Brain invasion by Borrelia burgdorferi, the agent of Lyme disease, results in an inflammatory and neurodegenerating disorder called Lyme neuroborreliosis. Rhesus monkeys infected with a neurotropic strain of B. burgdorferi showed immunohistochemical evidence of astrogliosis. We explored the hypothesis that mediators elicited by brain cells, particularly astrocytes and microglia, in response to bacterial lipoproteins, constitute a molecular basis for central nervous system (CNS) disease seen in neuroborreliosis. We established primary cultures of rhesus monkey astrocytes and stimulated the cells with recombinant lipidated outer surface protein A (L-OspA), a model B. burgdorferi lipoprotein. Pure astrocytic cultures produced the cytokines interleukin (IL-6) and tumor necrosis factor (TNF-alpha) in response to L-OspA. L-OspA could elicit a proliferative effect on astrocytes which was considerably reduced in astrocytes pretreated with anti IL-6 antibody prior to L-OspA stimulation. Interestingly, L-OspA could also induce apoptosis in astrocytes by virtue of its ability to induce TNF-alpha. Astrocyte proliferation with concomitant apoptotic cell death is commonly seen during gliosis. The synthetic lipopeptide Pam3hex, which mimics the lipid moiety present in all bacterial lipoproteins, also elicited both IL-6 and TNF-alpha in astrocytes L-OspA activated Extracellular Signal regulated Kinases Erk-1, Erk-2 and Mitogen Activated Protein Kinase (MAPK) p38 in astrocytes by virtue of its lipid moiety. Selective inhibition of these kinases with U0126 and SB 203580 respectively, indicated that TNF-alpha production in astrocytes as induced by bacterial lipoproteins is regulated by both Erk 1/2 and p38 MAP Kinases. IL-6 production in astrocytes as induced by L-OspA was unaffected by the blockage of either of the kinases individually but was diminished in the presence of both inhibitors. Further, the apoptotic mediator Fas was significantly upregulated (p = 0.004) in rhesus astrocytes when exposed to L-OspA To simulate a more in-vivo-like scenario we stimulated aggregate brain cultures and pure microglial cultures with L-OspA. We observed production of IL-1beta, and IL-12 in addition to IL-6 and TNF-alpha The findings of this in vitro model of the interaction of bacterial lipoproteins with the CNS indicate that bacterial lipoproteins have the potential to mount the inflammatory response seen in Lyme neuroborreliosis in the rhesus monkey / acase@tulane.edu

TNF-alpha has been proposed as a link between insulin resistance and obesity, however the molecular mechanism whereby TNF-alpha attenuates insulin signaling is not well understood. The aim of this thesis was to map the in-vivo mechanism by which TNF-alpha contributes to the pathogenesis of impaired insulin signaling, using obese Zucker rats in which TNF-alpha activity was inhibited through adenovirus (AdV)-mediated expression of a TNF inhibitor (TNFi). Control animals consisted of obese Zucker rats infected with the same titer of AdV carrying the lac-z cDNA. Both peripheral and hepatic insulin sensitivity were improved in obese Zucker rats following neutralization of TNF-alpha. Additionally, TNF-alpha neutralization led to a 2.5 fold increase in tyrosine phosphorylation (pY) of IR in skeletal muscle while this was unchanged in liver, suggesting that TNF-alpha is a mediator of insulin resistance in obesity and may modulate IR signaling in skeletal muscle and liver through different pathways. Further examination showed that pY of pp125FAK was significantly higher in TNF-alpha neutralized animals, indicating that TNF-alpha may induce insulin resistance in the liver by preventing insulin-stimulated pY of pp125FAK. Thus, TNF-alpha may induce insulin resistance in the liver by dephosphorylating pp125FAK or preventing its pY response to insulin treatment To explore the cellular mechanism whereby TNF-alpha prevents insulin-stimulated pY of pp125FAK in the liver, we measured c-Src kinase activity and the abundance of 3 major protein tyrosine phosphatases (PTPs)---PTP-1B, LAR and SH-PTP2---in liver homogenates from obese Zucker rats following TNF-alpha blockade. TNF-alpha neutralization did not alter hepatic c-Src kinase activity, but led to a 75% reduction in LAR protein levels while PTP-1B and SH-PTP2 levels remained unchanged. To further verify that TNF-alpha exerts direct effects on LAR expression, we treated HepG2 with TNF-alpha and found that LAR protein levels were elevated in a TNF-alpha dose-dependent manner while PTP-1B and SH-PTP2 abundance remained constant. Furthermore, treatment of HepG2 cells with TNF-alpha (0.8 mM, 72 hr) attenuated insulin stimulation of pp125FAK pY. Taken together, our data suggest that TNF-alpha may attenuate insulin action in the liver by preventing insulin-mediated pY of pp125FAK through increased LAR expression and that this probably represents the critical interface between TNF-alpha and insulin signaling / acase@tulane.edu

This study examines the molecular epidemiology of Plasmodium falciparum parasite infection in Malian children living in a village where malaria transmission is endemic. Previous epidemiological studies of malaria typically focused on symptomatic infection or clinical cases of malaria with a diagnoses confirmed by slide smears without a capacity to examine simultaneous infection with multiple parasites of the same species. Multiple malaria infections in the same individuals were detected by using molecular markers that target the genetic diversity in Plasmodium falciparum by examining three merozoite surface protein-1 (MSP-1) variants genetically characterized as the K1, MAD20, and R033 allotypes. Detecting these allotypes using PCR provided results that were used to calculate the molecular-based frequency of simultaneous infection with multiple malaria parasites. We report estimates of malaria parasite infection and clearance observed in a fixed cohort of 80 children aged 0--9 years from September 1995 to August 1996. Our findings are as follows; (1) infection rates exceed estimates based upon slide smears, (2) infection clearance and re-infection is common, and (3) seasonal shifts occur in allotype-specific rates of infection. Our data also suggest that competition may take place between two of the allotypes. We have concluded that the infection dynamics of malaria parasites in endemic regions merits more attention especially in the context of infection dynamics the potential role it may have for risk to disease The broader conclusions concerning this study are three-fold. First, we believe examining multiple infections with malaria parasite in humans is an important step toward understanding both immunity and susceptibility to malaria parasite infection and provides a way to examine the risk for disease. Second, results reported here validate the usefulness for using a molecular approach for examining the epidemiology of malaria parasite infection in humans. Finally, we suggest that our findings demonstrate that combining classical measurement strategies with molecular methods provides researchers with robust methods for conducting future epidemiological studies of malaria infection and that these methods can be applied to the study of other parasites and infectious pathogens / acase@tulane.edu

Breast cancer is an endocrine-responsive neoplasm which is primarily sensitive to the mitogenic effects of the steroid hormone estrogen. Estrogen exerts its effects by binding to the estrogen receptor (ER). As such, the presence or absence of ER in breast tumors is an important factor in the determination of the prognosis of the breast cancer patient, as well as in the determination of the treatment strategy used. The function of the wild type ER is well characterized, however little is known with regards to variant ERs in breast cancer. As a result, the presence of ER variants in human breast cancer has received much attention over the past several years, as researchers have endeavored to identify their function in the human breast. While ER variant proteins have yet to be identified in human breast tissue, tumor or otherwise, significant progress has been made towards the identification and characterization of ER mRNA variants. Studies have identified a plethora of variant ER transcripts, however the best characterized of these variant ER mRNAs may be those which lack either exon 5 or exon 7 Exon 5 and exon 7 both encode regions of the hormone binding domain of the ER, and the deletion of these exons putatively results in variant receptor proteins which are truncated within the hormone binding domain. Functional analyses of these variant proteins have shown that the exon 5 deletion variant ER is constitutively active, regulating the expression of estrogen-responsive genes. The exon 7 deletion variant ER is not able to regulate the transcription of estrogen-responsive genes, however it does function in a dominant-negative manner, inhibiting the actions of the wild type receptor when the two are coexpressed The research herein has employed a variety of approaches to characterize the expression patterns of wild type and exon 5 deletion variant ER mRNAs in human breast cancer cell lines and identify some of the consequences the expression of these variant ER transcripts has on the breast cancer cell. Growth studies indicate that MCF-7 human breast cancer cells which express an elevated percentage of the exon 5 deletion variant ER transcript proliferate at a faster rate, in the absence of estrogen, than those cells which express an elevated percentage of wild type ER transcript. MCF-7 cells which express an elevated percentage of the exon 5 deletion varian ER transcript are less responsive to the growth-stimulatory effects of estrogen than MCF-7 cells which express a higher percentage of wild type ER mRNA. The growth of MCF-7 stocks which express an elevated percentage of the wild type ER mRNA was significantly stimulated above control levels by day 3 of treatment, while the growth of MCF-7 cells which express an elevated percentage of the exon 5 deletion varian ER transcript were not significantly stimulated above control levels until day 7 of treatment. (Abstract shortened by UMI.) / acase@tulane.edu

The major limitations in studying the biochemical variables of phenylketonuria in human patients are as follows: (1) Multiple mutations in the phenylalanine hydroxylase locus resulting in different extent of residual enzymatic activity; (2) Limitations in the compliance to dietary management; (3) Limited number of specimens from various physiological fluids and tissues that could be obtained for studies The major advantage in using the murine model of phenylketonuria is that these restrictions do not exist and each of the variables could be evaluated separately In the present study, mice with a mutation in the phenylalanine hydroxylase enzyme (a missense mutation in Exon 7 at codon 263) were studied. A mutation at the same locus occurs also in human patients and in both mice and humans the mutation leads to undetectable activity of phenylalanine hydroxylase We evaluated the plasma amino acids and urinary metabolites of the alternate pathway of phenylalanine in PKU Mice (PahEnu2) and control mice from the same strain (BTBR) on unrestricted and restricted phenylalanine intakes. It was shown that plasma phenylalanine levels reflected the dietary phenylalanine intake. Plasma tyrosine levels were significantly higher in PKU mice on a restricted phenylalanine intake when compared to PKU mice on an unrestricted phenylalanine intake. Significant decreases in plasma glycine levels in PKU mice on an unrestricted phenylalanine intake were observed as compared to PKU mice on a restricted phenylalanine intake and to control mice It was also shown that the alternate pathway metabolites of phenylalanine (phenylpyruvic, phenylacetic, ortho-hydroxyphenylacetic, phenyllactic, and mandelic acids, and phenylacetylglycine) were excreted in parallel to the plasma phenylalanine levels. An interesting observation was that the phenylacetylglycine was shown to be the best predictor of the plasma phenylalanine levels in mice. This glycine conjugate has not been previously described in human patients In another group of mice and controls the phenylalanine intake was gradually increased to approximately four-fold that of the normal diet and than gradually decreased to a markedly restricted phenylalanine intake. A reverse ratio was documented between plasma phenylalanine and glycine levels in the PKU mice. Also, in PKU mice on restricted and unrestricted phenylalanine intakes, phenylacetylglycine was shown to be the best predictor of the plasma phenylalanine levels An interesting observation was the loss of the fur color in untreated PKU mice (which reversed with treatment) with intermediate changes in partially treated PKU mice and no changes in treated PKU mice Histological studies with light microscopy did not reveal any significant changes in control or PKU mice on unrestricted and restricted phenylalanine intakes Considering the analogy between the murine and human phenylketonuria this model could be further used to study the biochemical and nutritional problems in this disease / acase@tulane.edu

The cascade of pathophysiological events that ensue in response to vascular injury is analogous to generalized wound healing. Vascular injury in which the endothelial cell layer is denuded and the underlying vascular smooth muscle cell (VSMC) layer is disrupted results in an expansion of the neointima. The phases of the healing process are controlled in large part by growth factors released at the site of injury Once cells are made competent, progression factors such as insulin-like growth factor-1 (IGF-1) act to promote cells through the cell cycle and thus, allow for a full proliferative response. It was hypothesized that a reduction in serum IGF-1, characteristic of chemically-induced diabetes, would contribute to an impaired wound healing response such that VSMC proliferation, migration and ultimately, neointimal thickening in response to vascular injury would be attenuated Delayed reendothelialization following vascular injury has been suggested to have a permissive impact on VSMC proliferation. This is illustrated by the finding that in areas in which the endothelial lining rapidly regenerates following injury, neointimal thickening is less marked than areas where regeneration occurs later. It was also hypothesized that endothelial cell regrowth, morphology, and endothelium-dependent vasoreactivity following catheter injury are improved by the diabetic state and account, in part, for the decreased mitogenic response and the attenuated neointimal thickening in response to vascular injury It was found that alloxan-treated New Zealand White rabbits classified as diabetic (glucose ≥ 400mg/dL) had significantly decreased serum IGF-1 levels compared to untreated, euglycemic rabbits. Diabetic rabbits also had decreased I/M ratios (area of the intima divided by the area of the media multiplied by 100) 2, 4, and 8 weeks after aortic injury compared to euglycemic rabbits. The I/M for high hyperglycemic rabbits (glucose = 286--399 mg/dL) was comparable to diabetic animals yet their IGF-1 levels were not significantly different from control. Vascular IGF-1 content was similarly increased following catheter injury between diabetic and euglycemic rabbits Two weeks after catheter injury the percent of endothelial cell regrowth was significantly increased in diabetic animals compared to euglycemic animals In conclusion, this study provides evidence against a direct effect of IGF-1 in the impaired wound healing, VSMC proliferation and migration, and MAPK activity following balloon catheter injury of the alloxan-induced diabetic rabbit. Further, these results show that endothelial cell regrowth and morphology in diabetic rabbits is improved, however, the restoration of endothelium-dependent vasoreactivity is not. (Abstract shortened by UMI.) / acase@tulane.edu

Viruses can cause cancer in animals and humans. The C-type viruses are etiologic agents in animal carcinomas, sarcomas, leukemias, and lymphomas. Bovine leukemia virus can infect sheep, goats and monkeys and is capable of infecting human cells. Avian leukosis/sarcoma viruses can transform mammalian cells including those of humans and can induce tumor in primates Epidemiological studies have reported increased probability of acquiring specific types of cancer in certain human populations groups. Farmers have been found to have elevated risks for several specific types of cancer such as hematologic and lymphatic malignancies, lip, stomach, prostate and brain cancer. Veterinarians die more often of large intestine cancer, leukemia, Hodgkin's disease and skin cancer. Excess risk of cancer has been found more frequently in slaughterhouse workers and other meat workers. Exposure to animal virus is hypothesized The main objective of the present correlational study was to examine whether there is a relationship between the presence of animal populations which harbor oncogenic viruses and the occurrence of selected human cancers--hemopoietic/lymphatic tissue, lung, esophagus buccal cavity, colon, kidney, bladder--in North Carolina. Additional Monte Carlo simulation was done to speculate on the probability of high correlation between the presence of animals and human cancer deaths Results were consistent with previous findings. The main findings are as follows: (1) Monocytic/myeloid leukemias and lymphomas were correlated with the presence of bovine. (2) Myeloma and lung cancer were correlated with the presence of pigs. The pig/area index correlated with lung cancer. (3) There were suggested correlations between the presence of pigs and the occurrence of lymphatic leukemia, esophagus, bladder, kidney, buccal and pharynx cancer in humans. (4) Simulation results raised the possibility of significant higher correlation between human esophagus cancer with the presence of hogs. (5) Farming was highly correlated with all but three cancer types: lung, colon, and lymphomas. (6) Human colon cancer deaths were not correlated with any animal population / acase@tulane.edu

Platelet-derived growth factor (PDGF) regulates proliferation, chemotaxis and differentiation of connective tissue cells during embryogenesis and possibly in disease states such as pulmonary fibrosis. To assess the effects of PDGF on lung development and pulmonary fibrosis in vivo, transgenic mice were generated in which expression of the human PDGF-A or -B gene was targeted to distal lung epithelium under control of the human surfactant protein C (SPC) promoter In SPC-PDGFB transgenic mice, overexpression of PDGF-B resulted in an abnormal lung structure in both adult and juvenile mice, including enlarged airspaces, inflammation, and fibroproliferative lesions. The severity of this phenotype varied significantly on C57BL/6, SJL and B6SJL hybrid backgrounds and progressed with age. To determine the effect of overexpression of PDGF-B on the progression of pulmonary fibrosis, transgenic and nontransgenic mice were challenged with three different fibrotic agents, including bleomycin, silica, and asbestos. Lung fibrosis induced by bleomycin and silica was similar in SPC-PDGFB mice and nontransgenic mice. Asbestos induced more pronounced fibrotic lesions 2 days to 8 weeks after multiple asbestos exposures, but fibrotic lesions examined 10 months after multiple asbestos exposures were similar in transgenic and nontransgenic mice. These studies suggest that PDGF stimulates inflammation and fibroblast proliferation in acute fibroproliferative lesions but other factors are required for end-stage lesions to develop Overexpression of PDGF-A in transgenic mice from the SPC promoter resulted in perinatal lethality caused by respiratory failure. To examine the effects of PDGF-A during lung development, SPC-PDGFA embryos were obtained on embryonic days (E) 16.5 and 18.5. Transgene message was detected throughout epithelial cells of the terminal respiratory buds but not in bronchioles and blood vessels. Lungs of SPC-PDGFA transgenic mice were larger and more solid than those of their nontransgenic littermates. The weights of whole lung and heart en bloc, and of the left lung of transgenic mice were heavier than those of nontransgenic embryos on E16.5. Histologic analysis of transgenic lungs showed a dramatic increase in mesenchymal cells and terminal buds on both E16.5 and E18.5 with a decrease in conducting airways and airspaces of respiratory buds. The cellular proliferation rate in transgenic lung was higher than that in wild-type littermates on E16.5. In transgenic embryos, progression to the saccular stage of lung development was blocked and elastin deposition was impaired. The results of these studies indicate that the downregulation of PDGF-A expression, which normally occurs during progression to the saccular stage, is required for development of a functional lung. The studies reported here, in contrast to many experiments with cultured cells, suggest that PDGF-A can have more potent effects in vivo than PDGF-B / acase@tulane.edu

Despite over a decade of intense effort, a vaccine preventing HIV infection has not emerged and HIV remains a worldwide pandemic. HIV is transmitted primarily through sexual contact at vaginal, rectal, and oral mucosal surfaces to infectious genital secretions. Mucosal surfaces may function as physical barriers that restrict viral access to the host, since HIV-1 sequences in recently infected recipients are relatively homogeneous, while those present in the blood of their donors are heterogeneous. In addition, mucosal surfaces may function as selective barriers, for the majority of HIV-1 isolates in most newly infected individuals are macrophage-tropic while those in their corresponding donors are dual-tropic. Therefore, as mucosal surfaces provide the first line of defense against HIV-infection, characterizing viral genotypes associated with mucosal transmission of HIV/SIV is essential in designing HIV prevention strategies. Accordingly, the primary goals of this dissertation were to develop SIV:macaque animal models for mucosal transmission of SIV/DeltaB670 and to determine if the genetic selection observed in HIV-infected humans occurs in SIV-infected macaques Thirty-seven macaques were inoculated either intravenously, intravaginally, orally, intracolonically, or intrarectally with varying SIV/DeltaB670 dilutions. The intestinally inoculated macaques progressed the most rapidly to disease, followed by the intravenously, orally, and vaginally inoculated groups in the order listed ($\rho$ = 0.033). Comparable to human studies, a decline in CD4+ T-lymphocytes in infected macaques was predictive of clinical disease progression. Further, the presence of p27 antigenemia, regardless of the route of inoculation, identified animals at risk of disease progression A comparative genetic analysis of SIV/DeltaB670 genotypes transmitted across vaginal, oral, colonic, and rectal mucosal surfaces in SIV-infected animals was performed on peripheral blood mononuclear cells collected early postinoculation. In the majority of infected animals, multiple genotypes were identified independent of the route of infection with genotypes identified most frequently in the inoculum. Thus, these data indicate no obvious selection for particular genotypes from within the SIV/DeltaB670 quasispecies at any of these mucosal surfaces. These data support the hypothesis that the mucosal barrier may play a minor role in HIV genotypic selection at mucosal surfaces and emphasize the need to evaluate the viral diversity present within the mucosal secretions of chronically infected individuals / acase@tulane.edu

The mechanisms by which SL3-3 MuLV (SL3) induces thymic lymphoma are incompletely understood. We performed a longitudinal analysis of SL3 infection in the thymus and bone marrow to assess and quantify the molecular and cellular alterations that occur during progression to lymphoma. Studies were performed in parallel using a weakly pathogenic enhancer mutant of SL3, termed SL3DeltaMyb5, in order to discriminate between events that contribute to malignancy from those that occur as a result of viral infection alone. Semi-quantitative PCR reveals that proviral DNA is detectable in the bone marrow, thymus and spleen of SL3- or SL3DeltaMyb5-infected mice during the premalignant period (1--8 weeks post-inoculation). Infection with either virus results in a significant alteration in normal intrathymic T-cell subpopulation distribution, as evidenced by an increase in the proportion of double-negative (CD4-CD8 -) T-cells and decrease in the proportion of double-positive (CD4+CD8+) T-cells. In addition, a marked thymic atrophy is observed at 6 weeks post-inoculation (p.i.) only in SL3-infected animals, suggesting that thymic atrophy may be a necessary step in the malignant process mediated by this virus. Immunocytochemical staining for viral coat protein (gp70) in the bone marrow revealed that SU and SL3DeltaMyb5, show different patterns of infection in this tissue during the premalignant period. Colony forming assays and flow cytometric analysis of the bone marrow demonstrate a pattern of disrupted hematopoiesis in SL3- and SL3DeltaMyb5, animals at 2, 3 and 4 weeks p.i. A PCR-based protocol was used to determine sites and timing of appearance of two types of polytropic envelope recombinant viruses in animals infected with SL3 or SL3DeltaMyb5, Longitudinal analysis demonstrated that the initial site of recombinant virus formation in SL3-Infected mice is the thymus, which harbors both types of envelope recombinants as early as 2 weeks p.i. No recombinants were detectable in the bone marrow of SL3-infected mice until 6 weeks p.i, findings inconsistent with their role in the initial transforming event reported to occur in this tissue. Both types of recombinants were detectable in thymus, bone marrow and spleen of animals infected with the weakly pathogenic SL3DeltaMyb5, mutant, although with reduced frequency and delayed kinetics of appearance / acase@tulane.edu