Hepatitis C can be viewed as an acquired genetic disease and represents a major potential application of gene therapy. The development of successful gene therapy protocols requires a therapeutic gene capable of blocking viral gene expression or function, vectors that confer safe, efficient, and stable recombinant gene expression, as well as, animal models to test the toxicity and in vivo efficacy of anti-viral agents. To develop a primate model for liver directed gene therapy, we studied several gene transfer vehicles and routes in 8 rhesus monkeys. Adenovirus-mediated gene transfer to primate livers via the portal vein or saphenous vein was efficient, but resulted in transient expression and was accompanied by an immune response to both vector and transgene products and acute hepatitis, while lipofectamine-mediated transfer was inefficient. Immunosuppression prior to administration of adenovirus prolonged transgene expression and prevented hepatitis. Selective gene ablation with intracellular single-chain immunoglobulin (sFv) represents a novel approach to treatment of viral infection. For this purpose, recombinant variable regions of the human monoclonal antibodies CM3.B6 and DIO.E6 that react with the NS3 and NS4A proteins of HCV respectively, were introduced into adenoviral vectors. The sFv's were efficiently and stably expressed in hepatocytes and bound to their cognate antigens within cells. Significantly, expression of sFv to NS3 resulted in a decrease in HCV RNA in primary hepatocytes isolated from explanted livers of three patients with chronic HCV when measured by quantitative, competitive RT-PCR. In an attempt to establish a convenient animal model of HCV infection, we produced transgenic mice by introducing the polyprotein coding region of HCV genotype lb genome under transcriptional control of the liver-specific mouse albumin enhancer/promoter. One mouse expressed the transgene in the liver, however he failed to reproduce. Interestingly, this mouse developed hepatocellular carcinoma (HCC). More data is needed to make a connection between HCV transgene expression and HCC. This study demonstrates the therapeutic potential of adenovirus mediated expression of sFv's to HCV proteins. However, improved vectors or immunomodulatory adjunct therapy will be required to make this therapy feasible in humans. HCV transgenic mice may be a good model to test this antiviral approach / acase@tulane.edu

Cadmium is a heavy metal which is naturally found in soil and water. It has similar properties to copper, zinc and calcium, and it may disrupt essential metal homeostasis. The toxicity of cadmium is magnified by its extremely long biological half-life in the body, between 10 to 30 years. Although the primary body burden is found in the liver and kidneys, the testis is extremely sensitive to cadmium, and acute exposure results intesticular edema and hemorrhagic necrosis. While most animals with scrotal testes are susceptible to the testicular toxicity of cadmium, strain-related differences to this effect occur in mice. For this dissertation, the mechanism(s) of murine strain differences to cadmium-induced testicular toxicity was studied using sensitive (129/J) and resistant (A/J) mice. We found significantly decreased cadmium concentrations in the testis, epididymis and seminal vesicle 24 hr after subcutaneous cadmium exposure in the resistant A/J mice, as compared to the sensitive 129/J mice. The transport of cadmium across vascular barriers was also markedly different in the two strains. Influx rates of $\sp{109}$Cd into the brain, testis and epididymis were found to be significantly lower in A/J mice. To help identify the natural ligand for the transport system in these tissues, $\sp{65}$Zn influx rates were studied. Zinc is an essential element, and a transport system should exist to facilitate passage into tissues. Since cadmium is a nonessential and toxic metal, specific mechanisms would not be expected to be present to maintain homeostatic concentrations of this cation. Cadmium and zinc share many properties, and the two elements have been shown to compete for transport. Thus, cadmium may use the zinc transporter to enter tissues. The resistant A/J mice demonstrated a significantly reduced zinc entry into the testis and brain when compared to $\sp{129}$/J mice. Cadmium and zinc transport were significantly lower in A/J mice, suggesting that the resistant mice may have a decreased number of zinc transporters, or a mutation in the transporters that makes them less active. Therefore, the nature of murine strain resistance to the testicular effects of cadmium may be related to a relative deficiency or a mutation in the zinc transporter / acase@tulane.edu

The use of insecticide-treated bednets (ITNs) has proven to be a promising and affordable intervention for controlling malaria in endemic areas of sub-Saharan Africa. However, there is concern that sustained ITN use in areas of high malaria transmission may delay the acquisition of clinical malaria immunity, consequently shifting the burden of mortality from younger to older children. Furthermore, there is concern that the efficacy of ITNs may diminish following sustained use due to the potential of lowered malaria immunity among the target population. To address these concerns, monitoring of child mortality using a demographic surveillance system (DSS) was continued for an additional two years after ITNs were distributed to control villages at the completion of a two-year community randomized control trial that assessed the impact of ITNs in an area of intense perennial malaria transmission in western Kenya. Consequently, all-cause child mortality (1--59 months) was assessed among children living in villages where ITN coverage has been sustained for over four years, as well as within villages where ITNs had only recently been introduced within the past two years. An additional aim of this research was to provide an evaluation of the DSS at identifying births and death in children <5 years old using a two-sample capture-recapture method. Results from this research provide no evidence of a shift in mortality from younger to older children following sustained ITN coverage in this setting. ITNs were associated with saving approximately 355 post-neonatal infants and 468 children 1--59 months over the course of the four-year trial. Additionally, ITNs were shown to remain efficacious at reducing all-cause post-neonatal infant mortality, and to a lesser extent 1--59 month mortality, over the entire course of the trial. And lastly, capture-recapture methods appear to be a worthwhile tool for assessing differential ascertainment of vital demographic events between subgroups of the population monitored with a DSS / acase@tulane.edu

Glaucoma is a disease that affects over 1 million people in the United States, and is one of the three leading causes of blindness nationwide. Loss of vision from glaucoma is believed to be the result of damage to the axons of the retina ganglion cells as they pass through the lamina cribrosa, which spans the opening in the back of the eye wall called the scleral canal. It is known that elevations of intraocular pressure (IOP) are associated with this damage, but the exact mechanisms by which the level of IOP causes these changes are unknown and controversial The objective of this work is to investigate how the load-bearing connective tissues within and around the optic nerve head (ONH) respond to changes in intraocular pressure, and how these responses are altered when the tissues are damaged early in glaucoma. The connective tissues of the ONH provide support for the retinal ganglion cell axons as they pass through the wall of the eye, and are crucial in maintaining axonal health at the ONH. Since the ONH is the principal site of glaucomatous damage, understanding how these connective tissues respond to different loading conditions provide insight into the pathophysiology of the retinal axons in this disease Histologic measurements made in 4 mum serial sagittal sections show that acute increases in IOP can deform the load-bearing connective tissues of the ONH. This is true both when the IOP increase is from 0 to 10 mm Hg, and when the increase is from 10 to 30 or 45 mm Hg. Additionally, these measurements show that in eyes that have been given early experimental glaucoma, the magnitude of the deformations caused by a given IOP increase is larger than in normal eyes, and that these larger deformations have both a plastic and hyperelastic component. Using digitized three-dimensional reconstructions, these profound deformations can be visualized. Finally, using finite element modeling that incorporates these digital reconstructions, a better understanding was gained of how regional stresses and strains are related to these deformations, and how all of these factors are associated with the onset and progression of glaucoma / acase@tulane.edu

Cryptococcus neoformans is a ubiquitous, encapsulated fungus and is the causative agent of cryptococcosis, a life-threatening opportunistic infection. The most severe manifestation of infection with C. neoformans is the involvement of the central nervous system (CNS), called cryptococcal meningoencephalitis. In immunocompromised individuals, cryptococcal meningoencephalitis is often fatal; but even when treated with anti-fungal drugs, the infection persists requiring life-long secondary prophylaxis. With better understanding of the mechanisms of protection from this pathogen, it may be possible to develop therapies to augment or bolster the immune response in immunosuppressed individuals so that they may be able to clear this otherwise fatal infection. In this study, a murine model is used to analyze the components and mechanisms of a protective response to C. neoformans in the CNS. A cell-mediated immune response is induced in the otherwise susceptible mouse by immunization with a cell-free antigen. Characterization of the protective response in the CNS is performed. Survival studies demonstrate that the immune response induced by peripheral immunization is capable of protecting the mouse from an otherwise lethal intracerebral inoculation of C. neoformans. This protection is associated with the production of Th1-type cytokines including interferon-gamma (IFNgamma). The production of pro-inflammatory cytokines, are associated with protection, as is the early production of beta chemokines. CD4 + T cells are required for the protective response; furthermore, adoptive transfer experiments demonstrate that CD4+ T cells are sufficient for some protection. CD4+ T cells are also required for optimal cytokine and chemokine production during the protective response; and in immunized mice, CD4+ T cells themselves produce IFNgamma at the site of infection. Analyses of leukocytes in the brains of mice undergoing protective and non-protective responses to C. neoformans reveal that both resident macrophage-like cells and macrophages recruited to the CNS from the periphery may be involved in protection. Effector mechanisms may include the production of nitric oxide by inducible nitric oxide synthase (iNOS), which is upregulated in the protective response. This increased production of iNOS is CD4+ T cell-dependent. Overall, the analyses of this study support the hypothesis that a CD4+ T cell mediated response is capable of providing protection to cryptococcal infection of the murine CNS / acase@tulane.edu

The African blood fluke Schistosoma mansoni is the etiological agent of intestinal schistosomiasis, the most serious of the helminth parasitic diseases. LTR retrotransposons make up substantial portions of eukaryotic genomes and are important both for their roles in genome evolution and instability and for their potential use in various applications. At the outset of this study, we hypothesized that LTR retrotransposons exist in the genome of S. mansoni, that most but not all copies of these retrotransposons will be inactivated due to the accumulation of mutations, and that mechanisms must exist to prevent uncontrolled proliferation of these elements. In addition, we hypothesized that similar elements should exist in either closely related platyhelminth organisms, or in host species of schistosomes The first LTR retrotransposon to be characterized from the genome of Schistosoma mansoni, designated Boudicca, included direct LTRs 328 bp in length and three open reading frames encoding gag, pol, and an unknown gene. Boudicca was a member of the recently characterized CsRn1 subclade of Gypsy-like retrotransposons and was transcribed in the sporocyst, cercaria and adult developmental stages of S. mansoni. Boudicca's copy number was found to exceed 1,000, with the fully characterized copy resembling the sequences of mRNA transcripts produced by RT-PCR. Models of the secondary structure of the Boudicca transcript suggested a transcription or translation based method of regulation of Boudicca proliferation The second LTR retrotransposon characterized during these studies, Sinbad, belongs to one of five discreet subfamilies of Pao/BEL like elements, which were found to be present only in animal genomes. Sinbad was 6,287 bp long, was flanked by identical LTRs of 386 bp, and displayed genomic features characteristic of Pao/BEL elements. There are about 50 copies of Sinbad in the S. mansoni genome. ESTs representing transcripts of Sinbad were found in six developmental stages of S. mansoni. The LTRs of Sinbad, but not Boudicca, were able to drive luciferase expression in HeLa cells, indicating that they represent functional promotors. Sinbad's LTRs also appear to function bidirectionally Both Boudicca and Sinbad were shown by Southern hybridization to be present in the genome of the related schistosome, Schistosoma haematobium / acase@tulane.edu

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