No description available.

No description available.

No description available.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by polyclonal B-cell activation, autoantibody production and immune complex-mediated glomerulonephritis (GN). NZB/W F1 mice spontaneously develop SLE-like symptoms and have been widely used as an animal model for SLE. Even though the etiologic cause of autoimmunity in both human and murine lupus is not clearly understood, mounting evidence indicates the involvement of autoreactive B cells and T cells. Blockade of costimulatory pathways using CTLA4Ig fusion protein and anti-CD40 ligand monoclonal antibody (mAb) has been able to suppress autoantibody production and inhibit lupus nephritis in NZB/W F1 mice. In spite of these successes, protein- or mAb-based therapies are expensive, deliveries are problematic, and bolus injections result in transient and toxic levels of these reagents. In comparison, gene therapy offers an advantageous solution by achieving long-term, stable transgene expression and convenient gene delivery. Among the vectors used in gene therapy, recombinant adeno-associated virus (rAAV) has been proven to be a promising one because of its non-pathogenicity and non-immunogenicity. In this project, the hypothesis that rAAV serotype 8 (rAAV8)-mediated immunomodulator gene transfer was able to prevent and suppress lupus development in NZB/W F1 mice was tested. First, the transgene expression pattern following systemic delivery was studied. rAAV8-mediated gene delivery was able to achieve long-lasting and high-level transgene expression following a single intraperitoneal or intrasplenic injection. Second, rAAV8-mediated costimulatory blockade gene transfer prevented autoantibody production, delayed proteinuria onset, prolonged survival and protected kidneys from immune-complex induced tissue damage. Moreover, it successfully suppressed CD4+ T cell activation and also the transition from naïve to memory T cells. Among all early prevention strategies tested in newborn mice, the combination of murine CTLA4Ig (mCTLA4Ig) and murine CD40Ig (mCD40Ig) was more efficient than both single vectors, suggesting a synergistic effect between these two costimulatory blockades. Surprisingly, the late prevention in young lupus-prone mice achieved results similar to those observed in the early prevention. Furthermore, rAAV-mediated costimulatory blockade gene transfer did not suppress host humoral response to foreign antigens. Finally, in the treatment of lupus mice with high autoantibody titers, mCTLA4Ig alone, or in combination with mCD40Ig, was able to decrease autoantibody titers, delay proteinuria onset, and prolong survival. The therapeutic effects were evidenced to be dose-dependent. In general, this study indicates that rAAV8-mediated costimulatory blockade gene transfer is capable of preventing and suppressing lupus development, thereby suggesting the successful application of this autoimmune disease gene therapy both in research and in clinical settings to promote public health.

Alzheimers disease (AD), a common debilitating neurodegenerative disease, is a major public health problem in the United States because it affects almost 50% of the people after age 85. Although AD has been described since 1907, no cure or universally accepted effective treatment has been devised. Studying factors that play a role in risk and pathogenesis of LOAD may lead to development of new therapies and give an insight to the etiology of disease. Both genetic and environmental factors have been implicated in the etiology of AD. Twin studies have shown heredity to be a major causal factor in the development of LOAD. Despite the evidence for substantial genetic effect in the etiology of LOAD, the APOE*4 allele in the APOE gene is the only established risk factor in the development of LOAD. However, as the APOE*4 allele is neither necessary nor sufficient for the development of AD, this emphasizes the involvement of other genetic and/or environmental factors, which alone or in combination with APOE*4 can modify the risk of AD. Recently, linkage studies in multiplex families with LOAD have provided evidence for the existence of additional putative genes for AD on several chromosomes, including chromosome 10. A broad linkage peak encompassing >50 cM region between chromosome 10q21 and 10q25 has been implicated that influence both AD risk and age-at-onset (AAO). There are more than 300 genes in this broad genomic region of chromosome 10 and thus task for identifying the chromosome 10 gene is daunting. One approach is to focus on the known candidate genes in the region. There are number of promising candidate genes in this region that are involved in either in the production, processing or clearance of beta amyloid peptide and include choline acetyltransferase (CHAT), plasminogen activator, urinary (PLAU), insulin-degrading enzyme (IDE), hematopoietically expressed homeobox (HHEX), glutathione S-transferase, omega-1 (GSTO1), glutathione S-transferase, omega-2 (GSTO2), and protease, serine, 11 (PRSS11). In this study, we have examined the role of these genes under the linkage peak on chromosome 10 to assess their role with AD risk and AAO. Association studies for 14 markers were performed in a large case-control cohort comprising 1012 white LOAD subjects and 771 white control subjects. No significant associations were observed with any of the polymorphism examined in the IDE, HHEX, GSTO1, GSTO2 and PRSS11 genes. Of the 3 CHAT SNPs examined, we detected both allelic and genotypic association of the intron 9 polymorphism with AD risk. The rare AA genotype appears to confer a modest risk for the development of AD in a recessive fashion (OR: 2.37; p=0.007). In addition, we observed APOE-dependent effect of the CHAT exon 5 polymorphism with AD risk (OR=0.76; p=0.046). Although these associations are modest, they suggest the presence of putative functional variants in the CHAT gene or nearby genes. In the PLAU gene, we examined 3 tagSNPs and found a modest protective effect with one SNP in the 3 UTR (OR=0.71; p=0.02), which was confined to APOE*4 carriers (OR=0.58; p=0.02). In our analysis of the association of the candidate genes with AAO, suggestive association were observed only with the PLAU 3 UTR (p=0.10) and intron 9 (p=0.04) polymorphisms. In summary, our data on a large number of AD cases and controls suggest that genetic variation in two positional candidate genes on chromosome 10 (PLAU and CHAT) may affect the risk and AAO of LOAD.

In the United States, cancer is a leading cause of death, second only to heart disease (MOKDAD et al. 2004). Although it is estimated that in 2005, cancer of the oral cavity will account for only 2.1% of cancer cases and 1.3% of overall cancer deaths in the U. S. (JEMAL et al. 2005), high-risk behaviors such as smoking cigarettes, using smokeless tobacco, and consuming excessive alcohol have been shown to play a major role in OSCC development. Exposure to environmental agents, including tobacco products, alcoholic beverages, and/or viruses, such as human papillomavirus (FORASTIERE et al. 2001; HO and CALIFANO 2004; MORK et al. 2001) have a profound influence on cells within the oral cavity. These factors have been shown to induce genetic alterations including chromosomal alterations, DNA changes, and/or epigenetic alterations, such as changes in DNA methylation that affect genetic regulation. Genetic alterations in cells are useful biological markers that assist in early detection of cancer and response to therapy (SIDRANSKY 1995). Currently, however, there are no useful biomarkers to identify early changes involved in OSCC development. One genetic alteration observed in 45% of OSCC is amplification of chromosomal band, 11q13. This event has been shown to follow dysplastic cellular changes, but occur prior to development of carcinoma in situ (FORASTIERE et al. 2001). Therefore, 11q13 amplification may be a useful biomarker for detecting OSCC. In addition, understanding the molecular mechanisms that promote 11q13 gene amplification may provide valuable information for devising novel prevention measures and therapies. In the current study, we show that the primary mechanism promoting 11q13 gene amplification is BFB cycles. Furthermore, we suggest that breakage at the common fragile site, FRA11F, may be responsible for initiating 11q13 gene amplification. By determining the primary mechanism that leads to 11q13 amplification in OSCC, additional investigations focusing on the biological basis of this process may provide important information for developing successful measures and treatments that will increase the survival rate for individuals afflicted with oral cancer.

Programs such as routine follow-up phone calls are an important part of public health. These programs are developed as a means to enhance the medical care offered by physicians. It brings many services to the public, making them more accessible for everyone. Integrating outreach programs in order to provide services to help meet the needs of a chronically ill population is of great importance to the field of public health. It is vital for patients with sickle cell disease to adhere to comprehensive care in order to decrease the morbidity and mortality of the disease. Patients who do not adhere to the recommended routine of care set forth by their physicians run the risk of developing serious complications or even death. The Comprehensive Sickle Cell Center at Childrens Hospital of Pittsburgh has established an outreach program that is aimed at increasing attendance to comprehensive care clinic, as well as educating patients and their families about sickle cell disease. All active patients were routinely contacted via phone in order to remind them to schedule an appointment in the clinic and to provide a range of services over the phone, such as prescription refills, answering questions about sickle cell disease, and psychosocial support. The patients were also asked questions about their home care routine so as to follow-up with them. This outreach program has been successful in increasing attendance to comprehensive clinic and in increasing the number of patients who are compliant with annual screenings.

Caveolae (little caves) are 50 -100 nm invaginations seen on the plasma membrane of most cells. Caveolin-1, the marker protein of caveolae in most tissues, is the structural and functional unit of caveolae. Previous studies have demonstrated the tumor suppressor capability of caveolin-1. Our lab has already shown that expression of caveolin-1 induces premature senescence in cells, possibly including those with malignant potential. We proposed that this might explain the tumor suppressor function of caveolin-1. We have further shown that oxidative stress induces premature senescence through up regulation of caveolin-1. To further elucidate the molecular mechanisms underlying this process, we used H2O2 as a model to generate stress induced premature senescence (SIPS) and examined the response of caveolin-1 promoter under these conditions. Constructs with serially truncated segments of the mouse caveolin-1 gene promoter linked to a luciferase reporter gene were made and luciferase assays were carried out. These experiments demonstrated that the critical regions lay in two segments: -222/-372 and -91/-150 of the promoter region. A consensus Sp1 binding sequence was identified within each deleted segment. Gel shift analysis of protein binding from nuclear extracts to these caveolin promoter DNA sequences confirmed that transcription factors were binding to the Sp1 consensus elements as part of the transcriptional response to H2O2 induced senescence. Further deletion mutagenesis of the individual Sp1 consensus sites confirmed the identity of the transcription factor to be Sp1. These findings suggest that Sp1 mediates oxidant induced up regulation of caveolin-1 expression. In subsequent experiments, we examined the effect of inhibitors of p38 Mitogen activated protein kinase pathway on the levels of caveolin-1 expression during SIPS by western blot and luciferase assay. We found that this pathway plays a direct role in the up regulation of caveolin-1 during SIPS, possibly through modification of Sp1 to increase its activity. Public Health Importance: Cancer is a leading public health concern. Cav-1 has been shown to be a tumor suppressor gene involved in a large number of human tumors. Induction of premature senescence in cells with malignant potential is thought to be a vital tumor suppressor function. Our study aims to define the tumor suppressor capacity of Cav-1 by elucidating the pathway by which it induces premature senescence.

A BIPHASIC ROLE FOR THE VOLTAGE-GATED SODIUM CHANNEL SCN5LAB IN CARDIAC DEVELOPMENT OF ZEBRAFISH JEFFREY S. BENNETT Dissertation under direction of Professor Dan M. Roden, M.D. Voltage-gated sodium channels (VGSCs) play an important role in generation and propagation of action potentials in nervous and cardiac tissues. Less is known of a possible role for them in development of the heart. Homozygous deletion of the voltage-gated sodium channel Scn5a results in embryonic lethality and developmental defects in the mouse heart. Morpholino knockdown of the two cardiac VGSC isoforms in zebrafish, scn5Laa and scn5Lab, results in small dysmorphic ventricles and death by four days post-fertilization. Here, I examine a role for scn5Lab in multiple phases of cardiac development. I find that loss of scn5Lab results in impaired specification of pre-cardiac mesoderm, with specific loss of the cardiac transcription nkx2.5. Differentiation of myocardium from the second heart field is determined to be intact, genetically and morphologically. Further I find that proliferation of differentiated cardiomyocytes is absent in morpholino-injected embryos, an effect that appears to be independent of impaired cardiac function. Electrophysiologic studies suggest voltage-gated sodium channels are present, but not required for action potentials at this time. I conclude that scn5Lab has a biphasic role in cardiac development of zebrafish, likely independent of its role in sodium ion permeation

A member of the TGF-β superfamily of cytokines, BMP2 not only plays a critical role in pattern formation and morphogenesis during early embryonic development, but also promotes osteoblast differentiation and bone formation, making it a vital factor for the maintenance of bone health. Expanding on earlier studies of the regulatory landscape surrounding Bmp2 by BAC transgenesis in mice that had lead to the identification of the osteoblast enhancer ECR1, the work presented here further scrutinizes the particular role of ECR1 during osteogenesis and provides striking evidence for the hypothesis that despite its indispensable role for Bmp2 expression in osteoblasts -, it is in fact not entirely autonomous, but requires additional enhancer activity to control the full scope of osteoblast-specific expression. Subsequent analysis of the chromosomal conformation during transcriptional activation does not only support this hypothesis and substantiate the dynamic nature of looping interactions at the locus, but combined with additional epigenetic characteristics also serves to highlight several distant loci within the gene desert as excellent candidates for putative enhancer function.