyper-activation or increased signaling of the MAPK-ERK and PI3K-Akt-mTOR pathways. We have now identified two novel hyperactivated mediators of resistance to tamoxifen therapy that lie at the intersection of these key pathways, and we show their importance in resistance. The two effectors of tamoxifen resistance are the inhibitor of translation initiation factor eIF4E, known as 4E-BP1, and the phosphorylation of eIF4E by hyper- activation of its ERK associated kinase, MNK1. Both are therapeutic targets for existing experimental drugs developed for other purposes with good toxicity profiles. eIF4E comprises the basic translation component for loading ribosomes onto mRNAs. Many studies by my group and others have shown that increased phosphorylation of eIF4E is controlled by the ERK-MNK1 pathway, and its increased abundance is controlled by the mTOR/4E-BP1 pathway, which selectively upregulates translation of specific mRNAs required for survival, proliferation and metastasis of breast cancer cells.


Eye pathologies, such as cataracts and macular degeneration, are the leading causes of impaired vision and blindness in the world. Although these pathologies form as a natural process of aging, there are several environmental and lifestyle factors that increase the risk of these diseases, such as cigarette smoking, exposure to light and ultraviolet radiation, oxidative stress, steroid treatment and stress-induced DNA damage. We propose to investigate the role of the metal-responsive transcription factor 1 (MTF-1) in the transcriptional regulation of a suite of genes involved in eye development that may also contribute to environmental stress- related eye pathologies. The main objectives of this proposal are to determine what role MTF-1 plays in normal eye development and whether or not inappropriate activation of MTF-1 signaling by cellular stress in the eye can lead to misexpression of genes that promote eye pathologies. Zebrafish embryos and adults will be used as our model organism and comparative experiments will be performed in a human primary lens epithelial cell line. Targeted gene expression assessments by in situ hybridization, real-time PCR and global transcriptomic profiling via RNA-seq will be used to determine the effects of MTF-1 activation or inhibition on the regulation of eye-specific genes. We have chosen the toxic metal cadmium as our model environmental risk factor because it is one of the best known activators of MTF-1 signaling and because it is believed to be a direct causative factor in the increased risk of cigarette smokers to cataractogenesis and macular degeneration. ChIP-Seq analysis complementary to the microarray expression profiling will be used to determine DNA binding by MTF- 1 as confirmation of a direct role in the regulation of genes responsible for eye development and stress-related pathologies. Additional promoter characterization via transient transfection assays and chromatin immunoprecipitation will be used for final confirmation of MTF-1 regulation of the eye-specific genes in zebrafish and humans. The results obtained from these experiments will yield public health significance by providing novel insight into mechanisms of action by which environmental stressors contribute to eye pathologies. In addition to the discovery of a potential new regulatory pathway involved in eye development with new insight into cataractogenesis and macular degeneration, successful completion of the proposed research will further expand the current use of zebrafish as a model organism for eye disease-related research.


The long-term objective of the research program is to induce immune tolerance to allogeneic organ transplants such that immunosuppressive drugs are not required to maintain permanent graft acceptance. Previous studies in the program have shown that tolerance to heart allografts can be achieved using combined heart and bone marrow transplantation in wild-type MHC-mismatched murine hosts that have been conditioned with total lymphoid irradiation (TLI)and anti-thymocyte serum (ATS). The hosts become stable mixed chimeras without the development of graft versus host disease (GVHD). Tolerance induction and prevention of GVHD is dependent on host regulatory natural killer (NK)T cells that become the predominant residual T cell subset after TLI and ATS conditioning. Our recent studies show that tolerance and GVHD prevention is also dependent on the development of donor Treg cells and host Treg cells that are not NKT cells. A hypothesis that explains the results is that host NK T cells interact with host and donor APC's, and then augment/activate the non-NK Treg cells that provide alloantigen specificity for tolerance induction. Whereas the regulatory activity of the NK T cells is IL-4 dependent, that of the non-NK Treg cells is IL-4and IL-10 dependent. The hypothesis will be tested by adding back purified NK T cells and non-NK Treg cells from wild-type, Treg deficient, and cytokine deficient (i.e. IL-4"'", IL-10"'") host and donor type mice to appropriate TLI/ATS conditioned hosts. The conditioned hosts will receive combined MHC-mismatched organ and bone marrow transplants, and graft acceptance and GVHD will be monitored. The phenotype and cytokine dependence of the non-NK Treg cells will be determined as well as the dependence of NK T cell activation on interaction with APC's. Our recent studies show that the NK T cells in wild type mice are far more resistant to apoptosis induced by TLI/ATS conditioning than conventional T cells. However, the differential resistance is lost in p53"'" mice and in Bcl-2 transgenic mice. We will determine whether the ability to induce tolerance and prevent GVHD is also lost in the latter genetically altered mice.


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. The child obesity epidemic threatens a worldwide health care crisis. Prenatal, early infancy, and age at adiposity rebound are critical periods for development of persistent obesity and its co-morbidities. Early childhood growth patterns are associated with risk of obesity at later age. Interventions targeting the prevention of child obesity could be more effective if directed at the individual children who are likely to become overweight or obese later in life. Our aims are to characterize the trajectories of temporal change in age and sex adjusted BMI-z score, to explore the relationship of the pattern of trajectories with known correlates and consequences of child obesity. These aims will be achieved in two preliminary studies. In the first study, we will include children who were born between January 2003 and December 2005 and had the first well child visit at a Nemours clinic within the first month of birth and had at least one well child visit each year for the next 5 years. We will collect data related to demographics, socio-economic and co-morbidities from Nemours Electronic Medical Record (EMR). We will calculate age and sex adjusted BMI-z score for each subject and classify subjects in groups based on pattern of the correlation structure of change in BMI-z score of each individual. In study 2, we will select a sample of 600 patients from the stratified groups in study 1 and collect retrospective data on known correlates of childhood excess weight gain that are not available in the EMR. The proposed study will enable us to classify children in terms of their risk of excessive weight gain to ensure a stratified randomization in a future randomized control trial to evaluate three family based interventions targeting the prevention of excess weight gain in early childhood.


Congenital cataract is both an ophthalmologic and social problem. The role of hereditary has shown it forms 8.25% of all congenital cataracts. A genetic analysis in humans is not possible due to generation times and small numbers of progeny. Mouse congenital cataract mutants Cat-Fraser (Cat-Fr) and Lop provide an excellent system to study inheritance of cataract. They also provide a method to undertake in vivo and in vitro research on the growth, replication and gene expression in lenses of the cataractous mice. Breeding experiments are presently in progress to assign a specific chromosome for the dominant inherited Cat-Fr mouse. Chromosome 10 has already been mapped for the Lop cataract. Experimental chimeras formed from the aggregation of preimplantation embryos of cataractous and non-cataractous mice will attempt to "rescue" the abnormal lens defects. Blastocyst injection of cataractous cells will analyze the contribution of these cells to normal tissue. In vitro studies on clonal populations of mouse lens epithelial cells derived from Cat-Fr, Lop and normal lenses, will characterize morphological changes with replication and growth patterns, e.g. nuclear and cell size, distribution of microtubules, intermediate filaments and microfilaments, synthesis of specific lens proteins. Those in vitro parameters which are found to be associated with certan phases of the normal lens cells replication and/or growth pattern, will serve as a base line for similar studies on the lens epithelial cells derived from the cataractous mice. Somatic cell hybridization and micromanipulation techniques will be employed to study the possibility of manipulating the in vitro parameters.


This pilot study aims to evaluate the safety & toxicity & tolerance of combination cyclophosphamide, doxorubicin, vincristine, and prednisone (modified-chop) in combination w/triple antiretroviral therapy consisting of the protease inhibitor indinavir and 2 anti-retroviral transcriptase agents, 3tc and 4d4t.


The interaction of HDL apolipoproteins, particularly apoA-I, with the cell membrane transporter ABCA1 removes excess cellular cholesterol and protects against atherogenesis. This process is mediated by lipid- poor apolipoproteins generated by either de novo synthesis or dissociation from HDL particles. Thus, factors that impair the generation or lipid efflux activity of apoA-I could have profound atherogenic effects. Oxidative damage is implicated in the pathogenesis of atherosclerosis, a chronic inflammatory disease. 3- Chlorotyrosine and 3-nitrotyrosine, stable products of protein oxidation generated by phagocyte-derived hypochlorous acid (HOCI) and reactive nitrogen species (RNS), have been detected in human atherosclerotic lesions. However, the underlying factors that control tyrosine oxidation in proteins remain poorly understood. We found that oxidation of apoA-I by HOCI severely impairs its ability to remove cellular cholesterol by the ABCA1 pathway, consistent with the possibility that oxidation of HDL apolipoproteins in vivo would be atherogenic. We propose to test the hypothesis that site-specific oxidation of tyrosine residues by phagocyte-derived HOCI and RNS alters the biological and atheroprotective function of HDL. We will characterize the effects of site-specific oxidation of tyrosines in apoA-I on its ability to remove cellular cholesterol, identify protein motifs that direct site-specific tyrosine oxidation by HOCI and RNS, and determine if HDL is a physiological target for oxidative modification in the artery wall. This project will use HPLC and tandem mass spectrometry to locate and quantify the sites of tyrosine oxidation in apoA-I and model peptides, cell biology procedures to characterize the effects of apoA-I oxidation on lipid transport activity and interactions with ABCA1, tissue analysis to identify and characterize oxidized apoA-I in human atherosclerotic lesions, and mouse model approaches to test for the effects of macrophage-generated HOCI on apoA-I oxidation and atherogenesis in vivo. The proposed studies will provide insights into the structural features that direct oxidative modification of proteins, with important implications for the physiological significance of oxidative reactions in atherosclerosis and other inflammatory diseases.


The tick borne flaviviruses (TBFV) includes Tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Powassan virus and Langat virus (LGTV). The TBFV are listed among the NIAID category B and C lists of priority for research on pathogenesis, to identify novel targets for therapeutic and vaccine development. As their name suggests, these viruses are transmitted by ticks, and following infection of humans, cause encephalitis, meningitis or hemorrhagic fevers resulting in approximately 10,000 to 13,000 hospitalizations annually with mortality rates as high as 40%. The TBFV belong to the Family Flaviviridae, genus Flavivirus, which comprise some of the most medically significant emerging and re-emerging pathogens. Other members include the mosquito-borne West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). Hence, research into the pathogenesis of TBFV will reveal insight into the biology of this globally important group of viruses. The research in our laboratory aims to identify and understand interactions between the TBFV and their hosts (both the tick and the mammal) critical to virus replication and pathogenesis. We have been studying LGTV which is a naturally attenuated member of the TBFV that shares approximately 80% identity with TBEV at the amino acid level. This makes LGTV an excellent model to gain insight into the TBFV. The main studies ongoing in the laboratory are outlined below. 1. Study of virus interactions with the invertebrate host. Ticks represent the natural reservoir of TBFV and therefore are critical for virus persistence in nature and are the major source of infection for humans. We have investigated global transcriptional changes in Ixodes scapularis nymphs infected with LGTV using custom Agilent microarrays. The microarray was based on sequences from an mRNA library derived from salivary glands of Ixodes scapularis ticks. The aim of this work is to identify tick host proteins important for the replication or transmission of TBFV. These proteins can be used for the development of novel anti-tick vaccines that prevent virus transmission to the immunized mammalian host. We first examined transcriptional changes in uninfected ticks during feeding on mice. This revealed clusters of genes that were differentially regulated, particularly on day 0, 1 or 3 post tick attachment. Preliminary results from experiments with LGTV-infected ticks have identified a subset of these genes that are affected by infection. We are currently working to confirm these results and will then determine the immunogenic potential of those gene products. 2. Interactions between TBFV and host innate responses to infection. Following the bite from an infected tick, dendritic cells (DCs) resident in the skin of the mammalian host are among the first cell types infected by TBFV. These cells have important roles in innate immunity through the production of interferon (IFN), cytokines and chemokines, as well as in orchestrating adaptive immunity. Thus, early interactions between these cells and TBFV are likely to have a major influence on the outcome of infection. We have initiated a project in the lab to investigate the effects of TBFV infection on primary DCs and the ability of TBFV to modulate the signal transduction pathways involved in detecting virus infection and producing IFN. Following infection with LGTV, flow cytometry was used to distinguish between infected and uninfected bystander DCs. Maturation profiles revealed that infected DC, but not bystander cells, increased cell surface expression of MHC class II. However, infected DC did not upregulate CD80 and CD86, costimulatory molecules involved in activating T cell responses. Moreover, infected DC did not respond appropriately to stimulation with toll-like receptor (TLR) 3 or TLR4 ligands. The failure of LGTV-infected DC to express key molecules involved in anti-viral immunity in vivo may contribute to viral evasion of immune responses. Hence, examining these interactions will lead to an understanding of the mechanisms that contribute to TBFV pathophysiology. Type I and type II IFNs are crucial elements of the innate immune response to flavivirus infection, restricting virus replication, dissemination and lethality in mouse models. Type I IFN is a potential therapeutic candidate for flavivirus infection. However, such treatment often fails. We have previously shown that LGTV utilizes its nonstructural protein NS5 to interfere with IFN signaling by directly inhibiting Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal transduction. Over the past year, we have shown that NS5 from other flaviviruses including TBEV also suppress IFN responses. We also found that the virulent New York (NY99) strain of WNV suppressed IFN-dependent JAK-STAT signaling. In contrast, NS5 from Kunjin virus (KUN), a naturally attenuated subtype of WNV, was a poor suppressor of IFN responses. Importantly, mutation of a single residue in KUN NS5 to the analogous residue in WNV-NY99 NS5 (S653F) rendered this protein an efficient inhibitor signaling. Thus, a naturally occurring mutation is associated with reduced ability of NS5 to function as an IFN antagonist and hence may influence virulence of WNV field isolates. This work identifies NS5 as a potential virulence factor of WNV and has implications for live-attenuated vaccine design.


In order for sister chromosomes to be faithfully segregated to opposing daughter cells during cell division, each chromosome must possess a single centromere. The lack of a centromere or the creation of dicentric chromosomes (those possessing two centromeres) leads to chromosomal breakage and missegregation of chromosomes during mitosis. In turn, aberrant chromosome segregation may give rise to developmental abnormalities and neoplastic disease. Furthermore, a better understanding of the components of the human chromosome that govern transmission of genetic information may be important in the development of novel gene therapies. Specific Aim 1: By developing a transgenic mouse containing a conditional disruption of the endogenous mouse CENP-A gene, these studies seek to determine whether CENP-A protein is required for the establishment and maintenance of the mammalian centromere. Specific Aim 2: Furthermore, using a yeast two-hybrid screen, this study endeavors to identify proteins that interact with CENP-A that may be involved in centromere specification and kinetochore assembly. Specific Aim3: Finally, the studies proposed here are designed to determine whether CENP-A itself is sufficient to recruit centromere components and ultimately specify the centromere.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glycoxidation or advanced glycation endproducts (AGE) accelerate oxidative mechanisms, resulting in activation of transcriptional pathways, excessive proliferative/growth-related phenomena and sustained inflammation leading to clinical aging. These are further accelerated by diabetes or by conditions associated with increased production or decreased AGE clearance, eg. renal insufficiency. A major source of AGE precursors and oxidant-stress is identified to be the western diet. The turnover of AGE involves specific AGE receptors and depends on renal function. Advancing age is known to be associated with increased OS, increased prevalence of cardiovascular disease, impaired glucose tolerance, diabetes mellitus, renal decline, as well as with accumulation of AGE. While efforts have linked AGE-mediated OS and chronic complications in aging animals, human studies are strikingly lacking. We plan to test the hypotheses that: 1) older men and women (ages 60+) who consume standard (high AGE) diets will have higher serum AGE in conjunction with higher OS, and markers of vascular dysfunction or inflammation, compared to age-matched subjects consuming low AGE diet, and to younger (less than 35 years) subjects and 2) AGE-restriction, by dietary modification, will reduce the total AGE burden, oxidative and inflammatory mediators and attenuate the differences between older and younger groups. The Specific Aims are to determine: 1) the correlation of circulating levels of defined AGE and inflammatory markers (eg CRP, TNFa, and IL-6) in older and younger groups with the dietary intake of AGE. 2) The effect of dietary AGE restriction on AGE and AGE-dependent parameters of OS and inflammation. 3) The effect of environmental (dietary) glycotoxic burden on AGE receptor-mediated removal mechanisms in PBMN from older and younger persons. It is hoped that the data will lay the groundwork for future studies evaluating optimal methods of nutrient preparation as ways to ameliorate a major environmental promotor of pro-oxidant events, and thus aging.


PROJECT SUMMARY/ABSTRACT ? PROJECT 1 The CD226/TIGIT pathway is a novel co-stimulatory/co-inhibitory pathway that closely parallels the CD28/CTLA-4 pathway. Similar to CD28 and CTLA-4, CD226 and TIGIT share ligands (CD112 and CD155) and ligand engagement of CD226 co-stimulates T cell activation while engagement of TIGIT inhibits T cell responses. Over the last few years, TIGIT has emerged as an important co-inhibitory receptor. While initial experiments indicated that TIGIT inhibits T cell responses indirectly by promoting tolerogenic phenotype in dendritic cells, we, and others, have shown that TIGIT has T cell-intrinsic inhibitory functions. We have now found that TIGIT is co-expressed with other co-inhibitory receptors (PD-1,Tim-3,Lag-3) on CD8+ T cells that exhibit dysfunctional/exhausted phenotype in chronic diseases such as cancer and chronic viral infection. Importantly, we find that expression of TIGIT marks the most dysfunctional subset of CD8+ T cells in these conditions, thus raising the important issue of how TIGIT co-operates with other inhibitory receptors to drive severe dysfunctional phenotype in effector T cells. In addition to its role in effector T cells, we have recently identified that TIGIT marks a distinct subset of regulatory T cells (Treg) that exhibit heightened expression of known Treg effector molecules and increased suppressive capacity. Importantly, we find that TIGIT+ Treg are highly enriched in inflamed tissues and that they exhibit specialized function; selectively suppressing pro- inflammatory Th1/Th17 responses while sparing Th2 responses. However, how TIGIT may function in CD8+ T cells to dampen their responses while in Treg function to direct specialized Treg function is not known. Given the data supporting important roles for TIGIT in regulating effector T cells and Treg, it is surprising that nothing is known regarding the molecular signals that induce TIGIT expression. Our preliminary data indicate that the immunomodulatory cytokine IL-27 induces TIGIT expression both in vitro and in vivo. We have now leveraged this information to identify candidate transcription factors that are downstream of IL-27 and may drive TIGIT expression in effector T cells. Based on our data, we hypothesize that TIGIT has a dual role in regulating T cell responses: 1) TIGIT co- operates with other co-inhibitory molecules to regulate effector T cell phenotype and 2) TIGIT drives highly active and specialized Treg phenotype in tissue. We have generated a number of tools including agonist and antagonist antibodies and conditional knock-out mice to study the function of this emerging pathway in the regulation of T cell responses. Our specific aims are: 1) Dissect the T cell intrinsic and extrinsic roles of TIGIT in regulating T cell activation and dysfunction 2) Determine the role of TIGIT in directing Treg function in inflammatory conditions


This project involves a study of the regulation of histidine and threonine catabolism by concentrating on the control properties of the key enzymes histidase, urocanase and threonine dehydratase. The majority of the investigation deals with these enzymes in, or isolated from Pseudomonas putida. Threonine dehydratase of this organism is a dual function enzyme, being involved in both the biosynthesis of isoleucine and in the catabolism of threonine. Moreover, extensive mutant analyses indicate that the enzyme is required for efficient expression of the genes controlling the formation of the histidine utilizing enzymes. In the project we are attempting to confirm the existence of a covalently modified form of the enzyme by enzymological studies of the purified enzyme and by mutant construction and analysis. We propose that the covalent modification, probably a nucleotidylation, is regulated by the availability of an efficient nitrogen source and is employed by the cell as a means of directing threonine dehydratase activity towards isoleucine biosynthesis. Also planned are related studies on other control phenomena which may affect the biosynthesis of histidase, urocanase and other histidine-degrading enzymes. Of primary concern are the roles of cyclic AMP and glutamine synthetase in favoring transcription of the genes for these enzymes. Mutants altered in cyclic AMP production or glutamine synthetase activity are being isolated to facilitate these studies. The nature of allosteric regulation of histidase activity by pyrophosphate will also be examined with purified histidase. Further studies will be made on the urocanase of P. putida which has been recently found to be NAD positive dependent. Mechanistic analyses, primarily isotope exchange and NMR studies, will be conducted along with NAD positive binding measurements. Purification of beef liver urocanase will be attempted to permit generalization of the role of NAD positive as a coenzyme for urocanase.


DESCRIPTION: (from applicant's abstract) An important new hypothesis for understanding and treating drug addiction is that it may be an inappropriate form of learning. This hypothesis is based on studies demonstrating that glutamate, a transmitter critical for learning and memory, is required for the development of neuroadaptations believed to underlie addiction and relapse. We have shown that the repeated administration of psychostimulants (amphetamine or cocaine) causes profound alterations in glutamate systems. These include alterations in AMPA receptor expression and responsiveness in the nucleus accumbens (Nac), a brain region critical for the rewarding and addictive properties of psychostimulants. Medium spiny GABA-containing neurons, which represent 95% of all neurons in the Nac, receive convergent inputs from midbrain dopamine (DA) neurons and glutamate neurons originating in cortex and limbic regions. Our long-term goal is to understand how psychostimulants, which initially target the DA transporter, ultimately produce adaptations in glutamate transmission in the Nac. This is an important question, because glutamate systems are likely to mediate the neuronal plasticity underlying the transition from drug experimentation to drug dependency. Unfortunately, we know little about chronic interactions between DA and glutamate receptors in the Nac. A logical starting point is to determine how acute DA receptor stimulation can influence glutamate transmission. This application will use postnatal Nac cultures to test the hypothesis that DA receptors modulate the phosphorylation of the AMPA receptor subunit GluR1. We are focusing on AMPA receptors because they are the primary mediators of excitatory transmission in medium spiny neurons and on phosphorylation as a regulatory mechanism because GluR1 phosphorylation leads to marked enhancement of AMPA receptor-mediated currents. Phosphorylation of GluR1 will be detected using phosphorylation site-specific antibodies to GluR1 developed by Dr. Richard Huganir. One selectively recognizes GluR1 phosphorylated on ser-845 [protein kinase A (PKA) site]. The other recognizes GluR1 phosphorylated on ser-831 [protein kinase C (PKC)/Ca2+ -calmodulin dependent protein kinase type II (CaMKII) site]. The first Aim is to characterize conditions regulating basal phosphorylation of GluR1 and the kinases involved in its phosphorylation. First, control cultures will be compared with cultures incubated in the absence of Ca2+, the absence of synaptic activity (TTX, bicuculline, MK-801, CNQX), the absence of inhibitory activity (bicuculline), and the absence of excitatory activity (MK-801, CNQX). Second, we will use activators and inhibitors of PKA, PKC and CaMKII to determine which kinases phosphorylate GluR1 in our cultures. The second Aim is to determine if DA receptor stimulation modulates GluR1 phosphorylation. Experiments will be designed to detect both stimulatory and inhibitory effects of D1 and D2 DA receptor activation. The identity of the residue phosphorylated, combined with experiments involving selective protein kinase inhibitors, will help identify the kinases involved in DA receptor-mediated effects.


Serum-free conditioned media from several human tumor cell lines (e.g., epidermoid carcinomas, melanomas, bronchogenic carcinomas, and rhabdomyosarcomas) and from NIH/3T3 cells transfected with DNA from a human lung carcinoma were observed to produce a class of factors which inhibits growth of human melanoma and carcinoma cells in soft agar and in monolayer cultures. These inhibitors of tumor cell growth have been designated tumor inhibiting factors (TIFs). Normal human fibroblasts and epithelial cells, however, are stimulated to proliferate by these same factors. This project is directed towards the purification and characterization of this new class of factors that inhibits tumor cell growth. We have determined some of the biochemical and biophysical properties of TIF. We have also identified a rich source of TIF in certain normal human tissues. This should facilitate its purification and characterization. This source should yield sufficient TIF for sequencing, antibody production, and in vivo experiments.


Project Summary Studies on the health effects of sex tourism report that sex workers are one of the most vulnerable groups for acquiring HIV due to their high rates of sexual risk behaviors1 and their disadvantaged positions to negotiate safe sex because of social, economic, and cultural factors.2 In low prevalence countries with concentrated epidemics, HIV prevalence rates among sex workers are often 3 to 4 times that of the general population.3 This risk is often perpetuated by the availability and accessibility of illegal drugs in tourism zones. While studies have examined the association between HIV risk-related behaviors including drug use and sexual risk-taking and the social and physical environment that characterize neighborhoods;4, 8-11 there is a fundamental gap in the current scientific evidence-base about which extra-individual contexts, ?activity spaces? (spaces in which daily activities occur) and social interactions or networks interact, and how that impacts HIV risk behavior. The goal of the proposed research is to utilize innovative spatial-temporal and social network methodology to describe the relationship between activity spaces and social network characteristics of male migrant sex workers in tourism zones in the Dominican Republic (DR), and to assess how such daily activity patterns and networks jointly affect HIV risk behavior, testing and infection. Considering that the DR has one of the largest sex tourism industries in the Caribbean and is currently experiencing a dramatic increase in the use and transport of illegal drugs, spurred by a shift in smuggling routes to the United States,27, 28 the country provides a unique environment for examining how environmental and social structures shape HIV risk and related behaviors among sex workers. A deeper understanding of the multiple factors that influence day-to-day experiences of male migrant sex workers is paramount for reducing the HIV burden in the DR, and for preventing international transmission through tourism and migrant routes. This study has three specific aims: (1) To utilize novel spatial-temporal methodology to characterize the activity spaces of male migrant sex workers in the Dominican Republic by linking activity space GPS data to already collected, novel ethnographic mapping data from a current NIDA-funded R01, and to examine the impact of risky activity spaces on HIV risk and related behaviors, specifically drug use, risky sex, HIV testing, and self-reported HIV; (2) To describe the social networks (e.g. size, composition) of male migrant sex workers, and assess the role social networks have on HIV risk and related behaviors in this population; (3) To examine the interaction between daily activity space and social network characteristics on HIV risk and related behaviors. If activity spaces can be identified and play a significant role in drug use and other HIV-related behaviors?including their combined role with network characteristics?it will provide critical information for creating effective structural and interpersonal HIV prevention interventions. To address the current barriers to HIV prevention and treatment, it is paramount that we focus not only on multiple social determinants, but also on the social experiences and lives of at risk populations.


DESCRIPTION: This application is a request for continued support of an ongoing project. The overall goal of the project is to better understand the cognitive and brain bases of covert motor processes, particularly motor imagery. The project focuses on the activity of three cortical motor areas (primary, premotor, and supplementary) during overt movement and covert motor processes. The principal measures employed are based on event-related brain potentials. These measures have the temporal resolution to detect changes in brain activity occurring on the order of milliseconds and sufficient spatial resolution to monitor separately each of the above cortical areas. In the proposed new research, these measures will be combined with magnetic resonance images, in order to correct for distortions resulting from conduction of the electric signal through the skull and to locate sources of activation on an accurate map of the cortical surface. "Functional coupling" between the cortical areas will be examined through measures of correlation between their moment-by-moment activities. The experiments conducted in the project to date have monitored the three motor areas under a wide range of conditions in which covert motor processes are known to take place. The new experiments will monitor these and other cortical areas during cognitive events in which covert motor processes have been hypothesized to play a role. These include observation of a skilled performance prior to its imitation, use of an "articulatory loop "to maintain information in working memory, and illusions evoked by viewing a moving phantom image of one's own limb . Also examined will be the covert motor processes responsible for the timing and serial order of hierarchical movement sequences, as well as the continuity between motor imagery and gentle overt movements. Findings from the project may help to 1) improve the efficacy of mental practice, observational learning, or treatment to aid motor recovery, 2) diagnose disorders of the motor system, and 3) determine the direct consequences of activity in the studied brain areas for learning, remembering, or tuning movement. At a broader level, the work will enhance our understanding of the motor system at both a functional and physiological level. Further information concerning its less known covert side may lead to unexpected insights about the motor system in general, including its possible roles in cognition.


A number of 4-X-bioimidazoles are accessible by direct electrophilic substitution (nitro, halo, etc.); 2-X-bioimidazoles are far less accessible and can be obtained ,only by indirect and, often, very tortuous routes. Prior to our major efforts in this area, the majority of 2-X-bioimidazoles were unknown. By far the 2-substituted imidazoles most easily obtainable are 2-iodo and 2-(trifluoromethyl). The 2-iodo derivatives of histidine and histamine are of interest, not only because of their potent antimalarial activities, but because they can serve as valuable intermediates for the synthesis of other 2-X-bioimidazoles, as can the trifluoromethyl analogues. 'We are currently exploring the utility of such conversions to prepare photosensitive bioimidazoles and affinity labels for in vivo use. Over the past 15 years, we have found consistently that 2-X-bioimidazoles (especially fluoro and iodo) have a broad range of strong biological activities but ,the corresponding 4-X-bioimidazoles are essentially inactive. Various explanations for this remarkable selectivity in biorecognition have been invalidated on experimental grounds. Our analyses of (13)C NMR spectra have now revealed that the differentiation may be based solely on tautomer preference. While 4-X-imidazoles exist preferentially as the unnatural pi-tautomers, 2-X-bioimidazoles exist preferentially natural tau-tautomers. This discovery generates broad implications in many areas of drug design, e.g., selective antihistamines, imidazole-based antimalarials and antimicrobials, CVS and CNS-selective TRH analogues, and analogues based on pyrrole and triazole regioisomerism. Thus. novel synthetic methods are being explored to make available even more 2-X-bioimidazoles.


The long-term goals of this project are to understand the molecular mechanisms that underlie the degeneration and death of neurons in Parkinson's Disease (PD) and to use this information in turn to devise treatments to suppress the progression of this disorder in patients. The proposed studies will test the over-arching hypothesis that neuron degeneration and death in PD are due to inactivation of the key enzyme Akt which is required for nerve cell function and survival, and that this is mediated by induction of the stress-responsive protein Trib3, an inhibitor of Akt activation. The specific aims will assess four hypotheses: (1) that Trib3 expression is elevated in PD models and in PD; (2) that Trib3 mediates neuron degeneration and death in models of PD; (3) that Trib3 promotes neuron death by interfering with phosphorylation/activation of Akt; and (4) that induction of Trib3 in PD models and PD is mediated by the transcription factor ATF4. Multiple experimental approaches will be employed. These will include assessing Trib3 expression in toxin- and alpha- synuclein based cell culture models of PD, in animal models of the disease and in post-mortem brain tissues from PD patients and controls; determining whether loss of Trib3 expression, either via shRNAs or gene deletion, protects neurons from death in cell culture and animal models of PD; ascertaining (by over- expression or loss-of function studies) whether Trib3 is responsible for mediating the loss of Akt activity that occurs in cellular models of PD; and establishing whether manipulating expression of ATF4 in culture and animal models influences Trib3 induction in culture and animal models of PD. If successful, these studies will establish Trib3 as a required element in the mechanism that leads to neuron death and degeneration in PD and will provide insight as to how it does this, and how it is induced in this disorder. Such information will be exploited in the long term to formulate new approaches to suppress progression of PD in patients. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is characterized by progressive degeneration and death of specific types of nerve cells. Current treatments are aimed at ameliorating the symptoms of this disease rather than its progression. The proposed studies seek to uncover the molecular mechanisms underlying nerve cell degeneration and death in PD so as to lead to generation of new strategies to suppress disease progression.


All patients developing non-A, non-B hepatitis following open heart surgery will be followed with serial studies of ALT to determine the incidence of chronic liver disease and to compare this incidence with those having type B hepatitis. Liver biopsy will be obtained when the ALT is elevated for more than six months and then again at one and three years. Biopsy material will be obtained for flourescent and EM studies, as well as light microscopy with multiple stains.


Ubiquitination is a versatile post-translational modification that regulates a large number of different cellular processes including signal transduction, protein trafficking, DNA repair, gene transcription and regulated protein degradation. Typically, ubiquitin is appended to a target protein as a signal that is then received and interpreted by proteins carrying ubiquitin-binding domains (UBDs). UBDs are numerous and diverse, and, in most cases, little is known about how they function in the physiological context of a full-length protein. Several protein interaction SH3 domains were recently discovered to act as UBDs, whereas SH3 domains were previously thought to act almost exclusively by recognizing proline-rich peptides. The known ubiquitin-binding SH3 domains are components of the endocytic machinery. The first-discovered and perhaps best-characterized role of ubiquitin outside proteasome degradation is as a regulator of receptor endocytosis. Numerous components of the endocytic machinery that act to internalize cargo are ubiquitinated and/or carry UBDs. Furthermore, many endocytic cargo proteins are themselves modified by ubiquitin at the plasma membrane, where ubiquitin acts as a regulated internalization signal to direct these proteins into endocytic vesicles. Specific cargo proteins that are ubiquitinated and regulated by ubiquitin-dependent endocytosis include leptin receptors, growth factor receptors, glucose transporters, ion channels, the aquaporin-2 water channel and the anthrax toxin receptor. Impairment of ubiquitin-dependent endocytosis results in, or is linked to, diseases including hypertension, cancers, immune disorders and viral infections, as well as inflammation and bacterial toxin entry into cells. Three components of the endocytic machinery that bind to ubiquitin through SH3 domains have been identified: amphiphysin, CIN85 and the yeast homologue of CIN85, SlA1. Biochemical and genetic experiments proposed in this grant will define the function of ubiquitin in regulating these proteins, and will provide a general paradigm for how ubiquitin regulates the formation of the protein-protein interactions mediated by SH3 domains.


This investigation is a family study on the effect of heterozygosity for an inactivating mutation of the estrogen receptor gene on bone density and other parameters of estrogen action. Excluding one nuclear family, the lumbar spine bone densities suggest that carrier status is associated with decreased bone mineral density compared to wild type; the mean spine BMD z scores were -0.9 for heterozygotes vs. -0.1 in wild type individuals (p = 0.02). In this past year, there were no outpatient visits, but the Core Laboratory of the CRC performed determinations on the collected samples. The analysis of the data will be forthcoming.


A central issue in neuroscience is to understand how experience shapes brain function. Recent studies in visual cortex suggest that the maturation of intracortical inhibitory circuits is necessary to initiate and drive ocular dominance plasticity during the critical period. However, the maturation and regulation of the specific type of inhibitory circuit involved is not understood. Critical period plasticity is also modulated by visual deprivation and BDNF over-expression, although the cellular mechanisms remain unknown. Here we hypothesize that the functional maturation of a specific subtype of GABAergic neurons, parvalbumin (Pv) basket interneurons, are a component of the inhibitory mechanism underlying critical period plasticity, and a cellular target of experience deprivation and BDNF regulation. Using bacterial artificial chromosome transgenic (BAC) mice expressing GFP in Pv-interneurons, we will characterize the morphological and physiological development of Pv-interneurons using two photon laser scanning microscopy and electrophysiology in brain slices. We will then examine the effects of dark rearing on the maturation of Pv-interneurons. Finally, we will test the role of BDNF in experience-dependent maturation of Pv-interneurons by blocking trkB signaling using BAC transgenic mice expressing a dominant negative form of trkB receptor specifically in Pv-intemeurons. These results will reveal a mechanism by which experience shapes function of an identified inhibitory network and provide strong evidence that maturation of the Pv-interneuron circuit facilitates the coding of visual information necessary to drive ocular dominance plasticity. The knowledge gained will aid in the design of drug treatments in diseased states such as epilepsy and schizophrenia.


Complex cellular networks underlie the functional foundation of the mammalian central nervous system (CMS). Understanding the physiological dynamics of these networks, in other words understanding how signaling between interacting groups of cells produce and modulate meaningful physiological information, will directly contribute to our understanding of how the CMS functions in health and how it fails in disease. At present, our mechanistic understanding of the dynamics of neuronal and glial networks is very limited, even though we understand the molecular functional unit that underlies it (i.e. the synapse). One approach is to apply network theory to characterize neuronal and glial networks. Network theory is a branch of statistical mechanics that classifiescomplex networks independent of the physical details of the network and provides an understanding of its dynamical behavior. Applying network theory to neuronal and glial networks requires knowing their structure or topology. However, high throughput computationally intensive measurementsof molecular signaling between neurons and glia, and the extraction of quantitative information about their underlying network structure is not possible given current techniques. What is needed therefore, are algorithms and softwarethat will allow the high throughput characterization and analysis of physiological neuronal and glial networks. Here, we propose to develop computational tools that will allow us to map the spatial and temporal topology of functional neuronal and glial signaling networks, and classify and analyze them within the context of network theory. We present a detailed discussion on the algorithms and programming required to do so, and illustrate the operation and validation of a beta version of such a program. We propose that using this approach, neuronal and glial networks can be classified within known mathematical networktypes and behave as dictated by the quantitative properties of the network types they are classified into. We also present preliminary experimental data showing for the first time that calcium signaling in astrocyte networks, mapped using our software tools, have a previously unidentified toplogy. We propose that the networktopologies of healthy neurons and glia remodel following injury and underlie the induction and maintenance of neuropathological disease states, making the clinical significance of these findings and the development of the computational tools required to investigate them very important.


The crowded environments inside cellular compartments are very different from the typical dilute conditions of in vitro and in silico biophysical studies of biomolecular systems. The long-term objective of this project is to bridge the in vitro-in vivo gap, by quantitatively reconstructing the influences of cellular environments on the thermodynamic and kinetic properties of biomolecules. Exploiting tremendous opportunities opened by our postprocessing approach for modeling effects of crowded cell-like environments and other recent advances, in this project we will (1) advance FFT-based postprocessing to achieve high accuracy in modeling crowding; (2) quantitatively delineate temperature dependence of crowding effects; and (3) characterize conformational ensembles and binding of intrinsically disordered proteins under crowding. Through capitalizing on FFT-based postprocessing and carrying out our own wet-lab studies, we will closely integrate computation and experiment to overcome challenges toward gaining insights into in vivo biochemical processes. The ability afforded by this research to use dilute-solution experiments and simulations for predicting the conformational ensembles of intrinsically disordered proteins under cell-like conditions will move us forward in elucidating their cellular functions. The conceptual advance that macromolecular crowding in cellular environments may serve as an important factor for protein stability in thermophiles could have broad implications for protein evolution and design.


Need for new lead compounds against Leishmania and Trypanosoma intensifies the desirability of improved screening adapted to large-scale work. We wish to exploit a hardy Leptomonas of proven high sensitivity to several standard antitrypanosomatid agents; Crithidia fasciculata; and T. mega of amphibia -- a trypanosome easily cultivable on autoclaved media, and whose position hints it may share chemotherapeutic responses with stercorarians, e.g., T. cruzi and salivarians. Minimal media for the Leptomonas and T. mega would be applied to broaden the tolerances --at present apparently narrow -- in the published medium for T. brucei. Detection of novel agents acting as antimetabolites, and mode-of-action studies of likely targets of antimetabolites, make development of physiologically lean, highly reproducible media, desirable. By playing one flagellate against the other we hope: a) to detect lead compounds, and how best to deploy the few available effective drugs; b) use of these drugs to uncover new targets for antimetabolite therapy, especially polyamines, for in related work; the Leptomonas has already served to chart cross-resistance and collateral sensitivities to several established antitrypanosomatid agents. The organisms would be grown in physiologically minimal defined media at blood pH. They would be used to explore interrelationships among polyamines, their precursors, and Mg2 ion; the former compounds are likely targets, along with nucleic acids, of the strongly cationic trypanocides. These organisms would be used also to chart the effects of other agents on trypanosomatid growth, applied singly and in combination with cationic trypanocides. Such agents might significantly inhibit: a) biosynthesis of polyamines; 2) methylation of tRNA's; 3) heme metabolism. Work would focus on ethidium, crystal violet, Antrycide, pararosaniline, methylglyoxal-bis(guanylhydrazone) (a potent inhibitor of polyamine biosynthesis), and transmethylase inhibitors such as 6-dimethylaminonicotinamide. Effective drugs and combinations would be tested against our trypanocide-resistant Leptomonas strains. Inhibition analysis would be directed at identifying metabolites (e.g., polyamines) counteracting these drugs, then assembly of these metabolites into "rescue" cocktails to be tested on all 3 organisms in the presence of drugs.


Photodynamic therapy (PDT) utilizing hematoporphyrin derivative (HPD) and a cw argon-pumped dye laser tuned to 630 nm has been shown to be a promising treatment for a number of forms of cancer. However, because tissue penetration is maximized for wavelengths greater than 700 nm, substantial effort has been directed towards finding new dyes which absorb in the near-ir (670-900 nm). The tuning range of the recently developed Ti:sapphire laser overlaps this regions, thus providing an all-solid-state alternative to the dye laser. The Phase I effort will involve cell-level experiments using two new photosensitizers: sulfonated chloraluminum phthalocyanine (CASPc) and benzoporphryin derivative monoacid A (BPD) which absorb at 680 nm and 690 nm respectively. Although a Ti:sapphire system can be operated either pulsed or cw in this wavelength region, the initial experiments will be performed with a pulsed laser, since the cw Ti:sapphire would produce results that were very similar to the cw dye. Although the primary cause of cell necrosis under cw irradiation is the creation of singlet oxygen, the high intensity levels generated by the pulsed laser may photosensitize by non-oxygen-dependent mechanisms. The long term goal would be to develop a clinically qualified solid-state laser for PDT.


Influenza viruses are classic examples of antigenically variable pathogens, and have a seemingly endless capacity to evade the immune response. For example, since the influenza A(H3N2) subtype entered the human population circa 1968 the vaccine against it has had to be updated 24 times to track the evolution of the viral quasispecies and to remain effective. Most of the bioinformatics methods for analyzing viral evolution are based on genetic analyses; however, it is the phenotypic (antigenic) properties of the virus that determine its success at escaping prior immunity and causing infection. Indeed, the antigenic data are the primary criteria for selecting the virus strain used in the influenza vaccine, and which are important for much basic and applied research on influenza. However, there is no reliable method to determine quantitatively antigenic differences. Antigenic differences are typically determined using some form of binding assay (for influenza virus, the hemagglutination inhibition assay, for other pathogens a neutralization assay, ELISA, etc.). In such assays, typically a panel of antisera is titrated against a series of antigens and the data are organized in tabular form and analyzed by eye. This has been the procedure for over 50 years. These data are difficult to interpret quantitatively, and sometimes even for experts give an inconsistent picture. The primary reason for this difficulty is that the data contain irregularities, or paradoxes. On such irregularity is that one antiserum might detect a difference between two antigens, while another will not. Another irregularity is that heterologous titers are sometimes higher than homologous titers. Furthermore, it is often difficult to compare data from different laboratories. These, along with other irregularities result in binding assay data only being considered reliable enough to judge large antigenic differences?in the case of influenza virus differences of sufficient magnitude that they necessitate an update of the vaccine strain. By only being able to judge gross differences among the thousands of influenza strains characterized each year, one misses opportunities to optimize the vaccine strain choice, to detect signals in the evolution of the viral quasispecies that could give advance warning of the necessity to update the vaccine, to understand the epidemiology of influenza, to judge how vaccination affects the viral evolution, and to devise novel intervention strategies which have the potential to fundamentally change our options to control epidemic and pandemic influenza. Influenza virus is one example of an antigencially variable pathogen;others include human immunodifficiency virus and hepatitis C virus. The degree of antigenic diversity will increase as interventions increase selection pressure to generate escape mutants, and the characterization of these phenotype differences will thus only increase in importance.