Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo-induced antigen technology (IVIAT). Methods and Results: The convalescent-phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo-induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion-mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo-expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo-induced antigen technology (IVIAT). Methods and Results: The convalescent-phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo-induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion-mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo-expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.

Molecular biotechnology of marine algae in China

Molecular biotechnology of marine algae is referred to as the biotechnology on the identification, modification, production and utilization of marine algal molecules. It involves not only the manipulation of macromolecules such as DNA, RNA and proteins, but also deals with low molecular weight compounds such as secondary metabolites. In the last decade, molecular systematic researches to investigate the relationship and to examine the evolutionary divergence among Chinese marine algae have been carried out by Chinese scientists. For example, RAPD has been widely used in several laboratories to elucidate genetic variations of the reds, such as Porphyra, Gracilaria, Grateloupia and the greens such as Ulva and Enteromorpha. Some important data have been obtained. The study on molecular genetic markers for strain improvement is now in progress. In 1990s, genetic engineering of economic seaweeds such as Laminaria, Undaria, Porphyra, Gracilaria and Grateloupia has been studied in China. For Laminaria japonica, the successfully cultivated kelp in China, a model transformation system has been set up based on the application of plant genetic techniques and knowledge of the algal life history. Progress has been made recently in incorporating a vaccine gene into kelp genome. Evidence has been provided showing the expression of gene products as detectable vaccines. In the present paper, the progress of molecular biotechnological studies of marine algae in China, especially researches on elucidating and manipulating nucleic acids of marine algae, are reviewed.

Isolation and analysis of the vaccine potential of an attenuated Edwardsiella tarda strain

Edwardsiella tarda is an important aquaculture pathogen that can infect a wide range of marine and freshwater fish worldwide. In this study, a modified E. tarda strain, TX5RM, was selected by multiple passages of the pathogenic E. tarda strain TX5 on growth medium containing the antibiotic rifampicin. Compared to the wild type strain, the rifampicin-resistant mutant TX5RM (i) shows drastically increased median lethal dose and reduced capacity to disseminate in and colonize fish tissues and blood; (ii) exhibits slower growth rates when cultured in rich medium or under conditions of iron depletion; and (iii) differs in the production profile of whole-cell proteins. The immunoprotective potential of TX5RM was examined in a Japanese flounder (Paralichthys olivaceus) model as a vaccine delivered via intraperitoneal injection, oral feeding, bath immersion, and oral feeding plus immersion. All the vaccination trials, except those of injection, were performed with a booster at 3-week after the first vaccination. The results showed that TX5RM administered via all four approaches produced significant protection, with the highest protection levels observed with TX5RM administered via oral feeding plus immersion, which were, in terms of relative percent of survival (RPS), 80.6% and 69.4% at 5- and 8-week post-vaccination, respectively. Comparable levels of specific serum antibody production were induced by TX5RM-vaccinated via different routes. Microbiological analyses showed that TX5RM was recovered from the gut, liver, and spleen of the fish at 1-10 days post-oral vaccination and from the spleen, liver, kidney, and blood of the fish at 1-14 days post-immersion vaccination. Taken together, these results indicate that TX5RM is an attenuated E. tarda strain with good vaccine potential and that a combination of oral and immersion vaccinations may be a good choice for the administration of live attenuated vaccines. (C) 2010 Elsevier Ltd. All rights reserved.

鱼类疫苗的研究：I: 海水鱼类病原菌乳化口服疫苗的研究；II：迟缓爱德华氏菌和嗜水气单胞菌外膜蛋白疫苗的研究

本论文的目的是研究几种病原菌口服疫苗接种鱼类的免疫效果，并从常见病原菌株中筛选几个具有较好保护效果的蛋白抗原，利用口服免疫的方式，接种养殖动物，并检测免疫效果。 以10号白油为有机溶剂，采用搅拌与均浆方法制备鳗弧菌M3和SMP1的油乳化二价疫苗，用饵料包埋后以口服途径免疫养殖大菱鲆，评价免疫大菱鲆的免疫应答和疫苗的保护效果。结果显示，以油乳化和未油乳化疫苗分别连续口服免疫大菱鲆一周后，在后肠组织，乳化疫苗刺激产生的非特异性活力、特异性抗体水平均高于未乳化疫苗；而在血清，两种疫苗引起的两种酶的活力、SMP1抗体水平没有变化，但在乳化疫苗免疫的大菱鲆检测到明显高于未免疫对照大菱鲆的M3抗体水平。大菱鲆后肠组织原位杂交结果显示，口服免疫的大菱鲆后肠褶皱有IgM抗体的产生和分布。其中，乳化疫苗免疫大菱鲆的IgM抗体的产生和分布水平高于未乳化疫苗免疫的大菱鲆。攻毒实验显示，乳化疫苗免疫的大菱鲆对M3和SMP1的感染分别获得100%和50%的免疫保护率，而未乳化疫苗获得的免疫保护率分别为57.9%和0%，表明乳化疫苗比未乳化疫苗更有效地保护大菱鲆、抵抗病原的感染。在乳化疫苗免疫持续期的研究中，免疫的大菱鲆后肠在免疫后120天仍能检测到抗体效价，在免疫后90天还能观察到一定的免疫保护效果。免疫30天、60天、90天和120天的大菱鲆分别获得100%、66.7%、36.7%和13.3%的免疫保护力。 以鳗弧菌M3和SMP1、链球菌CF、迟缓爱德华菌SMW7作为细菌抗原制备油乳化多价口服疫苗和轮虫携带疫苗，口服途径免疫养殖大菱鲆与大菱鲆初孵仔鱼。结果显示，在免疫大菱鲆后肠可检测到抗M3抗体水平的提高（P<0.05），而在其胆汁、鳃、中肠、体表黏液、前肠与血清中抗体效价变化与对照组没有显示出差异；没有检测到免疫大菱鲆后肠抗SMP1、SMW7、CF抗体效价。M3浸泡攻毒实验显示，口服免疫的大菱鲆获得了100%的免疫保护力；在M3注射攻毒和SMP1、CF、SMW7浸泡攻毒大菱鲆的实验中，在每个攻毒组中，免疫组大菱鲆开始死亡的时间都要比对照组有不同程度的延迟，但攻毒大菱鲆都发生死亡，不能显示出与对照组的差异。轮虫携带免疫的结果显示，免疫的大菱鲆初孵仔鱼并未获得较好的保护效果，与对照大菱鲆没有体现出差异。 从致病性病嗜水气单胞菌（Aeromonas hydrophila）LSA34克隆并表达ahaI基因和gapA基因，从迟缓爱德华菌（Edwardsiella tarda）LSE40克隆并表达eseB，将所得蛋白分别通过腹腔注射途径免疫大菱鲆，检测蛋白的免疫原性和免疫保护。结果在免疫后7天就可以检测到AhaI、GapA蛋白免疫组大菱鲆产生的抗体，至第40天可以检测到明显的保护性抗体，之后抗体效价增加明显，直至第60天时达到最高值。EseB免疫的大菱鲆第一次免疫后15天就有较高的抗体效价产生，明显高于对照组大菱鲆血清抗体效价，到距第一次免疫60天时，抗体效价达到最高值。攻毒实验显示，与对照组相比，AhaI免疫组和GapA免疫组对LSA34感染的免疫保护力分别为80%和100%；AhaI免疫组和GapA免疫组对LSE40感染的免疫保护力分别为30%和10%。，而对照组牙鲆对人工攻毒不具有保护力。以AhaI和GapA作为疫苗免疫大菱鲆，使大菱鲆获得了对嗜水气单胞菌LSA34较高的免疫保护；而对迟缓爱德华氏菌LSE40的交叉保护能力没有明显提高。EseB免疫的大菱鲆在攻毒实验中并没有显示出较好的保护效果，与对照组相比，只是在死亡时间上有所延迟。 以从致病性嗜水气单胞菌中克隆的ahaI和gapA基因表达出的蛋白为蛋白抗原制备油乳化疫苗，用饵料包埋后以口服途径免疫养殖牙鲆，评价免疫牙鲆的免疫应答和疫苗的保护效果。结果显示，以油乳化和未油乳化疫苗分别免疫牙鲆一周后，在后肠组织，AhaI和GapA乳化疫苗免疫组牙鲆检测到抗体，且分别高于AhaI和GapA未乳化疫苗免疫的牙鲆；而在血清，GapA的两种疫苗引起的GapA抗体水平没有变化；但在AhaI乳化疫苗免疫的牙鲆第21天和第35天的血清中检测到高于未免疫对照牙鲆的AhaI抗体水平，AhaI未乳化疫苗免疫牙鲆血清对照组相比没有检测到AhaI抗体水平的变化。

The rpf gene of Micrococcus luteus encodes an essential secreted growth factor

Galina V. Mukamolova, Obolbek A. Turapov, Konstantin Kazarian, Miroslav Telkov, Arseny S. Kaprelyants, Douglas B. Kell and Michael Young (2002). The rpf gene of Micrococcus luteus encodes an essential secreted growth factor. Molecular Microbiology, 46 (3), 611-621. Sponsorship: BBSRC / Russian Foundation for Basic Research (grant 00-04-48691)/ WHO Global Programme for Vaccines and Immunization / Wellcome Trust RAE2008

Cobertura de la vacuna del virus del papiloma humano en la zona básica de Barañáin

El cáncer de cuello de útero o cáncer de cérvix es el segundo tumor más frecuente en las mujeres del mundo después del de mama y el quinto de todos los canceres. En España, es el sexto más frecuente, constituyendo el 4,8% de los canceres en la mujer. Este tipo de cáncer es el resultado final de una infección no resuelta por el virus del papiloma humano (VPH). Hoy en día, el VPH representa una de las infecciones de trasmisión sexual más común. El 70-80 % de hombres y mujeres sexualmente activos están o han estado expuestos al VPH. El cambio en la conducta sexual de las mujeres ha llevado a que las infecciones de trasmisión sexual sean ahora más frecuentes que en las generaciones anteriores. Hay un inicio más precoz de las primeras relaciones sexuales y un número más elevado de compañeros sexuales. Estos cambios han hecho aumentar la prevalencia del VPH en los últimos años en las mujeres jóvenes de España. La combinación de estrategias de prevención primaria (vacunación contra el VHP) y secundaria (cribado) permitiría reducir la incidencia y la mortalidad del cáncer de cuello de útero. Existen dos vacunas frente al VPH que son Gardasil ® y Cervarix ®. Al tratarse de dos vacunas nuevas, el éxito de la introducción dependerá de la comprensión apropiada de los riesgos y beneficios de la vacuna para prevenir la infección del VPH. Esto se consigue mediante programas efectivos de educación y formación sobre el VPH.

Vírus ao serviço da indústria farmacêutica

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Imunoterapia Tumoral com Células Dendríticas

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Novos sistemas terapêuticos de administração transcutânea

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Novos sistemas terapêuticos para administração nasal de fármacos

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

The Retail Market for Bednets in Kenya: How Well Is It Working?

Background Achieving the goals set by Roll Back Malaria and the Government of Kenya for use of insecticide treated bednets (ITNs) will require that the private retail market for nets and insecticide treatments grow substantially. This paper applies some basic concepts of market structure and pricing to a set of recently-collected retail price data from Kenya in order to answer the question, “How well are Kenyan retail markets for ITNs working?” Methods Data on the availability and prices of ITNs at a wide range of retail outlets throughout Kenya were collected in January 2002, and vendors and manufacturers were interviewed regarding market structure. Findings Untreated nets are manufactured in Kenya by a number of companies and are widely available in large and medium-sized towns. Availability in smaller villages is limited. There is relatively little geographic price variation, and nets can be found at competitive prices in towns and cities. Marketing margins on prices appear to be within normal ranges. No finished nets are imported. Few pre-treated nets or net+treatment combinations are available, with the exception of the subsidized Supanet/Power Tab combination marketed by a donor-funded social marketing project. Conclusions Retail markets for untreated nets in Kenya are well established and appear to be competitive. Markets for treated nets and insecticide treatment kits are not well established. The role of subsidized ITN marketing projects should be monitored to ensure that these projects support, rather than hinder, the development of retail markets.

Household Health and Cocoa Production: A Baseline Survey of Smallholder Farming Households in Western Region, Ghana

Background Chronic illness and premature mortality from malaria, water-borne diseases, and respiratory illnesses have long been known to diminish the welfare of individuals and households in developing countries. Previous research has also shown that chronic diseases among farming populations suppress labor productivity and agricultural output. As the illness and death toll from HIV/AIDS continues to climb in most of sub-Saharan Africa, concern has arisen that the loss of household labor it causes will reduce crop yields, impoverish farming households, intensify malnutrition, and suppress growth in the agricultural sector. If chronic morbidity and premature mortality among individuals in farming households have substantial impacts on household production, and if a large number of households are affected, it is possible that an increase in morbidity and mortality from HIV/AIDS or other diseases could affect national aggregate output and exports. If, on the other hand, the impact at the household farm level is modest, or if relatively few households are affected, there is likely to be little effect on aggregate production across an entire country. Which of these outcomes is more likely in West Africa is unknown. Little rigorous, quantitative research has been published on the impacts of AIDS on smallholder farm production, particularly in West Africa. The handful of studies that have been conducted have looked mainly at small populations in areas of very high HIV prevalence in southern and eastern Africa. Conclusions about how HIV/AIDS, and other causes of chronic morbidity and mortality, are affecting agriculture across the continent cannot be drawn from these studies. In view of the importance of agriculture, and particularly smallholder agriculture, in the economies of most African countries and the scarcity of resources for health interventions, it is valuable to identify, describe, and quantify the impact of chronic morbidity and mortality on smallholder production of important crops in West Africa. One such crop is cocoa. In Ghana, cocoa is a crop of national importance that is produced almost exclusively by smallholder households. In 2003, Ghana was the world’s second-largest producer of cocoa. Cocoa accounted for a quarter of Ghana’s export revenues that year and generated 15 percent of employment. The success and growth of the cocoa industry is thus vital to the country’s overall social and economic development. Study Objectives and Methods In February and March 2005, the Center for International Health and Development of Boston University (CIHD) and the Department of Agricultural Economics and Agribusiness (DAEA) of the University of Ghana, with financial support from the Africa Bureau of the U.S. Agency for International Development and from Mars, Inc., which is a major purchaser of West African cocoa, conducted a survey of a random sample of cocoa farming households in the Western Region of Ghana. The survey documented the extent of chronic morbidity and mortality in cocoa growing households in the Western Region of Ghana, the country’s largest cocoa growing region, and analyzed the impact of morbidity and mortality on cocoa production. It aimed to answer three specific research questions. (1) What is the baseline status of the study population in terms of household size and composition, acute and chronic morbidity, recent mortality, and cocoa production? (2) What is the relationship between household size and cocoa production, and how can this relationship be used to understand the impact of adult mortality and chronic morbidity on the production of cocoa at the household level? The study population was the approximately 42,000 cocoa farming households in the southern part of Ghana’s Western Region. A random sample of households was selected from a roster of eligible households developed from existing administrative information. Under the supervision of the University of Ghana field team, enumerators were graduate students of the Department of Agricultural Economics and Agribusiness or employees of the Cocoa Services Division. A total of 632 eligible farmers participated in the survey. Of these, 610 provided complete responses to all questions needed to complete the multivariate statistical analysis reported here.

An evaluation of reovirus as an effective therapeutic for cancer

Cancer is a global problem. Despite the significant advances made in recent years, a definitively effective therapeutic has yet to be developed. Oncolytic virology has fallen back into favour for the treatment of cancer with several viruses and viral vectors currently under investigation including vesicular stomatitis virus (VSV), adenovirus vectors and herpes simplex virus (HSV) vectors. Reovirus has an advantage over many viral vectors in that its wild-type form is non-pathogenic and will selectively infect transformed cells, particularly those mutated in the Ras pathway. These advantages make Reovirus an ideal candidate as a safe and non-toxic therapeutic. The aim of the first part of this study was to determine the effect, if any, of Reovirus on cell lines derived from cancers of the gastrointestinal tract. These cancers, particularly those of the oesophagus and stomach, have extremely poor prognoses and little improvement has been seen in survival of these patients in recent years. Reovirus as a single therapy showed promising results in cell lines of oesophageal, gastric and colorectal origin. Further study of partially resistant cell lines using a combination of Reovirus and conventional therapies, either chemotherapy or radiation, showed that a multi-modal approach to therapy is possible with Reovirus and no antagonism between Reovirus and other treatments was observed. The second part of this study focused on investigating a novel use of Reovirus in an in vivo setting. Cancer vaccination or the use of vaccines in cancer therapy is gaining momentum and success has been seen both in a prophylactic approach and a therapeutic approach. A cell-based Reovirus vaccine was used in both these approaches with encouraging success. When used as a prophylactic vaccine tumour development was subsequently inhibited even upon exposure to a tumorigenic dose of cells. The use of the cell-based Reovirus vaccine as a therapeutic for established tumours showed significant delay in tumour growth and a prolongation of survival in all models. This study has proven that Reovirus is an effective therapeutic in a range of cancers and the successful use of a cell-based Reovirus vaccine leads the way for new advancements in cancer immunotherapy.

Identification of novel innate immune mechanisms regulating inflammation in the gastrointestinal tract

The Gastro-Intestinal (GI) tract is a unique region in the body. Our innate immune system retains a fine homeostatic balance between avoiding inappropriate inflammatory responses against the myriad commensal microbes residing in the gut while also remaining active enough to prevent invasive pathogenic attack. The intestinal epithelium represents the frontline of this interface. It has long been known to act as a physical barrier preventing the lumenal bacteria of the gastro-intestinal tract from activating an inflammatory immune response in the immune cells of the underlying mucosa. However, in recent years, an appreciation has grown surrounding the role played by the intestinal epithelium in regulating innate immune responses, both in the prevention of infection and in maintaining a homeostatic environment through modulation of innate immune signalling systems. The aim of this thesis was to identify novel innate immune mechanisms regulating inflammation in the GI tract. To achieve this aim, we chose several aspects of regulatory mechanisms utilised in this region by the innate immune system. We identified several commensal strains of bacteria expressing proteins containing signalling domains used by Pattern Recognition Receptors (PRRs) of the innate immune system. Three such bacterial proteins were studied for their potentially subversive roles in host innate immune signalling as a means of regulating homeostasis in the GI tract. We also examined differential responses to PRR activation depending on their sub-cellular localisation. This was investigated based on reports that apical Toll-Like Receptor (TLR) 9 activation resulted in abrogation of inflammatory responses mediated by other TLRs in Intestinal Epithelial Cells (IECs) such as basolateral TLR4 activation. Using the well-studied invasive intra-cellular pathogen Listeria monocytogenes as a model for infection, we also used a PRR siRNA library screening technique to identify novel PRRs used by IECs in both inhibition and activation of inflammatory responses. Many of the PRRs identified in this screen were previously believed not to be expressed in IECs. Furthermore, the same study has led to the identification of the previously uncharacterised TLR10 as a functional inflammatory receptor of IECs. Further analysis revealed a similar role in macrophages where it was shown to respond to intracellular and motile pathogens such as Gram-positive L.monocytogenes and Gram negative Salmonella typhimurium. TLR10 expression in IECs was predominantly intracellular. This is likely in order to avoid inappropriate inflammatory activation through the recognition of commensal microbial antigens on the apical cell surface of IECs. Moreover, these results have revealed a more complex network of innate immune signalling mechanisms involved in both activating and inhibiting inflammatory responses in IECs than was previously believed. This contribution to our understanding of innate immune regulation in this region has several direct and indirect benefits. The identification of several novel PRRs involved in activating and inhibiting inflammation in the GI tract may be used as novel therapeutic targets in the treatment of disease; both for inducing tolerance and reducing inflammation, or indeed, as targets for adjuvant activation in the development of oral vaccines against pathogenic attack.