Thesis (M. Phil.)--University of Hong Kong, 1998.

Nociceptive afferent axons of the stomach send signals centrally to the brain and locally to stomach tissues. Nociceptive axons can be detected with a variety of different markers. In particular, the neuropeptide substance P (SP) is one of the most commonly used markers for nociceptive nerves in the somatic and visceral organs. In addition, the Local release of SP contributes to tissue protection and repair in response to noxious stimuli. However, the topographical distribution and morphological structure of SP-immunoreactive (SP-IR) axons and terminals in the whole stomach have not yet been well determined. In this study, we labeled SP-IR axons and terminals in flat-mounts and serial sections of the antrum, pyloric sphincter, and duodenum (APD) of the mouse stomach. The flat-mount stomachs included the external muscles (longitudinal and circular layers) and the myenteric ganglionic plexus, whereas APD serial sections also included the submucosa and mucosa. Tissue samples were scanned using confocal and Zeiss M2 imager microscopy to acquire detailed high-resolution images of SP innervation and produce montages of the whole stomach flat-mount and APD serial sections. Additionally, we double-labeled the samples with vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH), and injected the stomach walls with DiI-retrograde tracer to determine the connectivity map between SP-IR axons and their possible extrinsic origin. We found that: 1) SP-IR axons innervated all layers including the external muscles, myenteric ganglia, submucosa, submucosal ganglia, and mucosa. Many axons were immunoreactive for VAChT but not TH. 2) SP-IR axons and terminals formed an extensive network in the muscular layers and ran in parallel with the long axis of the external muscles. 3) SP-IR axons formed very dense terminal varicosities encircling individual neurons in the myenteric plexus 4) In the submucosa, SP-IR axons innervated blood vessels and submucosal ganglia and formed a nerve network around duodenal Brunner's glands. 5) In the mucosa, SP-IR varicose axons were organized into bundles in the muscularis mucosa and lamina propria at the base of the mucosa. Some SP-IR axons were found in the gastric subepithelial level and duodenal villi. 6) SP-IR axon density in the muscles and myenteric plexus was much higher than in the submucosa and mucosa. 7) The density of SP-IR axons and terminals in the flat-mounts varied in different stomach regions in the following order from high to low: antrum-pylorus, corpus, fundus, and cardia. 8) In the APD area, the muscular wall of the antrum and duodenum showed a higher SP-IR axon density than in the pyloric sphincter. 9) The extrinsic origin of SP-IR axons in the stomach derived mainly from the spinal T7-T11 dorsal root ganglia with a lesser contribution from the vagal nodose ganglia, but not from the celiac ganglia, the dorsal motor nucleus of vagus, or the nucleus tractus solitarius. Collectively, our data provide for the first time a map of the topographical distribution and morphology of SP-IR axons and terminals in the whole stomach with single-cell/axon/synapse resolution. This work will establish an anatomical foundation for functional mapping of the SP-IR nociceptive afferent innervation of the stomach and its pathological remodeling in gastrointestinal diseases.

Tumor Necrosis Factor-Like Ligand 1a (TL1A) is a cytokine encoded by Tumor Necrosis Factor Super Family 15 gene (TNFSF15) gene mostly in endothelial cells which binds to T-cells and foments the production of pro-inflammatory cytokines including TNF-α, IL-6, IL-1b, IFN- γ and IL-13. TL1A level is elevated in inflammatory diseases including Crohn's Disease (CD). Although Single Nucleotide Polymorphisms (SNPs) in TNFSF15 have been reported in CD, no studies have investigated the effect of these SNPs on TL1A, inflammation, and susceptibility to Mycobacterium avium subspecies paratuberculosis (MAP) infection. MAP is a strong candidate in CD pathogenesis. This study is designed to elucidate the combined effect of MAP and SNPs in TNFSF15 (rs4263839, rs7848647, rs6478108, or rs6478109) on TL1A secretion and downstream effect on pro-inflammatory cytokines. Peripheral blood from CD and healthy subjects was analyzed for MAP DNA, TNFSF15 genotyping, circulating TL1A level, and IFN- γ and TNF-α gene expression. Our data is first to report that rs4263839, rs7848647, rs6478108, and rs6478109 in TNFSF15 resulted in increase in circulating TL1A level in healthy and CD samples. Specifically, in CD samples with rs7848647, the average TL1A level was 146.9 pg/mL ± 124.5 compared 62.4 pg/mL ± 82.8 in normal samples. Similarly, TL1A level in CD samples with rs6478109 was 141.9 pg/mL ± 127.7 compared to 71.5 pg/mL ± 88.4 in normal samples (p < 0.05). All 4 SNPs resulted in significant elevation in TL1A level in healthy samples (p < 0.05). Moreover, IFN-γ expression was significantly higher, by approximately 1.6-fold in CD patients with SNPs relative to CD patients with no SNPs (p < 0.05). Interestingly, SNPs in TNFS15 had no significant effect on TNF-α expression. MAP was detected in the blood of 63% of CD compared to 6% healthy subjects (p < .001). The data did not support a correlation between MAP presence and circulating TL1A levels, and no correlation between SNPs in TNSF15 and MAP susceptibility. This study strongly suggests, that SNPs in TNFSF15 increase TL1A levels and may be a contributory factor to the inflammation experienced by CD patients. Over all, the study emphasizes the need for a pharmacogenomic approach in treatment delivery for patients with CD by using TNFSF15 SNPs to identify patients that would benefit from biologics targeting TL1A rather than TNF-α for more efficacious treatment regiments for CD patients.

Vibrio vulnificus, a natural inhabitant of brackish and estuarine environments, is a fatal opportunistic human pathogen with a mortality rate of over 50%. Unlike for other pathogenic Vibrio species, and despite its high mortality rate, there is no conclusive approach to indicate the pathogenic potential of V. vulnificus isolates from the environment. To this day, a single gene encoding a hemolysin, vvh, has been used to detect V. vulnificus, and one phenotype - indole production, has been used to assess the pathogenic potential and classification of a given strain. In this study, we use genomic-based approaches to identify distinct phenotypes that can characterize V. vulnificus strains with highest pathogenic potential (cluster 1 – C1), and to determine novel genes that can be used to accurately detect and identify V. vulnificus strains from natural reservoirs. Our phenotypic analyses indicate that strains from C1 utilize a more diverse range of carbon sources compared to strains less likely to emerge as pathogenic (cluster 2 – C2). We found that C1 and C2 prefer to inhabit different niches leading to behavioral separation. Physiological adaptations like motility in the presence of mucin and growth at different salinities indicate markedly different lifestyles for these clusters. These phenotypes can thus be used as markers to predict the pathogenic potential of unknown V. vulnificus isolates from the environment based on their clusters. Furthermore, we identified six candidate genes that can distinctly discriminate between the two clusters and are more sensitive in detecting V. vulnificus compared to existing typing techniques. The high degree of resolution offered by this simple, reproducible approach can thus be used to identify V. vulnificus strains from natural reservoirs, as validated in our study using environmental isolates from oysters and water.

<p>Patients presenting to the emergency department (ED) with chest pain suggestive of acute coronary syndrome (ACS) often wait long hours before a decision on their care is made. The recommended blood test to aid in diagnosing myocardial infarction (MI) is cardiac troponin I (cTnI) or cardiac troponin T (cTnT). However, other biomarkers representing acute processes and diseases related to ACS might also be useful for early identification. To that end, I evaluated whether a biomarker panel at presentation could improve the diagnostic performance of identifying patients at high risk for MI or any other related cardiac outcome as compared to using cardiac troponin alone. The patient population consisted of 102 patients who presented to the ED with chest pain. Sixteen biomarkers measured in serum obtained at presentation were ranked via receiver-operating-characteristic (ROC) curve analysis for a composite cardiac outcome within the first 72 hours following presentation to the ED. The top four biomarkers (soluble fms-like tyrosine kinase, Creatinine, monocyte chemoattraction protein-1, and NT-pro brain natriuretic peptide) were used to construct the panel test. The ROC derived cutoffs for each of the biomarkers were used to characterize abnormal concentrations with an overall biomarker score incorporating all 4 biomarkers used to classify patients that were either positive or negative for the biomarker panel. When used in conjunction with high-sensitivity cardiac troponin, the panel’s sensitivity and specificity were 100% (95%CI: 75-100%) and 54% (95%CI: 43-65%), respectively. This represented an improvement compared to high-sensitivity cardiac troponin I (hs-cTnI) or hs-cTnT alone which had a sensitivity/specificity of 92% (95%CI: 64-100%)/57% (95%CI: 46-68%) and 85% (95%CI: 55-98%)/55% (95%CI: 44-66%), respectively. In summary, a 4-biomarker blood-based panel used in conjunction with cardiac troponin at ED presentation may identify patients at risk for MI or related outcomes in the short term.</p> / Master of Science (MSc)

<p><em> Chlamydia pneumoniae</em> is a Gram-negative, intracellular bacterium which utilizes a type III secretion system for virulence. This system injects virulence associated proteins into a host cell and ultimately hijacks host intracellular machinery required for the bacteria to propagate and reproduce. Chlamydia outer protein N or CopN (<em>Cpn0324</em>), is a member of a family of proteins found in pathogenic bacteria, which inhibits premature secretion of effector proteins by plugging the base of the injectisome. The lack of a genetic system to manipulate the bacteria hampers the identification of proteins within the T3S field. The work presented in this thesis establishes the role of CopN as the plug protein of <em>Chlamydia pneumoniae</em>, and examines protein interactions within the individual CopN domains.</p> <p>The structure of CopN was first explored by limited proteolysis to establish the domain boundaries. We found three domains, an N-terminal, central domain, and C-terminal domain. Next, we used the full length protein as well as a series of truncations to examine the interactions within each domain. Using a subset of the known protein interactions between CopN and other T3S proteins, we found that the proposed chaperones Scc1 and Scc4 bind in the N-terminal region. There were no apparent interactions in the central domain, whereas FliI, FliF, CopD1_158-206 and Scc3 all bound within the C-terminal region of CopN. Finally the secretion of CopN in a HeLa cell model was addressed throughout the course of an infection. CopN was detected in the host cell immediately after infection, and then was not detectable again until late infection. Overall, I have characterized the individual domains of CopN and present data to support the role of CopN as the plug protein in the T3SS of <em>Chlamydia pneumoniae</em>.</p> / Master of Science (MSc)

<p>In spite of the tremendous body of cancer research, cancer remains a significant health issue requiring development of better therapeutics. The elucidation of the relationship between cancer and the immune system and the identification of tumour associated antigens together suggest that novel therapeutics using the immune system to target cancer is a promising avenue of research. Since immunological tolerance is a barrier to generating immune responses to self antigen, strategies to circumvent tolerance need to be investigated to target given antigens.</p> <p>Plac1 is a novel tumour associated antigen with expression restricted to placenta, testis and many tumour cells. Initial reports concerning the expression, immunogenicity and potential tumourigenic function of Plac1 suggest that it would be an ideal tumour antigen. Initial experiments in mice indicated that generating an immune response against the murine Plac1 would be difficult and the subsequent work sought to employ strategies to facilitate anti murine Plac1 immune responses and anti tumour efficacy in Plac1 expressing tumours.</p> <p>Another more studied tumour associated antigen is gp100. Unlike Plac1, immune responses against the murine gp100 can be generated through vaccination. These responses are unable to demonstrate any anti tumour activity in gp100 expressing cells. The bulk of the gp100 studies described here sought to modify the immune:tumour interaction such that the anti tumour activity of the anti gp100 responses could be improved.</p> <p>While the specific barriers to Plac1 vaccination and efficacy and gp100 vaccination and efficacy are different, they have in common that they represent likely issues in using therapeutic cancer vaccines clinically. In both cases investigating how these barriers can be overcome is important and relevant to the understanding of these barriers to success when they appear in the clinic.</p>

<p>ER stress in the kidney is associated with proteinuria. Clinical studies have linked proteinuria with the progression of chronic kidney disease (CKD) at all stages of GFR decline. We hypothesized that treatment with a chemical chaperone, 4-phenylbutyrate (4-PBA), would reduce the severity of CKD and proteinuria in salt sensitive hypertension. The differences in renal pathology between salt sensitive and insensitive hypertension when animals were fed an 8% NaCl (HS) diet were assessed. The Dahl salt sensitive (Dahl S) rat was used as a model of salt sensitive hypertension, while the spontaneously hypertensive rat (SHR) was used as a model of salt insensitive hypertension. The myogenic response of the arcuate artery was studied to determine whether the differences in renal pathology between these models of hypertension was due to an effect of salt on myogenic constriction. Myogenic constriction displayed salt sensitivity in the Dahl S as there was a significant reduction in blood vessel constriction with increasing intralumenal pressures. Myogenic constriction was reduced, but not completely abolished in the SHR with high salt (HS), providing a possible explanation of why this model of hypertension does not develop an equivalent level of renal damage with blood pressure increase as the Dahl S rat. ER stress induction with tunicamycin in arcuate arteries from normotensive animals resulted in an attenuation of the myogenic response. Myogenic constriction was protected from tunicamycin induced ER stress with 4-PBA. 4-PBA treatment (1g/kg/day) in HS fed Dahl S ameliorated proteinuria, renal intratubular protein casts, and renal fibrosis. This correlated with a protection of myogenic constriction and integrity of the glomerular filtration barrier. This suggests that myogenic constriction of the renal vasculature is an important mechanism to protect against salt sensitive hypertension-induced proteinuria. Further, that high salt feeding may inhibit this protective mechanism by inducing ER stress in the renal blood vessels.</p> / Master of Science (MSc)

<p>Successful pregnancy involves the accumulation of non-T, FcR-positive cells at the implantation sites of syngenic allogeneic of CTL (cytotoxic T cells) in vitro and in vivo by blocking the response to interleukin 2. It has been shown that xenogeneic embryos can be gestated successfully if enveloped in the trophoblast genotypically compatible with the pseudopregnant recipient (Rossant et al., 1982, J. Emb. Exp. Morph., 69: 141). We have recently demonstarted that the trophoblast plays a critical role in the localization of the deciduaassociated suppressor cell in pseudopregnant mice. We now show that supernatants genereated from trophoblast cell cultures and day 9.5 ectoplacental cone cultures were successful in the recruitment of the non-T, granulated suppressor cell which sediments at 3 ± 0.05 mm/h. These results suggest that the trophoblast elaborates a factor(s) which plays an important role in the accumulation of the deciduaassociated suppressor cells in the decidue which may protect the anti-genic fetus from maternal immue rejection.</p> / Doctor of Philosophy (PhD)

<p>In chickens afflicted with hereditary muscular dystrophy, the two major types of muscle present respond dissimilarly to the disease process: fast twitch glycolytic muscles possess and express the dystrophic gene overtly during ex ovo development while the genotypically dystrophic slow tonic muscles fail to express dystrophic phenotypes. Therefore, in chickens, muscular dystrophy is muscle fibre type specific.</p> <p>The primary goal of this thesis was to experimentally alter the genetically dystrophic slow tonic musle in an attempt to induce this muscle to express dystrophic phenotypes. Since motor nerves influence the phenotypes of skeletal muscles, it was decided to replace the motor innervation of a slow tonic muscle with that of a fast twitch muscle within a-dystrophic chicken. The surgical cross-reinnervation between the ALD muscle and the 'fast' nerve was performed at hatching by transposing the right ALD muscle to the left side of the back in order to prevent selective self-reinnervation by the severed ALD nerve. The experimental muscles were examined at various time intervals from 2 to 104 weeks postoperatively and compared to age-matched ALD muscles in genetically normal chickens. In addition, denervation of ALD muscles, with and without transposition served as control experiments.</p> <p>Selected histochemical and structural properties of unoperated ALD and fast twitch posterior latissimus dorsi (PLD) muscles of normal and dystrophic chickens were compared between 2 and 32 weeks ex ovo to provide criteria for the analysis of muscles cross-reinnervated by 'fast' nerves and to determine which of these parameters were altered as a result of the dystrophic process. Normal and dystrophic ALD muscles exhibited similar phenotypes: acid and alkaline stable myosin ATPase activity, 'en grappe' endplates, weak glycolytic and strong oxidative capacities, and peripheral location of nuclei. Furthermore, the growth rate, size, and shape of fibres in the normal and dystrophic ALD muscles were similar. In contrast, the myosin ATPase and innervation patterns of normal and dystrophic PLD muscles differed from those of ALD muscles: PLD fibres of either genotype exhibited alkaline stable, acid labile myosin ATPase activity and focal 'en plaque' innervation. Normal and dystrophic PLD muscles also exhibited different phenotypes: dystrophic muscles had a lower muscle weight, abnormal size, shape, and growth rate of fibres, increased number of scattered nuclei, add abnormal glycolytic and oxidative capacities.</p> <p>The results presented from this work indicate that the genetically dystrophic ALD muscles respond differently to cross-reinnervation than do normal ALD muscles. The cross-reinnervated muscles in normal birds demonstrated all of the characteristics of an unoperated ALD muscle with the exception of the presence of isolated groups of fibres exhibiting a fast twitch type of myosin ATPase response. This result suggests that the principle response of normal ALD muscles to a foreign 'fast" nerve is one of resistance to alteration. In contrast, data from the experimental ALD muscles of dystrophic genotype support the conclusion that these muscles are dramatically remodelled after cross-reinnervation. An augmented regenerative response within the dystrophic muscles resulted in hyperplasia which, in turn, led to a complete restructuring of these muscles. Therefore, the present cross-reinnervation experiments demonstrate, for the first time, that a phenotypically normal muscle of dystrophic genotype can be induced to express a dystrophic characteristic: an augmented regenerative response after an experimentally-induced injury. It is important to note that the initial induction of this dystrophic phenotype was demonstrated in the absence of the foreign 'fast' nerve and seemed to be due to the response of this muscle to the severe injury imposed upon the muscle during the inital operation.</p> / Doctor of Philosophy (PhD)