Improved methods for the in situ hybridisation detection of messenger RNA ( mRNA ) in sections of liver tissue, were derived by use of an experimental system. This involved the use of tritiated-poly ( dT ) probes to detect poly ( A ) sequences attached to the 3 ' end of mRNA in sections of mouse liver that had been processed in various ways. The improved - methods were applied to the detection of hepatitis B virus ( HBV ) - and hepatitis delta virus ( HDV ) - RNA. In situ hybridisation and immunostaining techniques were then applied to studies of the pathogenesis of HBV and duck hepatitis B virus ( DHBV ) infection. In situ hybridisation studies of liver biopsy tissue from HBV - infected immunosuppressed renal transplant patients demonstrated an anatomical association between piecemeal necrosis and HBV replication at the cellular level in some patients. However, widespread replicative infection of hepatocytes also occurred in some patients in the presence of normal hepatocyte morphology and mild inflammatory changes indicating that at the cellular level virus replication was not necessarily a direct cytopathic process. These findings supported the view that hepatocyte Injury may : ( i ) result from immune - mediated damage directed against cells undergoing replicative, but not restricted infection ; ( ii ) eliminate cells undergoing replicative infection and favour clonal regeneration of cells undergoing restricted infection. Localisation of interferon - alpha ( IFN - alpha ) expression in liver tissue chronically infected with HBV and HDV, identified mononuclear cells and fibroblasts ( but not hepatocytes ) as the main producers of IFN - alpha. IFN - alpha - positive cells were associated with areas of liver tissue containing cells supporting virus replication and exhibiting the greatest degree of liver damage, suggesting that locally produced IFN - alpha may be a natural regulator of virus replication in chronic liver disease. Experimental DHBV infection of Pekin - Aylesbury ducks showed that virus inoculated either intravenously or intraperitoneally, gained access to randomly distributed hepatocytes without first replicating in other cell types in the liver. Virus was seen to disseminate to contiguous cells following anatomical boundaries by the third day post - inoculation. Markers of DHBV infection in liver and serum showed reproducible kinetics, and duck hepatocytes in this system appeared to be highly permissive as large amounts of DHBV DNA and DHBsAg were produced intracellularly without the development of ongoing cytopathology. Hepatocytes were the major cell type responsible for early significant DHBV replication, in contrast to pancreas, kidney, spleen and circulating mononuclear cells where significant levels of infection were detected only after the first week of infection and the onset of viraemia. / Thesis (Ph.D.)--Department of Microbiology and Immunology, 1989.

A major product of HIV-infected and cytokine-stimulated monocytic-lineage cells is quinolinic acid (QUIN), a neurotoxic metabolite of the kynurenine pathway (KP) of L-tryptophan (L-Trp) metabolism. Despite the large number of neurotoxins found in HIV patients with AIDS Dementia Complex (ADC), only QUIN correlates with both the presence and severity of ADC. With treatment, cerebrospinal fluid (CSF) QUIN concentrations decrease proportionate to the degree of clinical and neuropsychological improvement. As endothelial cells (EC) of the blood-brain barrier (BBB) are the first brain-associated cell that a bloodborne pathogen would encounter, this project examined the BBB response to KP metabolites, as these are implicated in damage of the CNS associated with ADC. Using RT-PCR and HPLC/gas chromatographymass spectrometry (GC-MS), I found that cultured primary human BBB EC and pericytes constitutively expressed the KP. EC synthesised kynurenic acid (KA) constitutively, and after immune activation, kynurenine (KYN). Pericytes produced small amounts of picolinic acid and after immune activation, KYN. An SV40-transformed BBB EC showed no KP expression. By contrast, human umbilical vein EC only expressed low levels of KA after immune activation, however human dermal microvascular EC showed a similar constitutive and inducible KP to that in BBB EC. As T cells are central to primary HIV infection, I also examined KP expression in two CD4+ and one CD4- cell lines, but none showed either constitutive or inducible KP expression. I next examined how QUIN might interact with BBB EC. There was no binding of 3H-QUIN to cultured primary human BBB EC, however a biologically relevant concentration of QUIN induced changes in gene expression which adversely affected EC function, possibly mediated by lipid peroxidation and oxidative stress. The upregulated genes were of the heat shock protein family, and the downregulated genes were associated with regulation of cell adhesion, tight junction and cytoskeletal stability, metalloproteinase (MMP) regulation, apoptosis and G protein signaling. Immunofluorescence showed that QUIN induced morphological changes in BBB EC consistent with the changes in gene expression. Gelatin zymography showed that this was not mediated by MMPs, as constitutive MMP expression was unchanged. These data provide strong evidence for QUIN directly damaging the BBB in the context of HIV infection.

A major product of HIV-infected and cytokine-stimulated monocytic-lineage cells is quinolinic acid (QUIN), a neurotoxic metabolite of the kynurenine pathway (KP) of L-tryptophan (L-Trp) metabolism. Despite the large number of neurotoxins found in HIV patients with AIDS Dementia Complex (ADC), only QUIN correlates with both the presence and severity of ADC. With treatment, cerebrospinal fluid (CSF) QUIN concentrations decrease proportionate to the degree of clinical and neuropsychological improvement. As endothelial cells (EC) of the blood-brain barrier (BBB) are the first brain-associated cell that a bloodborne pathogen would encounter, this project examined the BBB response to KP metabolites, as these are implicated in damage of the CNS associated with ADC. Using RT-PCR and HPLC/gas chromatographymass spectrometry (GC-MS), I found that cultured primary human BBB EC and pericytes constitutively expressed the KP. EC synthesised kynurenic acid (KA) constitutively, and after immune activation, kynurenine (KYN). Pericytes produced small amounts of picolinic acid and after immune activation, KYN. An SV40-transformed BBB EC showed no KP expression. By contrast, human umbilical vein EC only expressed low levels of KA after immune activation, however human dermal microvascular EC showed a similar constitutive and inducible KP to that in BBB EC. As T cells are central to primary HIV infection, I also examined KP expression in two CD4+ and one CD4- cell lines, but none showed either constitutive or inducible KP expression. I next examined how QUIN might interact with BBB EC. There was no binding of 3H-QUIN to cultured primary human BBB EC, however a biologically relevant concentration of QUIN induced changes in gene expression which adversely affected EC function, possibly mediated by lipid peroxidation and oxidative stress. The upregulated genes were of the heat shock protein family, and the downregulated genes were associated with regulation of cell adhesion, tight junction and cytoskeletal stability, metalloproteinase (MMP) regulation, apoptosis and G protein signaling. Immunofluorescence showed that QUIN induced morphological changes in BBB EC consistent with the changes in gene expression. Gelatin zymography showed that this was not mediated by MMPs, as constitutive MMP expression was unchanged. These data provide strong evidence for QUIN directly damaging the BBB in the context of HIV infection.

Although the occurrence of ketosis is a postpartum phenomenon, recent studies have focused on the prepartum period as key in the development of the disorder. Indicators of prepartum energy status, such as depressed dry matter intake (DMI) and elevated plasma non-esterified fatty acid (NEFA) concentrations have been associated with the occurrence of ketosis. The objective of this study was to investigate the role of phosphoenolpyruvate carboxykinase (PEPCK) in the periparturient and ketotic cow. The enzyme PEPCK catalyzes the rate limiting step in gluconeogenesis in hepatocytes. Whereas, in adipocytes, it has been suggested that PEPCK functions in the synthesis of glycerol for the formation of triacylglycerol (TAG) when plasma glucose concentrations are low. Thirty-four pregnant multiparous Holstein dairy cows were fed a single prepartum ration that consisted of 50% oat hay, 18% corn silage and 32% grain mix (DM basis). The ration was formulated to meet or exceed NRC requirements of 14% CP and 1.6 Mcal/kg NE[subscript L]. At calving, cows were transitioned onto one of two postpartum diets: control (n=14) or 3.5% supplemental fat (n=20). The postpartum diets, fed from wk 1 to 3, were formulated to isonitrogenous and to meet NRC requirements. Both diets consisted of 25% alfalfa, 25% corn silage and 50% grain mix. The control and fat diets contained 17.2 and 17.6% CP and 1.67 and 1.74 Mcal/kg NE[subscript L] respectively. Liver biopsies from 28 cows and adipose tissue biopsies from 6 cows were collected at -14, 2 or 3 and 14 d relative to calving. Tissue samples were analyzed for PEPCK mRNA and activity. All results were analyzed by period: prepartum (-21 to -2 d), freshening (2 to 7 d) and postpartum (8 to 21 d). In a previous study in our lab, 25 and 75% cows on the control and fat diets, respectively, experienced ketosis. In the current study there a 40% occurrence of ketosis for both control and fat diet groups. The high occurrence in both diets may be attributed to the rapid transition from the dry cow ration (70:30 forage to concentrate ratio, DM basis) to the lactating cow ration (50:50 forage to concentrate ratio, DM basis). The cows on the fat diet had lower serum glucose at freshening. Cows with ketosis had higher prepartum body weights (788 kg) than non-ketotic cows (743 kg; P<.1). No prepartum differences were seen in body condition score, DMI, NE[subscript L] balance, NEFA, glucose or ��-hydroxybutyrate concentrations were detected between ketotic and non-ketotic cows. Expression of adipose PEPCK mRNA was not different between ketotic and non-ketotic cows. However, hepatic PEPCK mRNA expression was higher in non-ketotic cows at freshening when compared to ketotic cows. Cows that experienced ketosis had lower hepatic PEPCK activity prepartum (6.6 vs. 9.3 units /min/g protein) and postpartum (7.6 vs. 10.2 units/min/g protein; P<0.5) when compared to non-ketotic cows. Our data indicated that hepatic PEPCK is a useful prepartum predictor of a cows susceptibility to ketosis. / Graduation date: 1998

Three closely related isolates belonging to the A��� serotype of infectious pancreatic necrosis virus (IPNV) were selected for comparison, to provide insight into the nature of variation in the virulence of IPN viruses. Brook trout fry (Salvelinus fontinalis) were experimentally infected with the three isolates by immersion. Cumulative mortalities over a 62 day period for the three isolates were 67%, 78%, and 93%. The negative control was 3%. Virus titers from whole fish homogenates sampled at peak mortality for each isolate were statistically similar, indicating that quantity of virus does not account for virulence differences. For the two least virulent isolates, the virus titer was inversely correlated with fish weight, whereas for the most virulent isolate, no correlation was observed. Amino acid sequences of the viral capsid protein VP2 were determined using the reverse transcriptase polymerase chain reaction (RT-PCR). There were two amino acid changes at residue 217 and 288 between the two least virulent isolates and the most virulent isolate. These changes might provide a specific molecular basis for the variations in virulence among isolates. The progression of IPN virus infection in the experimentally infected fry was followed using histopathology, in situ cDNA hybridization, and alkaline phosphatase immunohistochemistry (APIH). While microscopic lesions were limited almost exclusively to necrosis of the pyloric caeca and pancreas, positive reactions with in situ hybridization and APIH were observed in tissues throughout infected fish. An IPNV infection appeared to be established in the fish by two routes: by entering the skin/lateral line and diffusing through the muscle, and from the oral region into the gastrointestinal tract by ingestion. In a second experiment, within a group of experimentally infected brook trout fry, external and behavioral signs of IPN disease in moribund fish disappeared, with the fish becoming healthy in appearance. Several of these fish were sampled, along with dead, moribund, and asymptomatic fish (never showed signs of IPN disease). Very few differences were observed among the fish sampled, using histopathology and in situ hybridization. Fish that appeared to recover after displaying signs of IPN disease died within a 2 week period. / Graduation date: 1997

The outer membrane protein OmpA is one of the major outer membrane proteins in many species of bacteria, including the Yersiniae. Our goal was to explore the role of OmpA in Y. pestis. This encompasses the ability of Yersinia to infect and survive within macrophages, as well as to resist antimicrobial compounds. Our laboratory found that a delta ompA mutant is impaired in a macrophage-associated infectivity assay. We also found that OmpA might play a role in the ability of the bacteria to resist antimicrobial peptides, specifically polymyxin B. Aditionally, we assessed the differences in OmpA of Y. pestis and E. coli, and determined that the characteristics we have observed in Y. pestis are unique compared to what has previously been described in E. coli. Our results indicate that Y. pestis OmpA might act through known pathways of antimicrobial resistance such as the PhoPQ two-component regulatory system, although further experiments are needed to determine the precise mechanism of function OmpA. Overall, our project characterizes the different functions of OmpA in Y. pestis, both as a key player in intracellular survival and as a necessary component in conferring resistance to antimicrobial peptides.

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) superfamily of proteins, circulates in blood in a complex with vitronectin (VN). These two proteins are also found localized together in the extracellular matrix in many different pathophysiological conditions. Both of these proteins are involved with a number of physiologically important processes. Though PAI-1 is a well-known inhibitor of serine proteases, more emphasis is now geared towards its protease independent functions. VN, on the other hand, is a binding protein that exists in the circulation in a preferred monomeric conformation. However, in the extracellular matrix, VN exists as multimer with altered conformation. Though the exact reason for such conformational alterations and compartmentalization is unknown, there are a number of biomolecules, including PAI-1 that are proposed to cause such alterations. In last few years, sufficient experimental evidence has been gathered to confirm this protease- independent effect of PAI-1 by which it induces multimerization of VN in a concentration-dependent fashion. It has been observed also that PAI-1 remains associated with this multimeric complex for several hours. A major focus of this dissertation work was to extend our understanding of the mechanism of the interaction between these proteins and to explore the physiological relevance of the multimeric complexes formed by their interaction on cellular adhesion and migration. In our study, emphasis has been given to the presence of an appropriate microenvironment so that the role of the multimeric complexes could be investigated in a relevant biological setting. Our findings indicate the importance of the surrounding microenvironment in establishing the specific role of the VN/PAI-1 complex in cell-matrix interactions. In a previous study from our lab, it was found that vitronectin knock-out mice were more resistant to Candida infection compared to wild type C57Bl/6 mice. One of the goals of this dissertation work was to provide a mechanistic explanation for their increased survival of the vitronectin knock-out mice upon Candida infection. Another important aspect of this work was to establish biophysical methods for understanding the structural changes that happen in PAI-1 naturally or due to ligand binding.

Reticuloendotheliosis virus (REV) is a retrovirus that causes a neoplastic disease in a wide range of avian hosts including chickens, turkeys, and ducks. In 1993, REV was detected in the endangered Attwater's prairie chicken (Tympanachus cupido attwateri), a subspecies of Tympanachus cupido. Subsequent infections of this prairie chicken have been identified at captive breeding facilities throughout Texas. The implications of these infections have severely hindered repopulation efforts by these facilities. This study focused on investigating REV infection of captive Attwater'€™s prairie chicken in order to better understand the disease affecting these endangered birds. The overall objective was to develop a means of eliminating this threat to the repopulation of the Attwater's prairie chicken. Several aspects of virus infection were investigated. Reagents capable of recognizing prairie chicken IgY and viral gag polypeptides were developed for use in assays for detection of antibody responses and titration of viral concentrations. Sequencing data of genomes collected from isolates of Texas prairie chickens and domestic chickens, as well as three REV prototype viruses, were compared to determine relationships among strains and identify the potential origin of the REV infecting Attwater'€™s prairie chicken. Additionally, a flow cytometry technique of segregating the lymphocyte population from peripheral blood mononuclear cells (PBMC) using a pan leukocyte monoclonal antibody was developed to more accurately measure changes within lymphocyte populations. This technique combined with intracellular labeling was used to deduce the target cells of REV infection. A nested polymerase chain reaction (PCR) test was developed for greater sensitivity in detecting infection in birds than the previous method of single amplification PCR. This greater sensitivity results in earlier identification of the virus in infected birds, which allows for earlier removal of infected birds to minimize transmission of the virus throughout the flock. The sensitivity of the nested PCR diagnostic test was determined in a dose response pathogenesis study, which was conducted on hybrid greater/Attwater's prairie chicken to observe the experimental development of disease in these birds. Finally, a vaccine was developed using plasmid DNA with REV encoded genes and tested on naturally infected prairie chickens to determine its efficacy in reducing viral load. Although no reduction in viral load was detected, the vaccine may be effective in providing prophylactic protection in future studies.

Human T-cell lymphotropic virus type 1 (HTLV-1) infects about 20 million individuals worldwide. This retrovirus induces two major types of pathologies: adult T-cell leukemia (ATL) or a neurodegenerative disorder called HAM/TSP (HTLV-associated myelopathy/tropical spastic paraparesis). The HTLV-1 transactivator protein (Tax) plays a central role in the development of these pathologies. We hypothesized that Tax activities are modulated through complexation with cellular proteins. Using the yeast two-hybrid method, we isolated two cellular Tax binding proteins, Gβ2 and MCM3, involved in cell signaling and DNA replication, respectively. We demonstrate that the interplay between Tax and Gβ2 modulates migration of infected T-lymphocytes toward chemokines. On the other hand, by interacting with MCM3, Tax accelerates DNA replication during the synthesis phase of the cell cycle and generates DNA damages responsible for cell transformation. We have thus identified two novel Tax partners potentially participating to the mechanisms by which the viral protein ensures viral persistence and leads to the development of HTLV-1 pathologies.

Pasteurella multocida causes diseases in many animal species including fowl cholera, a septicemic disease of poultry and other birds. Pathogenesis of the disease has been studied by many investigators by the systemic administration of the organism in poultry. However, only a few studies have been done as to the respiratory pathogenesis of the organism. The objective of the study was to investigate the fate of P. multocida after the intratracheal administration in turkeys The fate of four strains of Pasteurella multocida was studied after their intratracheal inoculation in young adult turkeys. Viable bacterial counts were made in respiratory tissues as well as in the liver, spleen and blood at 6 and 9 hrs after the inoculation of approximately 10⁹ viable organisms of each strain. A virulent, encapsulated strain, P-1059, invaded systemically by 6 hrs postinoculation (PI) and multiplied vigorously in all tissues and organs examined. A blue colony mutant of P-1059, T-325, which does not possess a thick layer of capsule, as well as CU vaccine strain, invaded the parenchymal organs, but did not show significant increase in viable counts at 9 hrs PI compared with at 6 hrs PI. Another vaccine strain, M-9, also invaded blood and internal organs by 6 hrs PI, however, its viable counts showed no significant change between 6 and 9 hrs PI, or in some tissues significant decrease at 9 hrs PI. The results indicate that all the four strains possess high capacity to invade respiratory tissues with varying capacity to persist in host tissues. The lesions caused by two strains of Pasteurella multocida (P-1059 and M-9) were observed after their intratracheal inoculation in young adult turkeys. The lesions were observed in the respiratory organs at 0, 0.25, 0.5, 1, 2, 3, and 6 hrs after inoculation of approximately 10⁹ viable organisms of each strain. Both virulent strain, P-1059 and non-virulent vaccine strain, M-9, have capacity to invade and multiply in the tissues examined. Macroscopicly, the lesions in the lung and in the airsac were found as early as 1 hr PI, including the infected lung was foamy and the airsac became cloudy. They became more severe by 2 to 6 hrs PI. Microscopicly, hecerophiles were present, occasionally, in the lung, trachea and airsac by 0 to 1 hr after inoculation. Then they became more severe by 2 to 6 hrs PI. By 6 hrs PI, there were diffuse heterophiles infiltration in the trachea, lung, anc airsac. The lung vascular was edema. The trachea ciliate and mucous gland was cystic or hyperplasia, and the airsac shewed increased in thickness and cloudiness. These results of study indicate that the lesion caused by P-1059 and vaccine strain, M-9, were not significantly different. / Graduation date: 1992