Dissertations / Theses on the topic 'Schistosoma japonicum'

Les recherches portent sur le developpement larvaire intramolluscal, la variabilite genetique et certaines relations de parente de schistosoma japonicum, agent de la bilharziose arterioveineuse humaine en chine continentale. Ces recherches demontrent que s. Japonicum presente, par rapport aux autres schistosomes de l'homme, de nombreux caracteres originaux concernant le lieu de penetration des miracidiums, la localisation des sporocystes primaires, la morphologie des sporocystes primaires et secondaires, la dynamique des populations larvaires, la strategie de colonisation de l'espace-mollusque, la chronobiologie des emissions cercariennes, la sensibilite des stades larvaires intramolluscaux au praziquantel. Le processus de replication des sporocystes secondaires est decrit pour la premiere fois dans le cycle de s. Japonicum; il permet un clonage indefini du parasite par transplantations micro-chirurgicales. Sur la base d'une comparaison entre s. Japonicum originaire des provinces de anhui et sichuan, il est montre qu'il existe un polymorphisme de compatibilite. Le rejet des parasites allopatriques par les mollusques peut se produire a des stades tardifs de la parasitose et provoquer des phenomenes de self-cure. Le polymorphisme de compatibilite est susceptible de renforcer les effets de l'isolement geographique, donc la divergence genetique entre populations; un aspect inattendu de cette divergence concerne les modalites de nage des miracidiums. Des essais de croisement entre s. Japonicum et s. Mansoni montrent que, malgre l'eloignement phyletique des especes, l'interaction entre males et femelles reste possible dans plusieurs processus de la sexualite, parmi lesquels la formation des couples; ces essais de croisement induisent une reproduction parthenogenetique

Schistosomiasis, a disease of humans caused by helminth parasites of the genus Schistosoma, kills 200 to 500 thousand people annually, endangering over 600 million people world-wide with 200 million people infected in 2003 [1, 2]. Three species of schistosome are primarily responsible for human infections, namely, Schistosoma haematobium, endemic to Africa, India, and the Middle East, S. mansoni, endemic to Africa / South America, and S. japonicum endemic to China and the Philippines [3]. The major pathological effects of schistosomiasis result from the deposition of parasite ova in human tissues and the subsequent intense granulomatous response induced by these eggs. There is a high priority to provide an effective sub-unit vaccine against these schistosome flukes, using proteins encoded by cDNAs expressed by the parasites at critical phases of their development. One technique that may expedite this gene identification is the use of microarrays for expression analysis. A 22,575 feature custom oligonucleotide DNA microarray designed from public domain databases of schistosome ESTs (Expressed Sequence Tags) was used to explore differential gene expression between the Philippine (SJP) and Chinese (SJC) strains of S. japonicum, and between males and females. It was found that 593, 664 and 426 probes were differentially expressed between the two geographical strains when mix sexed adults, male worms and female worms were compared respectively. Additionally, the study revealed that 1,163 male- and 1,016 female-associated probes were differentially expressed in SJP whereas 1,047 male- and 897 female-associated probes were differentially expressed in SJC [4]. Further to this, a detailed real time PCR expression study was used to explore the differential expression of eight genes of interest throughout the SJC life cycle, which showed that several of the genes were down-regulated in different life cycle stages. The study has greatly expanded previously published data of strain and gender-associated differential expression in S. japonicum. Further, the new data will provide a stepping stone for understanding the complexities of the biology, sexual differentiation, maturation, and development of human schistosomes, signaling new approaches for identifying novel intervention and diagnostic targets against schistosomiasis [4].

Schistosoma japonicum, a multi-host parasite, remains highly endemic in China and has recently re-emerged in previously controlled regions. One reason may be, given the current human- and bovine-based control policy, due to a serious lack of knowledge of the potential role for other species of mammals, including dogs, cats and small rodents, in the transmission. This thesis aims to contribute to our understanding of transmission dynamics of S. japonicum by investigating the implications of different reservoirs in the transmission across two contrasting geographical regions/settings: the marshland with the disease persistence versus the hilly region where the disease was once controlled. Longitudinal characterization of S. japonicum infection at both definitive host and intermediate host levels was performed throughout 2006-2007, with the highest prevalence and infection intensity observed in rodents in the hilly region and in the agriculturally important domestic animals (cattle and goats) in the marshland. Three chronobiological trials of cercarial emergence were performed to identify any host (with nocturnal vs diurnal activity)-associated biological traits of the parasite. A late afternoon shedding pattern was observed in the hilly region, compatible with a nocturnal rodent reservoir, and a morning-afternoon dual shedding pattern within marshland areas, consistent with a diurnal bovine major reservoir. Characterization of the parasite population genetic diversity, using microsatellite markers, at both larval stages, also indicated cattle to be the main definitive host reservoir species in the marshland, which was further confirmed by sibling relationship analyses. In the hilly regions, however, epidemiological, biological and molecular data indicated that, in addition to the role of rodents as the main reservoirs to maintain the disease, dogs, with their higher mobility, may also play a significant role in S. japonicum transmission in these areas. The implications of these results, in terms of parasite strain sub-structuring and targeted disease control, were discussed.

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Schistososmiasis is a chronic endemic infection caused by parasites of the genus Schistosoma, and it occurs in 74 countries in Africa, South America and Asia. The three main agents of this infection in humans are: Schistosoma mansoni and Schistosoma japonicum, that cause the hepatic-intestinal disease, and Schistosoma haematobium, responsible for the genitourinary infection. Despite the effective treatment like praziquantel, schistososmiasis remains as the second most prevalent parasitic disease in the world. Diagnosis of the intestinal schistososmiasis is achieved through the direct visualization of the eggs in fecal samples. The current method recommended by the World Health Organization in epidemiological studies is the Kato-Katz method. Despite it being simple and cheap, in areas of low endemicity this technique loose sensibility, leading to the occurrence of false-negative cases and underestimation of the prevalence in the studied area. Helmintex™ is a coproparasitological method highly sensitive that allows the isolation of Schistosoma eggs from 30 grams of feces, based in the interaction between the eggs and paramagnetic microspheres in a magnetic field. However, this method demands time and specialized equipment, being of difficult manipulation in work field. The mechanism that promotes the interaction between the paramagnetic spheres with the Schistosoma eggs is not known. Considering the necessity of sensitive diagnostic tools of easy applicability in epidemiological studies in low endemicity areas, this work has the purpose to study the surface physical-chemical characteristics of S. mansoni and S. japonicum eggs, in order to enhance the efficiency of the Helmintex™ method. S. mansoni and S. japonicum eggs were isolated from livers of experimentally infected mice. The eggs were submitted to morphological and structural analysis using Scanning and Transmission Electron Microscopy and elemental analysis using Energy Disperssion Spectroscopy. The magnetic susceptibility was determined using SQUID (Superconducting Quantum Interference Device) and the concentration of the chemical elements was determined through Atomic Emission Spectroscopy. Experiments to elucidate the interaction properties of the eggs of the eggs and the microspheres were conducted incubating the eggs from both species with different paramagnetic microspheres. The results show that the egg surface of both species is recovered by a dense layer of microspines, being those shorter and less spaced in S. mansoni. The eggs spontaneously bind the particles, with a greater preference for magnetic material. S. japonicum eggs have a higher affinity for paramagnetic microspheres than S. mansoni eggs. The presence of streptavidin in the surface of the microspheres enhances the affinity of both species for non-magnetic material, however it decreases the affinity for paramagnetic microspheres. Despite the presence of iron in the eggshell of S. mansoni and S. japonicum, the origin of the interaction does not seem to be magnetic, but, based in the difference of electrostatic charges present in the surface of the eggs and the microspheres. The continuity of this study is important to determine the physical-chemical characteristics of eggs from human feces, and it can lead to the upgrading and optimization of the Helmintex™ method. Studies using Atomic Force Microscopy are in progress.
A esquistossomose é uma infecção crônica endêmica causada por parasitos do gênero Schistosoma, e ocorre em países 74 países na África, América do Sul e Ásia. Os três principais agentes desta infecção em humanos são: Schistosoma mansoni e Schistosoma japonicum, causadores da doença hepato-intestinal, e Schistosoma haematobium, responsável pela infecção genitourinária. Apesar de haver tratamento efetivo como o praziquantel, a esquistossomose permanece como a segunda infecção parasitária mais prevalente no mundo. O diagnóstico da esquistossomose intestinal é feito através da direta visualização dos ovos em amostras fecais. O método atualmente recomendado pela Organização Mundial de Saúde em estudos epidemiológicos é o método de Kato-Katz. Apesar de simples e barato, em áreas de baixa endemicidade esta técnica perde sensibilidade, levando à ocorrência de casos falso-negativos e subestimação da prevalência da área estudada. O Helmintex® é um método coproparasitológico altamente sensível que permite o isolamento de ovos de Schistosoma à partir de 30 gramas de fezes, baseado na interação entre os ovos e esferas paramagnéticas em um campo magnético. Entretanto, este método demanda tempo e equipamentos especializados, sendo de difícil manipulação em estudos de campo.O mecanismo que promove a interação das esferas paramagnéticas com os ovos de Schistosoma não é conhecido. Tendo em vista a necessidade de ferramentas diagnósticas sensíveis e de fácil aplicabilidade em estudos epidemiológicos em áreas de baixa transmissão, este trabalho tem por objetivo estudar características físico-químicas da superfície dos ovos de S. mansoni e S. japonicum, afim de aprimorar a eficiência do método Helmintex®. Ovos de S. mansoni e S. japonicum foram isolados de fígados de camundongos experimentalmente infectados. Os ovos foram submetidos à analise morfológica e estrutural utilizando Microscopia Eletrônica de Varredura e Transmissão e análise elementar utilizando Espectroscopia por Dispersão de Energia. A susceptibilidade magnética foi determinada utilizando-se o SQUID (Superconducting Quantum Interference Device) e a concentração dos elementos químicos foi determinada através de Espectroscopia por Emissão Atômica. Experimentos para elucidar as propriedades de interação dos ovos e das microesferas foram conduzidos incubando ovos de ambas as espécies com diferentes microesferas paramagnéticas. Os resultados mostram que a superfície do ovo de ambas as espécies é recoberta por uma camada densa de microespinhos, sendo estes mais curtos e menos espaçados em S. mansoni. Os ovos espontaneamente ligam-se às partículas, com maior preferência por material magnético. Os ovos de S. japonicum possuem maior afinidade pelas microsesferas paramagnéticas do que os ovos de S. mansoni. A presença de estreptavidina na superfície das microesferas aumenta a afinidade de ambas as espécies por microesferas não-magnéticas, porém diminui a afinidade por microesferas paramagnéticas. Apesar da presença de ferro na casca do ovo tanto de S. mansoni quanto de S. japonicum, a origem da interação não parece ser magnética, e sim, baseada na diferença de cargas eletrostáticas presentes na superfície dos ovos e das microesferas. A continuidade deste estudo é importante para determinar as características físico-químicas de ovos provenientes de fezes humanas, e pode levar ao aprimoramento e otimização do método Helmintex®. Estudos utilizando-se Microscopia de Força Atômica encontram-se em andamento.

La bilharziose reste un problème de santé majeur. L'équipe du Pr Dessein a montré que les infections élevées étaient déterminées par un locus majeur en 5q31 et que des polymorphismes dans un gène à ce locus,IL13, aggravent l'infection. Notre premier objectif était d'évaluer si des variants d'autres gènes de la voie de l'IL13 intervenaient dans le contrôle de l'infection. Nous avons observé une association entre le SNP rs324013, dans le promoteur de STAT6,et les niveaux d'infection à S. haematobium. Ce polymorphisme a un effet additif avec le polymorphisme IL13rs1800925. Ce SN modifie la fixation de facteurs nucléaires au niveau du promoteur de STAT6. L'équipe du Pr Dessein avait également montré que les fibres hépatiques avancées et sévères étaient déterminées par un autre locus majeur localisé en 6q23. Notre deuxième objectif fut d'évaluer dans le laboratoire du Pr Dessein et en étroite collaboration avec le laboratoire du Pr Li(Yueyang Institute of Parasitic disease)deux gènes candidats(IFNGR1 et CTGF) situés dans cette région chromosomique. Nous avons observé une association entre les deux polyporphismes(rs17066192 er rs673156)localisés dans le promoteur du gène. Nous avons observé une association entre les deux polymorphismes(rs17066192 et rs673156)localisés dans le promoteur du gène IFNGR1 et la fibrose hépatique: le génotype rs673156A/A et rs17066192C/C sont associés à un risque 7.3 fois et 1.5 fois plus élevé, respectivement, de fibrose avancée. Nous avons également montré que les variants rs9402373 et rs12526196 du gène CTGF sont indépendamment associés à la fibrose chez les fermiers et pêcheurs chinois infectés par S.japonicum. Sur la population chinoise d'étude, les risques relatifs associés aux polymorphismes rs9402373 et rs12526196 sont de 2.8 et 3
Schistosomiasis remains one of the world’s most prevalent diseases. It comprises a group of chronic diseases caused by helminths of the Schistosoma genus. Schistosoma haematobium causes obstructive nephropathy that can be aggravated by urinary bacterial infections. S.japonicum and S.mansoni cause hepatic fibrosis associated with portal blood hypertension, which can be lethal. In previous studies, our laboratory had shown that worm burden in S.haematobium infections were aggravated by IL13 variants and that severe hepatic fibrosis (HF) was controlled by gene(s) located on 6q23. The present study is to further evaluate other IL-13 pathway genes (STAT6) in the control of infection in Malian farmers and to test candidate genes in the 6q23 region in hepatic fibrosis (HF) in S.japonicum infected Chinese fishermen and farmers. First we have developped an improved FTA® technology technique to perform SNP genotyping. This technique allows us to use saliva samples for genotyping SNPs. Subsequently, this improved FTA® technology was used in our study on HF.Our work on a Malian sample infected with S. haematobium indicated that a polymorphism (rs324013) in the promoter of STAT6 gene was associated with the control of S. haematobium infection levels and has an additive effect with IL13rs1800925, a polymorphism previously associated with infection in this same population. Both SNPs modify the binding of nuclear factors to the promoter regions of their respective genes. Thus, both SNPs may play a crucial role in controlling S. haematobium infection levels. In order to study HF in S.japonicum infections, we have participated actively in the study that recruited of a large sample of Chinese fishermen and farmers who had been exposed to the infection for most of their life. HF was evaluated by ultrasound and covariates that could affect HF were evaluated by interviews. Then, we tested two genes (IFNGR1, CTGF) of the 6q23 region that were good candidates for the control of HF on these samples. Both genes encode molecules that were shown in animal and human studies to have strong effect on extracellular matrix proteins deposition and turnover. We found that two polymorphisms (rs17066192 and rs673156) in IFNGR1 promoter were associated with HF: the rs673156A/A genotype was associated with a 7.3-fold increased risk of advanced HF; and rs17066192C/C genotype with a 1.5-fold increased risk of HF. These results must now be confirmed in another population sample. We also found that variants of CTGF rs9402373 and rs12526196 were independently associated with HF in Chinese fishermen and farmers, in Sudanese, and in Brazilians infected with either S. japonicum or S. mansoni. Our results provide additional evidence for a protective role of IL-13 in schistosome infections, and they also demonstrate that TGFβ / CTGF pathway plays a key role in HF and should be targeted by chemotherapy. Ongoing studies evaluate whether CTGF variants could be used in the prognosis of the HF caused by schistosomes and also by other infectious agents

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Schistososmiasis is a chronic endemic infection caused by parasites of the genus Schistosoma, and it occurs in 74 countries in Africa, South America and Asia. The three main agents of this infection in humans are: Schistosoma mansoni and Schistosoma japonicum, that cause the hepatic-intestinal disease, and Schistosoma haematobium, responsible for the genitourinary infection. Despite the effective treatment like praziquantel, schistososmiasis remains as the second most prevalent parasitic disease in the world. Diagnosis of the intestinal schistososmiasis is achieved through the direct visualization of the eggs in fecal samples. The current method recommended by the World Health Organization in epidemiological studies is the Kato-Katz method. Despite it being simple and cheap, in areas of low endemicity this technique loose sensibility, leading to the occurrence of false-negative cases and underestimation of the prevalence in the studied area. Helmintex? is a coproparasitological method highly sensitive that allows the isolation of Schistosoma eggs from 30 grams of feces, based in the interaction between the eggs and paramagnetic microspheres in a magnetic field. However, this method demands time and specialized equipment, being of difficult manipulation in work field. The mechanism that promotes the interaction between the paramagnetic spheres with the Schistosoma eggs is not known. Considering the necessity of sensitive diagnostic tools of easy applicability in epidemiological studies in low endemicity areas, this work has the purpose to study the surface physical-chemical characteristics of S. mansoni and S. japonicum eggs, in order to enhance the efficiency of the Helmintex? method. S. mansoni and S. japonicum eggs were isolated from livers of experimentally infected mice. The eggs were submitted to morphological and structural analysis using Scanning and Transmission Electron Microscopy and elemental analysis using Energy Disperssion Spectroscopy. The magnetic susceptibility was determined using SQUID (Superconducting Quantum Interference Device) and the concentration of the chemical elements was determined through Atomic Emission Spectroscopy. Experiments to elucidate the interaction properties of the eggs of the eggs and the microspheres were conducted incubating the eggs from both species with different paramagnetic microspheres. The results show that the egg surface of both species is recovered by a dense layer of microspines, being those shorter and less spaced in S. mansoni. The eggs spontaneously bind the particles, with a greater preference for magnetic material. S. japonicum eggs have a higher affinity for paramagnetic microspheres than S. mansoni eggs. The presence of streptavidin in the surface of the microspheres enhances the affinity of both species for non-magnetic material, however it decreases the affinity for paramagnetic microspheres. Despite the presence of iron in the eggshell of S. mansoni and S. japonicum, the origin of the interaction does not seem to be magnetic, but, based in the difference of electrostatic charges present in the surface of the eggs and the microspheres. The continuity of this study is important to determine the physical-chemical characteristics of eggs from human feces, and it can lead to the upgrading and optimization of the Helmintex? method. Studies using Atomic Force Microscopy are in progress.
A esquistossomose ? uma infec??o cr?nica end?mica causada por parasitos do g?nero Schistosoma, e ocorre em pa?ses 74 pa?ses na ?frica, Am?rica do Sul e ?sia. Os tr?s principais agentes desta infec??o em humanos s?o: Schistosoma mansoni e Schistosoma japonicum, causadores da doen?a hepato-intestinal, e Schistosoma haematobium, respons?vel pela infec??o genitourin?ria. Apesar de haver tratamento efetivo como o praziquantel, a esquistossomose permanece como a segunda infec??o parasit?ria mais prevalente no mundo. O diagn?stico da esquistossomose intestinal ? feito atrav?s da direta visualiza??o dos ovos em amostras fecais. O m?todo atualmente recomendado pela Organiza??o Mundial de Sa?de em estudos epidemiol?gicos ? o m?todo de Kato-Katz. Apesar de simples e barato, em ?reas de baixa endemicidade esta t?cnica perde sensibilidade, levando ? ocorr?ncia de casos falso-negativos e subestima??o da preval?ncia da ?rea estudada. O Helmintex? ? um m?todo coproparasitol?gico altamente sens?vel que permite o isolamento de ovos de Schistosoma ? partir de 30 gramas de fezes, baseado na intera??o entre os ovos e esferas paramagn?ticas em um campo magn?tico. Entretanto, este m?todo demanda tempo e equipamentos especializados, sendo de dif?cil manipula??o em estudos de campo. O mecanismo que promove a intera??o das esferas paramagn?ticas com os ovos de Schistosoma n?o ? conhecido. Tendo em vista a necessidade de ferramentas diagn?sticas sens?veis e de f?cil aplicabilidade em estudos epidemiol?gicos em ?reas de baixa transmiss?o, este trabalho tem por objetivo estudar caracter?sticas f?sico-qu?micas da superf?cie dos ovos de S. mansoni e S. japonicum, afim de aprimorar a efici?ncia do m?todo Helmintex?. Ovos de S. mansoni e S. japonicum foram isolados de f?gados de camundongos experimentalmente infectados. Os ovos foram submetidos ? analise morfol?gica e estrutural utilizando Microscopia Eletr?nica de Varredura e Transmiss?o e an?lise elementar utilizando Espectroscopia por Dispers?o de Energia. A susceptibilidade magn?tica foi determinada utilizando-se o SQUID (Superconducting Quantum Interference Device) e a concentra??o dos elementos qu?micos foi determinada atrav?s de Espectroscopia por Emiss?o At?mica. Experimentos para elucidar as propriedades de intera??o dos ovos e das microesferas foram conduzidos incubando ovos de ambas as esp?cies com diferentes microesferas paramagn?ticas. Os resultados mostram que a superf?cie do ovo de ambas as esp?cies ? recoberta por uma camada densa de microespinhos, sendo estes mais curtos e menos espa?ados em S. mansoni. Os ovos espontaneamente ligam-se ?s part?culas, com maior prefer?ncia por material magn?tico. Os ovos de S. japonicum possuem maior afinidade pelas microsesferas paramagn?ticas do que os ovos de S. mansoni. A presen?a de estreptavidina na superf?cie das microesferas aumenta a afinidade de ambas as esp?cies por microesferas n?o-magn?ticas, por?m diminui a afinidade por microesferas paramagn?ticas. Apesar da presen?a de ferro na casca do ovo tanto de S. mansoni quanto de S. japonicum, a origem da intera??o n?o parece ser magn?tica, e sim, baseada na diferen?a de cargas eletrost?ticas presentes na superf?cie dos ovos e das microesferas. A continuidade deste estudo ? importante para determinar as caracter?sticas f?sico-qu?micas de ovos provenientes de fezes humanas, e pode levar ao aprimoramento e otimiza??o do m?todo Helmintex?. Estudos utilizando-se Microscopia de For?a At?mica encontram-se em andamento.

Les infections humaines par Schistosoma japonicum et Schistosoma mansoni provoquent des pathologies hépatospléniques conduisant à la fibrose hépatique sévère chez 5 à 30% des sujets infectés vivant en zone endémique. Plusieurs études ont montré que le développement de cette fibrose était régulé par des cytokines mais aussi par des chimiokines. Les chimiokines sont des cytokines chimioattractantes produites par une variété des cellules immunes et non immunes, et qui ont été impliqués dans la régulation de l'inflammation granulomateuse et la fibrose pulmonaire et hépatique chez la souris et chez l'homme. Nous avons donc étudié la modulation des chimiokines et récepteurs dans le foie et la rate des patients hépatospléniques exposés à l'infection par S.japonicum. Notre étude démontre que les transcrits de chimiokines de type CXC et CC ainsi que des récepteurs sont augmentés dans le foie des patients hépatospléniques, lesquels ne sont pas significativement augmentés dans la rate au cours de l'infection. Cette augmentation des transcrits de chimiokines n'est pas restreinte aux chimiokines inflammatoires, une augmentation des transcrits des chimiokines homéostatiques CCL19 et CCL21 est aussi observée dans le foie des patients hépatospléniques. Nous avons également observé une corrélation des niveaux d'expression des ligands CXCR3 entre eux, dans le foie des sujets hépatospléniques. Ces observations suggèrent que les chimiokines régulent l'inflammation hépatique induite par les œufs de schistosomes et jouent probablement un rôle dans la fibrose hépatique
Human infections with Schistosoma japonicum and Schistosoma mansoni causes hepatosplenic diseases leading to severe hepatic fibrosis in 5 to 30% of infected subjects living in endemic areas. Several studies demonstrated that the development of this fibrosis was regulated by cytokines but also by chemokines. Chemokines are the chemoattractant cytokines produced by a variety of immune and non-immune cells, and have been involved in the regulation of inflammation and granulomatous pulmonary and hepatic fibrosis in mice and humans. We therefore studied the modulation of chemokines and receptors in the liver and spleen of hepatosplenic patients exposed to infection with S.Japonicum. Our study demonstrates that the transcripts of CXC and CC chemokines and their receptors are increased in the liver of hepatosplenic patients, which were not significantly increased in the spleen during infection. This increase of transcripts of chemokines is not restricted to inflammatory chemokines, an increase of transcripts of homeostatic chemokines CCL19 and CCL21 is also observed in the liver of hepatosplenic patients. Moreover, the proportion of CD3+ lymphocytes but not CD14+ monocytes/macrophages is increased in the liver. We also observed a correlation of expression levels of CXCR3 ligands between them, in the liver of hepatosplenic subjects. These observations suggest that chemokines regulate hepatic inflammation induced by schistosoma eggs and probably play a role in liver fibrosis ensuing

The goal of this study was to develop a passive, low-cost, low-technological, rapid surveillance method for detecting schistosome cercariae in schistosomiasis endemic regions, compared to other available methods. Schistosomiasis is a parasitic disease that is linked to abdominal pain, enlarged liver, and multiple organ failure, yet it is highly preventable as individuals are infected through dermal contact with contaminated waters. In areas where transmission mitigation efforts have been successful, a combination of control initiatives were employed, such as mass drug treatment, hygiene improvement, and monitoring of transmission foci. The environmental detection methods currently in use are labor- and time- intensive or cost-prohibitive in rural developing areas with high transmission rates and very limited resources. Three aims were formulated in order to develop and evaluate the device. The first aim was to examine the chemotactic response of Schistosoma mansoni and S. japonicum cercariae to media containing oleic acid (OA) and explore how cercarial age plays a role in that response. The second aim was to apply the findings from the first aim to the development of an environmental surveillance device for schistosome cercariae (ESDSC) and to laboratory optimize the device to maximize cercarial capture. After examining the performance of the device, the third aim was to compare the ESDSC to other environmental surveillance methods that are currently available for the detection of schistosome cercariae through a review of the literature. While both S. mansoni and S. japonicum cercariae exhibited highest attachment to media when freshly shed, they preferentially attached to different media types: S. mansoni to beeswax with 0.3 g/mL OA and S. japonicum to plain beeswax. An ESDSC was designed from inexpensive, easily sourced materials (acrylic and aluminum sheets) and a S. mansoni-specific ESDSC was laboratory tested by utilizing beeswax with 0.3 g/mL of OA to attract cercariae. The cercarial capture capability of the ESDSC was tested in different orientations, depths, distances (from origin of cercariae), and with or without the application of a heat source. The highest median cercarial capture rate of 3.3% was observed with the device in a horizontal orientation, submerged, and within 25 cm of the cercarial point of origin. There were no instances in which the ESDSC failed to capture any cercariae, even at a distance of 70 cm. Through a review of the literature, the ESDSC was compared to other environmental surveillance methods based on five attributes: time and labor, technical training, cost, infrastructure and capital investment, and sensitivity. Compared to the other surveillance methods, the ESDSC requires minimal time and labor, technical training, cost and resources; however, it exhibits only a moderate level of sensitivity. The value of the device will have greatest realization in endemic regions where health and economic resources are limited. What may work in one endemic region may not work best in another when factors such as availability of infrastructure, resources, and skilled technicians are considered. The ESDSC has the potential to function as a preliminary screening to detect contaminated sites so that complex and costly surveillance methods can be targeted more efficiently.

Law Pui-ki.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (leaves 98-105).
Abstracts in English and Chinese.
Statement --- p.i
Acknowledgments --- p.ii
Abstract --- p.iii
Table of contents --- p.viii
List of figures --- p.xv
List of tables --- p.xvii
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Schistosomiasis --- p.1
Chapter 1.1.1 --- Disease burden --- p.1
Chapter 1.1.2 --- Causative agents --- p.1
Chapter 1.1.3 --- Transmission --- p.2
Chapter 1.1.4 --- Pathology of the disease --- p.3
Chapter 1.1.5 --- Control and therapy --- p.3
Chapter 1.2 --- Schistosomiasis in China --- p.5
Chapter 1.3 --- Schistosoma japonicum --- p.6
Chapter 1.3.1 --- Life cycle of S. japonicum --- p.6
Chapter 1.3.2 --- Biology of S. japonicum cercaria --- p.9
Chapter 1.3.3 --- Transformation of cercaria to schistosomulum --- p.10
Chapter 1.3.4 --- Cercarial stage-specific antigens --- p.12
Chapter 1.4 --- Aim of study --- p.14
Chapter Chapter 2 --- Materials and methodology --- p.15
Chapter 2.1 --- Materials --- p.15
Chapter 2.1.1 --- Schistosoma japonicum cercaria cDNA library --- p.15
Chapter 2.1.2 --- Cercarial stage-specific clones --- p.15
Chapter 2.1.3 --- Adult worm and cercaria RNA --- p.18
Chapter 2.1.4 --- "Snail intermediate host, Oncomelania hupensis" --- p.18
Chapter 2.1.5 --- Bacterial strains --- p.18
Chapter 2.1.6 --- Chemicals --- p.19
Chapter 2.1.7 --- Kits and reagents --- p.21
Chapter 2.1.8 --- Nucleic acids --- p.22
Chapter 2.1.9 --- Solutions --- p.22
Chapter 2.1.10 --- Enzymes --- p.24
Chapter 2.1.11 --- Primers --- p.25
Chapter 2.1.12 --- Antibodies --- p.26
Chapter 2.2 --- Methodology --- p.27
Chapter 2.2.1 --- Identification of cercarial stage-specific genes --- p.27
Chapter 2.2.1.1 --- Sequence analysis of cercarial stage-specific clones --- p.27
Chapter 2.2.1.2 --- "Confirmation of stage-specific expression of the selected gene, 20H8 and sjCa8, by RNA dot blot" --- p.29
Chapter 2.2.1.2.1 --- Cloning of S. japonicum glyceraldehyde-3-phosphate dehydrogenase (sjGAPDH) (GenBank accession no. U75571) --- p.29
Chapter 2.2.1.2.1.1 --- Reverse transcription and PCR amplification of sjGAPDH --- p.29
Chapter 2.2.1.2.1.2 --- Cloning of sjGADPH in pBluescript II KS (-) --- p.29
Chapter 2.2.1.2.1.3 --- Sequence verification of cloned sjGAPDH --- p.30
Chapter 2.2.1.2.2 --- Synthesis of Digoxigenin (DIG)-labeled probe --- p.31
Chapter 2.2.1.2.2.1 --- Synthesis of DIG-labeled probe by PCR --- p.31
Chapter 2.2.1.2.2.2 --- Estimation of concentration of DIG-labeled probe --- p.32
Chapter 2.2.1.2.3 --- RNA dot blot --- p.33
Chapter 2.2.1.2.3.1 --- Transferring RNA to the membrane using the BIO-RAD blotting manifold --- p.33
Chapter 2.2.1.2.3.2 --- Hybridization of DIG-labeled probe to detect sjGAPDH --- p.33
Chapter 2.2.1.2.3.3 --- Detection of the chemiluminescent signal --- p.33
Chapter 2.2.1.2.3.4 --- Stripping membrane for reprobing --- p.34
Chapter 2.2.2 --- Characterization of the cercarial stage-specific gene and gene product of 20H8 --- p.35
Chapter 2.2.2.1 --- Cloning of full-length cDNA of 20H8 --- p.35
Chapter 2.2.2.1.1 --- 5'RACE of 20H8 --- p.35
Chapter 2.2.2.1.2 --- Cloning of full-length 20H8 into pBluescript II SK(-) --- p.36
Chapter 2.2.2.2 --- Analysis of DNA sequence and deduced amino acid sequence of 20H8 --- p.37
Chapter 2.2.2.3 --- Demonstration of the immunogenicity and antigenicity of 20H8 --- p.38
Chapter 2.2.2.3.1 --- Expression of 20H8 in E. coli --- p.38
Chapter 2.2.2.3.1.1 --- "Cloning of 20H8 in an E coli expression vector, pET32a+" --- p.38
Chapter 2.2.2.3.1.2 --- Expression of recombinant 20H8 protein in E. coli --- p.39
Chapter 2.2.2.3.2 --- Purification and concentration of recombinant 20H8 protein --- p.40
Chapter 2.2.2.3.3 --- Production of antiserum --- p.40
Chapter 2.2.2.3.4 --- Evaluation of immunogenicity of recombinant 20H8 protein --- p.41
Chapter 2.2.2.3.5 --- Evaluation of antigenicity of recombinant 20H8 protein --- p.42
Chapter 2.2.2.4 --- Immunolocalization of 20H8 in cercaria --- p.43
Chapter 2.2.2.4.1 --- Collection of S. japonicum cercaria --- p.43
Chapter 2.2.2.4.2 --- Immunofluorescence staining of cercaria --- p.43
Chapter 2.2.3 --- Characterization of the cercarial stage-specific gene and gene product of sjCa8 --- p.45
Chapter 2.2.3.1 --- Analysis of DNA sequence and deduced amino acid sequence of sjCa8 --- p.45
Chapter 2.2.3.2 --- Demonstration of the immunogenicity and antigenicity of sjCa8 --- p.46
Chapter 2.2.3.2.1 --- Expression of sjCa8 in E. coli --- p.46
Chapter 2.2.3.2.2 --- Purification and concentration of recombinant sjCa8 protein --- p.46
Chapter 2.2.3.2.3 --- Production of antiserum --- p.46
Chapter 2.2.3.2.4 --- Evaluation of the immunogenicity of sjCa8 --- p.47
Chapter 2.2.3.2.5 --- Evaluation of the antigenicity of sjCa8 --- p.47
Chapter 2.2.3.3 --- Demonstration of calcium-binding property of sjCa8 --- p.48
Chapter 2.2.3.3.1 --- Calcium-dependent electrophoretic mobility shift --- p.48
Chapter 2.2.3.3.2 --- Ruthenium red assay --- p.48
Chapter 2.2.3.4 --- Immunolocalization of sjCa8 in cercaria --- p.49
Chapter Chapter 3 --- Results --- p.50
Chapter 3.1. --- Identification of cercarial stage-specific genes --- p.50
Chapter 3.1.1 --- Identification of cercarial stage-specific genes by microarray --- p.50
Chapter 3.1.2 --- "Confirmation of stage-specific expression of the selected gene, 20H8 and sjCa8, by RNA dot blot" --- p.54
Chapter 3.2. --- Characterization of the cercarial stage-specific gene and gene product of 20H8 --- p.57
Chapter 3.2.1 --- Cloning of full-length cDNA of 20H8 --- p.57
Chapter 3.2.2 --- Analysis of DNA sequence and deduced amino acid sequence of 20H8 --- p.59
Chapter 3.2.3 --- Demonstration of the immunogenicity and antigenicity of 20H8 --- p.62
Chapter 3.2.3.1 --- Expression of 20H8 in E. coli --- p.62
Chapter 3.2.3.2 --- Purification and concentration of recombinant 20H8 protein --- p.64
Chapter 3.2.3.3 --- Production of antiserum --- p.65
Chapter 3.2.3.4 --- Evaluation of immunogenicity of recombinant 20H8 protein --- p.66
Chapter 3.2.3.5 --- Evaluation of antigenicity of recombinant 20H8 protein --- p.67
Chapter 3.2.4 --- Immunolocalization of 20H8 in cercaria --- p.68
Chapter 3.3. --- Characterization of the cercarial stage-specific gene and gene product of sjCa8 --- p.70
Chapter 3.3.1 --- Analysis of DNA sequence and deduced amino acid sequence of sjCa8 --- p.70
Chapter 3.3.2 --- Demonstration of the immunogenicity and antigenicity of sjCa8 --- p.75
Chapter 3.3.2.1 --- Expression of sjCa8 in E. coli --- p.75
Chapter 3.3.2.2 --- Purification and concentration of recombinant sjCa8 protein --- p.76
Chapter 3.3.2.3 --- Production of anti-sjCa8 serum --- p.77
Chapter 3.3.2.4 --- Evaluation of immunogenicity of sjCa8 protein --- p.77
Chapter 3.3.2.5 --- Evaluation of antigenicity of sjCa8 protein --- p.78
Chapter 3.3.3 --- Demonstration of calcium-binding property of sjCa8 --- p.79
Chapter 3.3.3.1 --- Electrophoretic motility shift --- p.80
Chapter 3.3.3.2 --- Ruthenium red binding assay --- p.80
Chapter 3.3.4 --- Immunolocalization of sjCa8 in cercaria 81 --- p.81
Chapter Chapter 4 --- Discussion --- p.83
Chapter 4.1 --- Identification of cercarial stage-specific genes --- p.83
Chapter 4.1.1 --- Identification of cercarial stage-specific genes by microarray --- p.83
Chapter 4.1.2 --- "Confirmation of stage-specific expression of the selected genes, 20H8 and sjCa8" --- p.85
Chapter 4.2 --- Characterization of the cercarial stage-specific gene and gene product of 20H8 --- p.86
Chapter 4.2.2 --- Analysis of DNA sequence and deduced amino acid sequence of 20H8 --- p.86
Chapter 4.2.3 --- Demonstration of the immunogenicity and antigenicity of 20H8 --- p.88
Chapter 4.2.4 --- Immunolocalization of 20H8 in cercaria --- p.90
Chapter 4.3 --- Characterization of the cercarial stage-specific gene and gene product of sjCa8 --- p.91
Chapter 4.3.1 --- Analysis of DNA sequence and deduced amino acid sequence of sjCa8 --- p.91
Chapter 4.3.2 --- Demonstration of the immunogenicity and antigenicity of sjCa8 --- p.93
Chapter 4.3.3 --- Demonstration of calcium-binding property of sjCa8 --- p.94
Chapter 4.3.4 --- Immunolocalization of sjCa8 in cercaria --- p.94
Chapter 4.4 --- Conclusions --- p.95
References --- p.96

Li Chi Ho.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (leaves 137-162).
Abstracts in English and Chinese.
Statement --- p.iii
Acknowledgments --- p.iv
Abstract --- p.vi
Abstract (Chinese version) --- p.viii
Abbreviations --- p.x
Table of contents --- p.xii
List of Figure --- p.xviii
List of Table --- p.xx
Chapter Chapter One --- Literature Review
Chapter 1.1 --- The Genus Schistosoma --- p.1
Chapter 1.2 --- Biology of Schistosoma japonicum --- p.3
Chapter 1.2.1 --- The History of discovery of Schitosoma japonicum --- p.3
Chapter 1.2.2 --- Life cycle of Schistosoma japonicum --- p.3
Chapter 1.2.2.1 --- Egg --- p.6
Chapter 1.2.2.2 --- Miracidia --- p.6
Chapter 1.2.2.3 --- Sporocysts --- p.7
Chapter 1.2.2.4 --- Cercaria --- p.9
Chapter 1.2.2.5 --- Schislosomula --- p.10
Chapter 1.2.2.6 --- Adult worms --- p.10
Chapter 1.2.3 --- Genetics of Schistosoma japonicum --- p.11
Chapter 1.2.3.1 --- Genome analysis --- p.12
Chapter 1.2.3.2 --- Schistosome genome --- p.13
Chapter 1.2.4 --- Tegumental membrane of Schistosomes --- p.14
Chapter 1.3 --- Pathology of Schistosomiasis --- p.15
Chapter 1.3.1 --- Acute Schistosomiasis --- p.15
Chapter 1.3.2 --- Intestinal Disease --- p.16
Chapter 1.3.3 --- Hepatosplenic Disease --- p.16
Chapter 1.3.4 --- Cerebral Schistosomiasis --- p.16
Chapter 1.4 --- Treatment of Schistosomiasis --- p.17
Chapter 1.4.1 --- Chemotherapy --- p.17
Chapter 1.4.2 --- Schistosoma Vaccine --- p.17
Chapter 1.5 --- GPI anchor --- p.19
Chapter 1.5.1 --- Function of GPI anchored proteins --- p.19
Chapter 1.5.2 --- Synthesis of GPI anchor --- p.21
Chapter 1.5.3 --- "Phosphatidylinositol Glycan, Class N (PIG-N)" --- p.26
Chapter 1.6 --- The role of GPI anchor proteins in Schistosome --- p.26
Chapter 1.7 --- Aim of study --- p.29
Chapter Chapter two --- Materials and Methods
Chapter 2.1 --- Materials --- p.31
Chapter 2.1.1 --- Cell lines and Bacterial Strains --- p.31
Chapter 2.1.2 --- Chemicals --- p.32
Chapter 2.1.3 --- Kits and Reagents --- p.34
Chapter 2.1.4 --- Nucleic acids --- p.34
Chapter 2.1.5 --- Reagents for Cell culture --- p.34
Chapter 2.1.6 --- Solutions --- p.35
Chapter 2.1.7 --- Enzymes --- p.37
Chapter 2.1.8 --- Primer List --- p.37
Chapter 2.1.9 --- Antibodies --- p.39
Chapter 2.2 --- Methods
Chapter 2.2.1 --- Screening of the S. japonicum cercaria stage cDNA library --- p.40
Chapter 2.2.1.1 --- λ phage plating --- p.40
Chapter 2.2.1.2 --- Single plaque isolation --- p.40
Chapter 2.2.1.3 --- Conversion of Lambda TriplEx to pTriplEx --- p.41
Chapter 2.2.1.4 --- preparation of plasmid DNA --- p.41
Chapter 2.2.1.5 --- cycle DNA sequencing --- p.42
Chapter 2.2.2. --- RT-PCR --- p.44
Chapter 2.2.2.1 --- Isolation of Total RNA by Guandidium Thiocyanate - Cesium Chloride ultracentrifugation --- p.44
Chapter 2.2.2.2 --- Synthesis of Fist Strand cDNA by reverse transcriptation reaction --- p.45
Chapter 2.2.2.3 --- PGR amplification of RT product --- p.46
Chapter 2.2.3 --- Rapid Amplification of cDNA Ends (RACE) --- p.46
Chapter 2.2.3.1 --- Synthesis of first strand cDNA for RACE reaction --- p.46
Chapter 2.2.3.2 --- 5 ´ةRACE for Sj-PIG-N gene --- p.47
Chapter 2.2.3.3 --- 3´ة RACE for Sj-PIG-N gene --- p.48
Chapter 2.2.3.4 --- Purification of DNA fragment from agarose gel --- p.48
Chapter 2.2.3.5 --- Ligation of purified PCR fragments and pBluescriptII KS(+) T-vector --- p.49
Chapter 2.2.3.6 --- Preparation of DH5a competent cells --- p.49
Chapter 2.2.3.7 --- Transformation of recombinant plasmid --- p.50
Chapter 2.2.4 --- Mammalian cell transfection --- p.50
Chapter 2.2.4.1 --- Stable transfection --- p.50
Chapter 2.2.4.2 --- Transient transfection --- p.51
Chapter 2.2.5 --- Biological function studies of Sj-PIG-N gene --- p.52
Chapter 2.2.5.1 --- Flow Cytometry (FCM) analysis --- p.52
Chapter 2.2.5.2 --- In Vitro Mannose Labeling of Microsomes and Characterization of Glycolipids --- p.53
Chapter 2.2.6 --- Intracellular localization assay --- p.54
Chapter Chapter Three --- Results
Chapter 3.1 --- Random sequence analysis of cercaria EST from S. japonicum Cercaria stage cDNA library --- p.55
Chapter 3.1.1 --- Sequencing results --- p.57
Chapter 3.1.2 --- BlastX Search results --- p.61
Chapter 3.2 --- The expression of Sj-PIG-N gene in both adult worms and cercaria --- p.68
Chapter 3.2.1 --- Spectrophotometric analysis of total RNA --- p.68
Chapter 3.2.2 --- Detection of Sj-PIG-N gene expression in both adult worm and cercaria stages --- p.70
Chapter 3.3 --- Cloning of the full length of Sj-PIG-N gene --- p.72
Chapter 3.3.1 --- Amplification of the full length of Sj-PIG-N gene --- p.72
Chapter 3.3.2 --- Obtaining the full length sequence of Sj-PIG-N gene --- p.74
Chapter 3.4 --- Sequence analysis of Ml length Sj-PIG-N cDNA --- p.80
Chapter 3.5 --- Analysis of Sj-PIG-N protein structure by computer programs --- p.91
Chapter 3.6 --- Construction of Sj-PIG-N gene into mammalian cell expression vector pEGFP-Nl and pEGFP-Hyg --- p.97
Chapter 3.6.1 --- Construction of pBluescriptII KS(+)-Sj-PIG-N-B --- p.97
Chapter 3.6.2 --- Construction of pBluescriptll KS(+)-Sj-PIG-N-E --- p.101
Chapter 3.6.3 --- Construction of pTRE2-Sj-PIG-N --- p.106
Chapter 3.6.4 --- Construction of pEGFP-Hyg-Sj-PIG-N --- p.109
Chapter 3.6.4.1 --- Construction of pEGFP-Hyg --- p.109
Chapter 3.6.4.2 --- Construction of pEGFP-Hyg-Sj-PIG-N --- p.114
Chapter 3.7 --- Molecular analysis of Sj-PIG-N gene --- p.117
Chapter 3.7.1 --- Functional analysis of Sj-PIG-N --- p.117
Chapter 3.7.1.1 --- FACS analysis of surface expression of GPI-anchored protein - Thyl --- p.117
Chapter 3.7.1.2 --- FACS analysis of surface expression of GPI-anchor protein - CD24 --- p.119
Chapter 3.7.1.3 --- In Vitro Mannose Labeling of Microsomes and Characterization of Glycolipids --- p.121
Chapter 3.7.2 --- Localization of Sj-PIG-N with immunofluorescent staining --- p.123
Chapter Chapter Four --- Discussion
Chapter 4.1 --- S. japonicum cercaria EST analysis --- p.125
Chapter 4.2 --- Structure analysis of Sj-Pig-N gene --- p.128
Chapter 4.3 --- Molecular analysis of Sj-PIG-N --- p.131
Chapter 4.4 --- Further study --- p.134
Chapter 4.5 --- Conclusion --- p.136
References --- p.137

Lok Chui-Lin.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (leaves 142-157).
Abstracts in English and Chinese.
Statement --- p.I
Acknowledgement --- p.II
Abstract --- p.IV
Chinese Abstract (摘要） --- p.VI
Abbreviation --- p.VIII
Table of Contents --- p.XIII
List of Tables --- p.XVII
List of Figures --- p.XVIII
Chapter Chapter One : --- Literature Review --- p.1
Chapter 1.1 --- The Schistosoma Species --- p.1
Chapter 1.1.1 --- The Schistosoma Gene Discovery --- p.3
Chapter 1.1.2 --- Schistosome Transcriptome --- p.4
Chapter 1.2 --- Schistosomiasis --- p.4
Chapter 1.2.1 --- Immunopathology of Schistosomiasis --- p.5
Chapter 1.2.2 --- Diagnosis of Schistosomiasis --- p.7
Chapter 1.2.3 --- Treatment and Control for Schistosomiasis --- p.7
Chapter 1.2.4 --- Vaccine Development for Schistosomiasis --- p.8
Chapter 1.3 --- "The Species, Schistosoma japonicum" --- p.9
Chapter 1.3.1 --- The Life Cycle of Schistosoma japonicum --- p.10
Chapter 1.3.1.1 --- "The Egg, Miracidium Phase of the Life Cycle" --- p.12
Chapter 1.3.1.2 --- Developmental Cycle within Mollusc Host --- p.12
Chapter 1.3.1.3 --- The Cercaria Phase of Life Cycle --- p.13
Chapter 1.3.1.4 --- Adult Schistosome in Definitive Host --- p.14
Chapter 1.4 --- Invasion by Schistosome Cercariae --- p.15
Chapter 1.5 --- "The Anti-inflammatory Protein, Sml6" --- p.16
Chapter 1.5.1 --- Discovery of Sm 16 --- p.16
Chapter 1.5.2 --- Cloning and Expression of Gene-encoding Sm 16 --- p.17
Chapter 1.5.3 --- Potential Anti-inflammatory Therapy using Sm 16 --- p.18
Chapter 1.6 --- Innate Immunity and Adaptive Immunity --- p.18
Chapter 1.6.1 --- Macrophage --- p.18
Chapter 1.6.2 --- Major Histocompatiblity Complex (MHC) --- p.20
Chapter 1.6.3 --- Adaptive Immunity to Parasites --- p.20
Chapter 1.7 --- Inflammation --- p.21
Chapter 1.7.1 --- Cells of the Inflammatory Process --- p.23
Chapter 1.7.2 --- Cytokines --- p.24
Chapter 1.7.2.1 --- Interleukin-1 (IL-1) System --- p.26
Chapter 1.7.2.2 --- Interferon (IFN) System --- p.27
Chapter 1.7.3 --- Anti-inflammatory Therapy --- p.28
Chapter 1.8 --- Aim of Study --- p.29
Chapter Chapter Two : --- Materials and Methods --- p.30
Chapter 2.1 --- Materials --- p.30
Chapter 2.1.1 --- "Cell Lines, Mouse Strain and Bacterial Strains" --- p.30
Chapter 2.1.2 --- Plasmids --- p.31
Chapter 2.1.3 --- Chemicals --- p.31
Chapter 2.1.4 --- "Kits, Nucleic Acids and Reagents" --- p.34
Chapter 2.1.5 --- Antibodies and Immunoglobins --- p.35
Chapter 2.1.6 --- Cell Culture Reagents --- p.35
Chapter 2.1.7 --- Solutions --- p.36
Chapter 2.1.8 --- Solutions of Reaction Kits --- p.39
Chapter 2.1.9 --- Enzymes --- p.41
Chapter 2.1.10 --- Major Equipments and Materials --- p.41
Chapter 2.1.11 --- Primers --- p.43
Chapter 2.1.11.1 --- Sequencing and Sj 16 Gene-coding Specific Primers --- p.43
Chapter 2.1.11.2 --- Primers for Cytokines --- p.43
Chapter 2.2 --- Methods --- p.45
Chapter 2.2.1 --- Amplification of Sjl6 cDNA from Schistosoma japonicum Cercariae --- p.45
Chapter 2.2.1.1 --- Isolation of Cercariae total RNA by Guanidinium Thiocyanate - Cesium Chloride Ultracentrifugation --- p.45
Chapter 2.2.1.2 --- Reverse Transcription - Polymerase Chain Reaction (RT-PCR) --- p.46
Chapter 2.2.1.2.1 --- Reverse Transcription (RT) --- p.46
Chapter 2.2.1.2.2 --- Polymerase Chain Reaction (PCR) --- p.46
Chapter 2.2.2 --- Cloning and Subcloning of Sj 16 --- p.47
Chapter 2.2.2.1 --- Preparation of DH5a Competent Cells --- p.47
Chapter 2.2.2.2 --- Purification of Plasmid DNA --- p.48
Chapter 2.2.2.3 --- Restriction Enzyme Digestion of DNA --- p.49
Chapter 2.2.2.4 --- Purification of DNA Fragments from Agarose Gel --- p.50
Chapter 2.2.2.5 --- Ligation of Purified DNA Fragments --- p.51
Chapter 2.2.2.6 --- Transformation of Recombinant Plasmid --- p.52
Chapter 2.2.2.7 --- Selection of Transformed Clones --- p.52
Chapter 2.2.2.7.1 --- Screening by X-gal and IPTG : a-complementation --- p.52
Chapter 2.2.2.7.2 --- Screening by Polymerase Chain Reaction --- p.53
Chapter 2.2.2.8 --- Cycle Sequencing --- p.53
Chapter 2.2.3 --- Expression of the rSj 16 in Eukaryotic System --- p.55
Chapter 2.2.3.1 --- Transfection of pSecTag2B/Sj 16 Plasmid into Animal Cells --- p.55
Chapter 2.2.3.2 --- PCR Screening of Transfected Cells --- p.56
Chapter 2.2.3.3 --- Analysis of mRNA Transcript by RT-PCR --- p.56
Chapter 2.2.3.4 --- Concentration of the Condition Medium --- p.57
Chapter 2.2.3.5 --- Western Blot analysis of rSjl6 Expression --- p.58
Chapter 2.2.4 --- Expression of rSjl6 in Bacterial System --- p.59
Chapter 2.2.4.1 --- Transformation of pET30a+/Sjl6 Plasmid into BL21 --- p.59
Chapter 2.2.4.2 --- Optimization of rSj 16 Expression --- p.60
Chapter 2.2.4.3 --- Solubility of the rSjl6 --- p.60
Chapter 2.2.4.4 --- Estimation of rSj 16 Concentration --- p.62
Chapter 2.2.4.5 --- Western Blot Analysis of rSj 16 --- p.62
Chapter 2.2.5 --- Recombinant Protein Purification --- p.63
Chapter 2.2.5.1 --- Affinity Chromatography of Recombinant Protein --- p.63
Chapter 2.2.5.2 --- Dialysis of Eluted Recombinant Protein in PBS --- p.64
Chapter 2.2.5.3 --- Estimation of Recombinant Protein Concentration --- p.65
Chapter 2.2.6 --- Demonstrate the Anti-inflammatory Activity of rSj 16 --- p.65
Chapter 2.2.6.1 --- Thioglycollate Induced Macrophage Recruitment --- p.65
Chapter 2.2.6.2 --- Cytospin and Hemacolor Staining of PECs --- p.66
Chapter 2.2.6.3 --- FACS Analysis of PECs --- p.67
Chapter 2.2.6.4 --- Isolation of total RNA by TRIZOL Reagent --- p.67
Chapter 2.2.7 --- Immunogenicity and Antigenicity of rSjl6 --- p.68
Chapter 2.2.7.1 --- Western Blot of rSjl6 with Schistosoma japonicum infected rabbit serum --- p.69
Chapter 2.2.7.2 --- Preparation of Anti-Sj 16 Serum --- p.69
Chapter 2.2.7.3 --- Western Blot of rSjl6 with immunized mice serum --- p.70
Chapter 2.2.8 --- FACS analysis of MHC (I) Expression --- p.71
Chapter 2.2.9 --- Anti-proliferative Assay using BrdU Kit --- p.72
Chapter Chapter Three : --- Results --- p.73
Chapter 3.1 --- Amplification of Sj 16 cDNA from Schistosoma japonicum Cercariae total RNA --- p.73
Chapter 3.2 --- Construction of pBluescript II SK(-) / Sjl6 --- p.75
Chapter 3.3 --- Analysis of Sj 16 Nucleotide and Amino Acid Sequence --- p.78
Chapter 3.3.1 --- Blastn Search Analysis --- p.80
Chapter 3.3.2 --- Blastx Search Analysis --- p.82
Chapter 3.3.3 --- Structural Analysis --- p.84
Chapter 3.4 --- Subcloning of Sjl6 cDNA into pET30a+ and pSecTag2B Expression Vector --- p.88
Chapter 3.5 --- Expression of the rSj 16 --- p.92
Chapter 3.5.1 --- Animal Cell Expression --- p.92
Chapter 3.5.1.1 --- Analysis of mRNA Transcript by RT-PCR --- p.93
Chapter 3.5.1.2 --- Western Blot of Condition Medium --- p.95
Chapter 3.5.2 --- Bacterial Cell Expression --- p.97
Chapter 3.5.2.1 --- Optimization of rSjl6 Expression --- p.97
Chapter 3.5.2.2 --- Estimation of rSjl6 Concentration --- p.98
Chapter 3.5.2.3 --- Solubility of rSj16 --- p.99
Chapter 3.5.2.4 --- Western Blot Analysis of rSjl6 --- p.100
Chapter 3.6 --- Purification of Recombinant Protein --- p.101
Chapter 3.6.1 --- Purification of rSj16 --- p.101
Chapter 3.6.2 --- Purification of rSjCa8 --- p.104
Chapter 3.7 --- Anti-inflammatory Activity of rSj 16 --- p.107
Chapter 3.7.1 --- Analysis of PECs in Thioglycollate Induced Inflammation --- p.107
Chapter 3.7.2 --- Hemacolor Staining of PECs --- p.110
Chapter 3.7.3 --- FACS Analysis of PECs --- p.110
Chapter 3.7.4 --- RT-PCR of RNA Isolated from PECs --- p.115
Chapter 3.8 --- Immunogenicity and Antigenicity of rSjl6 --- p.117
Chapter 3.8.1 --- Immunogenicity of rSj 16 --- p.117
Chapter 3.8.2 --- Antigenicity of rSj16 --- p.117
Chapter 3.9 --- Inhibitory Effect of rSj 16 on rMuIFN-a4 Induced Up-regulation of MHC(I) Expression --- p.120
Chapter 3.9.1 --- Time Course of rMuIFN-α4 Induced Up-regulation of MHC(I) Expression --- p.120
Chapter 3.9.2 --- Inhibitory Effect of rSjl6 on rMuIFN-α4 Induced MHC (I) Up-regulation --- p.120
Chapter 3.9.3 --- "Anti-proliferation Effect of rMuIFN-a4, rSj 16 and rSjCa 8" --- p.124
Chapter 3.9.4 --- Effect of Signal Transduction Inhibitors on rMuIFN-a4 Induced MHC (I) Up-regulation --- p.126
Chapter Chapter Four : --- Discussion and Conclusion --- p.129
Chapter 4.1 --- Discussion --- p.129
Chapter 4.1.1 --- Overview --- p.129
Chapter 4.1.2 --- Molecular and Structural Analysis of rSj 16 --- p.130
Chapter 4.1.3 --- Relationship between Sml6 and Sjl6 --- p.131
Chapter 4.1.4 --- Anti-inflammatory Activity of rSj 16 --- p.132
Chapter 4.1.5 --- Immunogenicity and Antigenicity of rSjl6 --- p.137
Chapter 4.1.6 --- Inhibitory Effect of rSjl6 on rMuIFN-a4 Induced Up-regulation of MHC (I) Expression --- p.138
Chapter 4.1.7 --- Relation between Sj 16 and the Innate Immune System --- p.139
Chapter 4.1.8 --- Further Study and Significance --- p.140
Chapter 4.2 --- Conclusion --- p.141
References --- p.142

Schistosomiasis, a disease of humans caused by helminth parasites of the genus Schistosoma, kills 200 to 500 thousand people annually, endangering over 600 million people world-wide with 200 million people infected in 2003 [1, 2]. Three species of schistosome are primarily responsible for human infections, namely, Schistosoma haematobium, endemic to Africa, India, and the Middle East, S. mansoni, endemic to Africa / South America, and S. japonicum endemic to China and the Philippines [3]. The major pathological effects of schistosomiasis result from the deposition of parasite ova in human tissues and the subsequent intense granulomatous response induced by these eggs. There is a high priority to provide an effective sub-unit vaccine against these schistosome flukes, using proteins encoded by cDNAs expressed by the parasites at critical phases of their development. One technique that may expedite this gene identification is the use of microarrays for expression analysis. A 22,575 feature custom oligonucleotide DNA microarray designed from public domain databases of schistosome ESTs (Expressed Sequence Tags) was used to explore differential gene expression between the Philippine (SJP) and Chinese (SJC) strains of S. japonicum, and between males and females. It was found that 593, 664 and 426 probes were differentially expressed between the two geographical strains when mix sexed adults, male worms and female worms were compared respectively. Additionally, the study revealed that 1,163 male- and 1,016 female-associated probes were differentially expressed in SJP whereas 1,047 male- and 897 female-associated probes were differentially expressed in SJC [4]. Further to this, a detailed real time PCR expression study was used to explore the differential expression of eight genes of interest throughout the SJC life cycle, which showed that several of the genes were down-regulated in different life cycle stages. The study has greatly expanded previously published data of strain and gender-associated differential expression in S. japonicum. Further, the new data will provide a stepping stone for understanding the complexities of the biology, sexual differentiation, maturation, and development of human schistosomes, signaling new approaches for identifying novel intervention and diagnostic targets against schistosomiasis [4].

博士
國立陽明大學
熱帶醫學研究所
96
Schistosomiasis is continuing a serious problem in public health around the world. This parasitic disease is mainly caused by the release of a large number of eggs by adult worms. Schistosoma japonicum infection induces severe pathological lesions because the large produce the most counts of eggs. Although a large amount of resources have been invested in various control and research programs, transmission remains in most of the endemic areas. Some characteristics of the worm are still unclear and have long perplexed investigators intent on controlling this parasitic disease. In the present thesis, we have conducted extensive studies and discussions on multifaceted aspects of genomics, immunology, and actual applications of Schistosoma. The entire thesis includes 3 sections as provided below. The first and main section reveals that macrophages play crucial roles in immune responses as they can initiate, modulate, and also serve as final effector cells during course of schistosomal infections. For better understanding the innate immune responses during host-parasite interactions, the global gene expression profiles of macrophages from the spleen of mice at different infection stages were compared by microarray-based analysis. Concurrently, we not only used flow cytometric analysis to investigate the immunocompetent changes in the macrophages, but also evaluated the population-differentiated proportion of the immune phenotype in a unique T1/T2 double transgenic mouse model. Hierarchical clustering analysis demonstrated that a significant switch in gene transformation associated with a type-1 response and linked with a type-2 cytokine phenotype occurs between 4.5 and 8 weeks post-infection. Moreover, the gene profiles at 3 later time-points following egg challenge were similar in complexity and magnitude. These data also showed that there are mostly inhibtion in gene expression related TLRs, IFN, MHC and TNFrsf at the switch between 4.5 and 8 weeks post-infection, and it is suggested that these immunomodulatory genes may be downregulated in resistance against S. japonicum eggs and granuloma pathology. In particular, the gradual upregulation of genes encoding YM1 and YM2 in infected mice indicated the role of alternatively activated macrophages (AAM�珺) in immune responses after schistosoma egg production. The induction of AAM�珺 in the spleen could be important for dampening the level of inflammation in hepatic granulomas and contributing to a long-term decrease in violent cytotoxicity and tissue-damaging immune responses that are associated with type-1 immunity. The genes expressions involved in repair/remodeling during liver fibrosis were also observed after eggs production. Histological analysis demonstrated that cellular infiltration and granuloma formation were initiated quickly after 7-week post-infection as a result of S. japonicum eggs deposited into the liver, and the pathology of disease gradually progressed with time. Flow cytometric analysis showed that macrophages from mice infected with S. japonicum were decreased at 3-5 weeks, and the number re-increased during eggs production at 7 weeks p.i. The surface markers MHC class II (IA/IE) and CD8α+ of the macrophages also exhibited a dramatic change before and after the egg-production time points. The transgenic mice experiments further demonstrated the proportion of CD4+ T cells and B cells shifted and differentiated into type 1 and type 2 regulatory subpopulations over the course of infection. The percentage of CD4+ Th2 cells was observed to be much higher than that of Th1 cells, especially after stimulation with S. japonicum eggs, whereas type 1 cells increased only at 3 and 5 weeks post-infection. The differentiation of B cells showed a similar trend. These results suggested that the infection of mice with S. japonicum resulted in a final Th2-skewed immune response that was associated with the phenotypical changes in the macrophages and the influence of macrophages that were alternatively activated by cytokine cross-modulation. All of these analyses elucidated a correlation between macrophage differentiation in systemic immune responses and different stages of schistosoma infection, thus proving the existence of variation in innate and adaptive immune mechanisms at the different infection stages. Understanding these immune mechanisms related to parasite resistance, pathology, and growth with regard to the disease will be helpful in further studies on S. japonicum. In the second part, we applied recombinant Sjc26GST protein for serodiagnosis of S. japonicum infection in water buffalo. The gene encoding Sjc26GST was cloned and expressed in Escherichia coli as a fusion protein with a His-tag. The purified reSjc26GST was used as an antigen for an enzyme-linked immunosorbent assay (ELISA) and for immunoblotting to detect S. japonicum antibodies in water buffaloes. Our results showed that mean the O.D. values of specific serum IgG antibodies from egg-positive buffaloes were 3.37-fold higher than that found in egg-negative buffaloes from non-endemic areas. The data also showed that the O.D. value of the endemic egg-negative group reached as high as 1.69 times that found in the non-endemic areas. The positivity rate of egg-positive buffaloes was 100%, but was 30.3% in the endemic egg-negative group. Infected bovine antisera also recognized reSjc26GST, a 27 kDa protein as determined by Western blot. These results suggest that the recombinant GST expressed in E. coli would be an effective diagnostic reagent for the detection of antibodies against S. japonicum in buffaloes. Due to its easy production, excellent sensitivity, and high specificity, the reSjc26GST protein described in this study can be considered as a candidate protein for immunological diagnosis of bovine schistosomiasis. In the third section, we attempted to establish an in vivo schistosomal mouse infection model to study the immunomodulatory roles of Chinese herbal medicine. The unique chronic characteristic of this parasite are suited to elucidate gentle curative efficacy of Chinese herbs. In this study, we tested the in vivo effects of polysaccharides derived from A. camphorata (AC-PS) on the immune function by detection of cytokine expression, and evaluation of the immune phenotype in inbred mice or the T1/T2 doubly transgenic mouse model. The protective effect of AC-PS in mice was finally tested by infection with S. mansoni. The induction of large amounts of IFN-γ, IL-2, and TNF-α mRNA were detected after 2 and 4 weeks of oral AC-PS administration in BALB/c and C57BL/6j mice. After 6 weeks of oral AC-PS administration to BALB/c mice, the number of splenic dendritic cells, macrophages as well as the surface expression of CD8α+ and MHC class II I-A/I-E on dendritic cells increased. The CD4+/CD8+ T cells ratio and number of B cells among the splenocytes were also augmented. In transgenic mice, 3- to 6-weeks of oral AC-PS administration increased the proportion of CD4+ T cells and B cells in the spleen. More specifically, there was an increase in Th1 CD4+ T cells and Be1 cells among the spleen cells as observed by detection of type1/type2 marker molecules. Using a disease model for parasitic infection, we found that AC-PS treatment inhibited S. mansoni infection in BALB/c and C57BL/6j mice. AC-PS appears to influence the development of Th1 responses in the immune system and has the potential for preventing S. mansoni infection. Taken together, this thesis had led to a profound understanding of schistosome-host interactions on a genomic and immunological basis, especially with regard to innate immunity of macrophages. This thesis also provides a new diagnostic method for domestic schistosomiasis and a unique in vivo schistosomal infection model for evaluating the curative efficacy of drugs or health food. We believe that these extensive studies result in improvements and provide direction of further schistosome investigations.

碩士
國立陽明大學
熱帶醫學研究所
95
Schistosomiasis is the secondary most important tropical disease listed by the World Health Organization; it has been estimated over 200 million people infected. It is believed that vaccination is the most effectively way to control and restrict this disease. Our laboratory had successfully expressed 26kDa GST protein in Escherichia coli strain M15 and indicated that it could induce a good immune response and confer a significant protection by reducing the worm burdens. It is suggesting that recombinant 26kDa GST might be a good candidate for Schistosoma vaccine development. In this study, we constructed a DNA vaccine of 26kDa GST and evaluated the effectiveness of its vaccination. We also evaluated antibody titer in sera of mouse and quantified the amounts of mRNA for various cytokines. The result showed that the level of anti-26kDa GST IgG antibody in immunized mice with GST DNA vaccine was significantly higher than normal controls. The subclass IgG2a antibody was induced by immuning DNA vaccine with IL-12. IgG1 and IgG2b antibodies were also produced in mice treated mutually with GST protein and the DNA vaccine. The mice immunized with GST DNA vaccine showed a worm reduction of 62% as compared with control group. The reduction rates were 68.90% or 72.30%, if mice were immunized with vaccine containing IL-12 plasmid or GST protein, respectively. The reduction rate was increased to 74.10% if the mice were immunized with DNA vaccine with both GST protein and IL-12 plasmid. The results of RT-PCR showed that expression of IL-2, IL-12, INF-r and TNF-α mRNAs from mice immunized with GST DNA vaccine were increased, and IL-4, IL-10 mRNAs were induced on mice with GST DNA vaccine and GST protein. The present study indicates that GST DNA plasmid is a distinctive and effective vaccine, and it is more effective to resist S. japonicum with the combination of IL-12 and GST protein.

碩士
國立陽明大學
寄生蟲學研究所
88
Schistosomiasis, a chronic granulomatous immune disease, is one of the most important human parasitic infections. Africa is the largest endemic area of schistosomiases. Among the 75 endemic countries in the world, there are 40 countries prevalent with more than two species of schistosomes. It has been reported that several species of schistosomes may coexist in the same human host. Moreover, experimental hybridization of human and animal strains of schistosomes prevalent in Africa has been studied extensively. However, only little information in the hybridization of Schistosoma japonicum with the other species/strains. The antibodies of the host cause formation of granuloma, an important pathogenic finding in schistosomiasis. Since species-specific glycoprotein has been demonstrated in S. mansoni and S. japonicum, it is generally considered that glycoproteins are the major antigens on the eggs of schistosomes. In the present study, hybridizations were carried out between S. mansoni and S. japonicum. Although most of the eggs in the F1 generation were immature, dead, or calcified, the morphology of the rare mature ones was demonstrated to be similar to the mother species. By analysis of the structure of the glycoproteins on the egg shell, S. mansoni eggs was shown to have a xylosylated 6-fucosylated trimannosyl core whereas S. japonicum eggs with a xylosylated 3-,6-difucisylated trimannosyl core. In addition, the glycoprotein structure of the F1 eggs was similar to that of the mother species. Although the male stimulation may not be necessary to the egg production in S. mansoni, the same phenomenon was not observed in S. japonicum. These findings indicate that offspring from the interspecific pairings between S. mansoni and S. japonicum may be due to parthenogenesis. Moreover, it also explains the similarity in the morphology and glycoprotein structure in the eggs.

Schistosome is the causative agent of schistosomiasis which is one of the world's most prevalent tropical diseases. In the skin of infected host, significant inflammatory response to the parasite is not observed. Previous studies from Schistosoma mansoni showed that this subdued inflammatory response was due to a 16-KDa protein, Sm16, which is present abundantly in the secretions of schistosomulae. Provided that Schistosoma japonicum shares the same infective pathway as S. mansoni by penetrating the skin, it seems logical that S. japonicum has a protein with a similar role to Sm16 to down-regulate host immune responses. According to the cDNA sequence of Sm16, a corresponding gene (designated Sj16) of Sm16 has previously been amplified and cloned from the cercarial cDNA of S. japonicum. Sequence analysis showed that Sj16 shares 99% identity with Sm16 in its nucleotide sequence, and 100% identity in its protein sequence. While previous studiers reported their failure in obtaining the soluble recombinant protein of Sm16, we expressed and purified the recombinant Sj16 (rSj16) from E. coli in the present study. Western blot and ELISA analysis showed that S. japonicum-infected rabbit sera could not recognize rSj16, indicating that native Sj16 might fail to induce circulating antibodies during S. japonicum infection. In the in vivo study, rSj16 dramatically suppressed not only the recruitment of leukocytes to the peritoneal cavity of BALB/c mice injected with thioglycollate, but also the maturation of thioglycollate-induced peritoneal macrophages. The suppression effect was accompanied by a marked up-regulation of IL-10 and IL-1RA transcripts, and down-regulation of IL-12p35, IL-1beta and MIP-2 transcripts in peritoneal cells. Further analysis revealed that rSj16 also inhibited both humoral and cellular immune responses to heterologous antigens. In addition, rSj16 was found to induce macrophage differentiation of the murine myeloid leukemia WEHI-3B (JCS) cells, and regulate the differentiation of mouse hematopoietic cells towards the macrophage lineage. Although previous studies indicated the involvement of endogenous IL-1alpha, IL-1beta and TNF-alpha in the macrophage differentiation of JCS cells, the results from this study suggested that rSj16-induced JCS cell differentiation do not rely on the endogenous production of these three cytokines. This is the first study to successfully express and purify sufficient soluble rSj16, and demonstrate the anti-inflammatory and immunomodulatory effects of the rSj16.
Hu, Shaomin.
Adviser: Ming Chiu Fung.
Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0210.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 139-154).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.

碩士
國立陽明大學
熱帶醫學研究所
93
Artemether, a derivative of artemisnin, was demonstrated to be an effective drug against schistosomes. It has been shown that the polysaccharides （Gl-PS）contained in Ganoderma lucidum can significantly induce the production of cytokines as well as antibodies in mice, thereby enhancing immunity. Whether artemether can promote the host immune response which leads to the killing of worms remain unknown. The purpose of this study is to investigate the efficacy of artemether alone or in combination with G. lucidum on the immune response induced and the probable mechanism of action mediated via induced changes in the expression profile of gene, and antibodies. The weight change and worm reduction rate of treated mice were also investigated. A single dose artemether at different time course alone or combined with Gl-PS was administered to schistosome-infected mice. The distinct weight loss observed in infected mice was found at day 35, which coincided with parasite maturation and the initiation of egg production in the acute stage. The results have shown that the greatest remedial effect was observed when artemether was given at day 7, particularly on the female worm reduction. The number of schistosomula were significantly reduced when administered with Gl-PS. The IL-2, IL-4, IL-10 and iNOSmRNA gene expressions increased markedly after arthmether treatment, however, only IL-10mRNA reached maximum when combined with Gl-PS. On the other hand, the levels of anti-SWAP and anti-rGST IgG antibodies in mice were significantly higher than in the normal control, which especially marked differences for IgG1 and IgG2b subclasses. These results indicated that administered artemether could enhance the expression of Th2 immune responses, thereby promoting the humoral immunity which may lead to the killing of schistosomes. This is the first study to investigate the immunomodulatory functions of artemether in conjunction with application of G. lucidum.

碩士
國立臺灣科技大學
化學工程技術研究所
86
A Schistosoma japonicum glutathione S-transferase (GST) and its four different mutants were studied to investigate the effect of a C-terminal addition of a metal affinity tag, 6His Tag, on the stability of the enzyme and the effects of cysteine (Cys)oxidation on the activity and the stability of the one. These mutants are GST/His (a fusion protein of GST and 6 His Tag), Cys85→Ser GST/His (a GST/His mutant where Cys 85 was replaced with Ser), Cys138→Ser GST/His and Cys 178→Ser GST His. The results showed that: 　　(1)GST/His expressed using T7 promoter was better produced than GST expressed using tac promoter. 　　(2)For GST/His and its mutants, the purification with Ni2+-NTA gels had better stability and application than the purification with glutathione (GSH) Sepharose 4B gels. 　　(3)Without the presence of reducing agents, the structures of GST and GST/His could be changed due to the oxidation of their Cys residues. 　　(4)The results of the kinetic studies showed that the Cys 85 and Cys 138 residues on GST/His had some contributions to the function and the structure of the G-site and the H-site of the enzyme, respectively. 　　(5)Under a denaturing condition where denaturant existed, the structures of GST and GST/His underwent reversible changes due to unfolding. 　　(6)At higt denaturant concentrations, GSH could protect GST and GST/His from strucrural changes and inactivation due to unfolding. 　　(7)The thermoinactivation rates of GST/His and its mutants were reduced when they were immobilized on Ni2+-NTA gels.

碩士
國立陽明大學
熱帶醫學研究所
93
Glutathione S-transferase (GST) is generally recognized by WHO as one of the major candidate vaccine antigens against Schistosome infections. The dendritic cells (DCs) and macrophages (Mψ) are antigen -presenting cells (APC), especially dendritic cells, acquire antigens in peripheral tissues and migrate to lymphoid organs where they present processed peptides to T cells, and induce innate and then acquired immune responses. In order to understand the interaction and immuno -modulation of dendritic cells, macrophages and 26KDa rGST vaccine antigen of Schistosoma japonicum, Flow cytometry and confocal laser scanning microscope were used to perform this study. The results showed that DC from mice immunized with 26KDa rGST antigen expressed strongly class II MHC and the CD8α+, but lesser on macrophages. The presentation of location of GST on DC is overlaped with class II MHC based on the obserotion of confocal laser scanning microscope. This result demonstrates that DC stimulated with rGST could process via incorporation of class II MHC molecule and present as GST antigen. Furthermore, we found that DCs of mice immunized with GST were re-stimulated rapidly and activated apparently while eggs produced, after seven weeks of infection. the CD8α+ marker started expressed on DCs continuously to chronic stage. In addition, the class II MHC increased markedly on macrophage after five and seven weeks of infection. These results suggested the antigen presenting ability of DC induced by rGST vaccine antigen come mainly from the GST of eggs, which lead to effective increase of host immune response against schistosome infection.

碩士
國立陽明大學
寄生蟲學研究所
87
Abstract In 1996 we reported our successful cloning of the cDNA of 26 KDa GST from a zoophilic Taiwan strain of Schistosoma japonicum by the RT-PCR technique, expressing the recombinant protein in Escherichia coli. and proving this GST to be a good immunogen. In the present study we have tried to further enhance the immunity of the GST protein. We used IL-12 as an adjuvant for recombinant 26 KDa GST and investigated its immuoefficacy against S. japonicum infection. We evaluated antibody titers in mouse sera, the production of mRNA for various cytokines, and the influence on host protective immunity. Results of experiments showed that when IL-12 was used as an adjuvant for 26 KDa GST the IgG titers increased. The mice immunized with 26 KDa GST containing FCA showed a worm reduction of 43.8% as compared with the control group which received FCA alone. If mice were immunized with 26 KDa GST containing FCA and IL-12, the reduction rate reached 50%; however, the rate reached a much higher 70.8% if the mice were given IL-12 once again after cercarial infection. This RT-PCR study showed that if IL-12 was given the mRNAs for IFN-g, IL-2 and TNF-a could be increased, but IL-4 and IL-5 were suppressed. The present study indicates that IL-12 used as an adjuvant for recombinant 26 KDa GST can increase the protective immunity against S. japonicum infection.

碩士
國立陽明大學
寄生蟲學研究所
88
Schistosomiasis is one of the most important tropical diseases and over 200 million people are estimated to be infected. It is believed that the development of a vaccine is the most effective means to the control this disease. The cDNA of 26KDa GST from the total RNA of adult worm of a Taiwanse strain of Schistosoma japonicum was synthesized and amplified by using the RT-PCR technique, followed by the subcloning in our laboratory. The recombinant proteins were then expressed in Escherichia coli. This recombinant protein has been demonstrated to be a good candidate vaccine. In this study, the immunoefficacies of using CpG motifs as an adjuvant for rGST26 against S. japonicum was determined. Worm reduction rates of 45% and 51% comparing with the adjuvant control group were obtained from mice immunized with rGST26 containing CpG motifs and with rGST26 containing CpG motifs and FIA respectively. Moreover, in the CpG motifs treated groups, the specific IgG antibody titles and cytokine mRNA expressions for IFN-g, IL-2, IL-12, TNF-a, and iNOs significantly increased, while those for IL-4 and IL-10 were suppressed. These results showed that CpG motifs as an adjuvant for rGST26 might be effective to increase the protective immunity of the host against S. japonicum infection by boosting the immune response.

碩士
國立臺灣科技大學
化學工程技術研究所
86
Application of a metal affinity tag constituted of six histidines, 6xHis tag, in immobilized metal affinity chromatography (IMAC) could simplify and integrate the purification and immobilization of recombinant protein together. We have engineered the 6xHis tag to the C-terminus of Schistosoma japonicum glutathione-S-transferase (GST) and created a fustion protein GST/His. The production, activtity, purity, proteolysis, and metal affinity of GST/His were extensively studied, as well as solid state refolding of GST/His The results showed that: 　　(1) GST/His expression using using T7 promoter was better produced than GST expression using tac promoter. 　　(2)The 6xHis tag had no effect on the activity and immunity of GST. 　　(3) The optimization condition to purify GST/His with Ni2+-NTA gel was to include 20 mM imidazole in native washing buffer and 10 mM imidazole in denatruing washing buffer. Beause TALON get had better specificity to GST/His than to GST, no imidazole was required in either washing buffer. 　　(4)The optimal immobilization concentration of GST/His on Ni2+-NTA gel was 5~50 駪/ml, resulting the best specific activity. 　　(5)After storage at either 4℃ or 25℃ for one month, the specific activity of immobilized GST/His did not decay. However, the specific activity of immobilized GST/His decayed 60% after 50 days storage at 25℃. 　　(6)We could recove and purify GST/His from inclusion bodies by solid state refolding procedure, where remove of high concentration of denaturing agent was performed stepwise with GST/His immobilized on Ni2+-NTA gel. 　　(7)The proteolysis of the free form of GST/His began from the C-terminus, while that of immobilized GST/His began from the N-terminus.

碩士
國立陽明大學
寄生蟲學研究所
89
Abstract Schistosomiasis is listed as the second most important tropical parasitic disease by World Health Organization. Among the three major human schistosomes, Schistosoma japonicum is the most pathogenic. The cDNA of 26KDa GST from the total RNA of adult worm of a Taiwanese strain of S. japonicum was synthesized and amplified by using RT-PCR technique, followed by the subcloning in our laboratory. The recombinant proteins were then expressed in Escherichia coli and demonstrated to be a good candidate vaccine. In this studies , the suitability of using 26KDa GST as a vaccine candidate against different strains of S. japonicum was evaluated. The results showed that the cDNA sequences of 26KDa GST among different strain of S. japonicum are similar and encode the same amino acid sequences. The worm reduction rate and cross protective immunity in mice immunized with native and recombinant GST26 from Taiwanese and Hupei strain of the parasite had similar results, indicating that the 26KDa GST can be a vaccine condidate against the different strains of S. japonicum. Passive transfer experiment using sera from the mice immunized with recombinant GST26, as well as monoclonal antibodies, showed a worm reduction rate of 50.2% and 40% respectively, indicating the close relationship between the protective immunity induced by GST26 and antibodies. The level of anti 26KDa GST IgG2b antibody in immunized mice with Freund's adjuvant was significantly higher than in the mice using Alum adjuvant. Also, poor reductive rate in mice group was obtained from the Alum adjuvant. This results showed that different adjuvant combined with subunit vaccine might produce different subclasses of antibodies which will lead to different protective effect.

碩士
國立陽明大學
寄生蟲學研究所
92
Glutathione-S-transferase （GST）is generally recognized as one of the major candidate vaccine antigen. The purpose of this study is to use a transgenic mice model to further investigate the immunoregulatory mechanisms and protective immunity brought about by the 26 KDa GST vaccine antigen from a Taiwanese strain of Schistosoma japonicum. The adoptive immunity induced were investigated with CD4+ T and B cells purified by MACS technique. The results showed that anti 26 KDa GST total IgG and IgG2b antibodies in immunized mice were significantly higher than those in control mice. The CD4+ T cells increased significantly at wk 5 after infection, whereas, the CD8+ T cells showed no statistically significant differences during the experiment. The Th1 subset of CD4+ T cells began to increase at wk 1 while the Th2 subset CD4+ T cells began to rise at wk 12. B cells increased markedly and reached a maximum at wk 4、5 and 12. The Be1 subset of B cells increased significantly from early to acute stages. NK cells began to rise at acute stages. The NK1 subset of NK cells increased significantly at wk 5 while the NK2 subset of NK cells started to increase at chronic stages. The results of adoptive immunity indicated that CD4+ T and B cells induced by SjT 26 KDa rGST could confer a significant protection by reduction in worm burdens.