Assessment of hypermucoviscosity as a virulence factor for experimental Klebsiella pneumoniae infections: comparative virulence analysis with hypermucoviscosity-negative strain

Background: Klebsiella pneumoniae displaying the hypermucoviscosity (HV) phenotype are considered more virulent than HV-negative strains. Nevertheless, the emergence of tissue-abscesses-associated HV-negative isolates motivated us to re-evaluate the role of HV-phenotype. Results: Instead of genetically manipulating the HV-phenotype of K. pneumoniae, we selected two clinically isolated K1 strains, 1112 (HV-positive) and 1084 (HV-negative), to avoid possible interference from defects in the capsule. These well-encapsulated strains with similar genetic backgrounds were used for comparative analysis of bacterial virulence in a pneumoniae or a liver abscess model generated in either naive or diabetic mice. In the pneumonia model, the HV-positive strain 1112 proliferated to higher loads in the lungs and blood of naive mice, but was less prone to disseminate into the blood of diabetic mice compared to the HV-negative strain 1084. In the liver abscess model, 1084 was as potent as 1112 in inducing liver abscesses in both the naive and diabetic mice. The 1084-infected diabetic mice were more inclined to develop bacteremia and had a higher mortality rate than those infected by 1112. A mini-Tn5 mutant of 1112, isolated due to its loss of HV-phenotype, was avirulent to mice. Conclusion: These results indicate that the HV-phenotype is required for the virulence of the clinically isolated HV-positive strain 1112. The superior ability of the HV-negative stain 1084 over 1112 to cause bacteremia in diabetic mice suggests that factors other than the HV phenotype were required for the systemic dissemination of K. pneumoniae in an immunocompromised setting

Mechanism Study of Lung Tumorigenesis Induced by Human Vegf Over-Expression in Transgenic Mice Using Mouse Cdna and Cpg Islands Microarray

Vascular endothelial growth factor (VEGF) family members consist of VEGF-A, VEGF-B,VEGF-C, VEGF-D, VEGF-E and placental growth factor (PlGF). The VEGF-A isoform plays acentral role in vascular development. VEGF-A and its receptor are involved in carcinogenesis,invasion and distant metastasis as well as tumor angiogenesis. Clara cell secretory protein (CCSP)is a protein that is secreted by nonciliated, nonmucous clara cells in the pulmonary airway. CCSPplays a protective role against pulmonary inflammatory response. CCSP is a potent naturalimmunosuppressor and anti-inflammatory agent. In this study, a transgene of CCSP-Vegf-A165-SV40poly(A) was constructed for the production of lung-specific overexpressing VEGF-A165 transgenicmice. The previous results showed that human VEGF-A overexpression in the lung tissue willinitiate a tumor growth in the transgenic mice. The object of this research proposal is to investigatethe mechanism of pulmonary tumorigenesis induced by human VEGF-A overexpression in the lungtissue of transgenic mice using cDNA microarray and CpG islands genechip. The whole project wasorganized to be finished within 3 years. In the first year, the study will focus on the establishment ofdifferent lines of transgenic mice with high- and low-level expression of VEGF-A protein in lungClara cells. The time-course of pulmonary adenocarcinoma development will be dissected bypathological analysis. The different transgenic mice lines will be used to determine the humanVEGF-A mRNA and protein expression level. The molecular properties of vascular angiogenesis inthe tumor mass will be studied. In the second year, a home-made animal in vivo image system willbe performed to observe the synergetic effect of lung tumorigenesis after nicotine-derived NNKtreatment in the VEGF-A transgenic mice. In addition, the genome-wide gene expression profiles oftransgenic lung tumor will be analyzed by the 43K mouse cDNA microarray hybridization. Thetumorigenic pathways including cell survival, oncogenesis, anti-apoptosis, cell proliferation andmigration will be examined based on microarray information. In the third year, the whole genomeof epigenetic changes during VEGF-A induced lung adenocarcinoma in the transgenic mice will befurther investigated using a mouse CpG islands genechip. Hypermethylated tumor suppressor geneswill be isolated to evaluate their function contributing in the inhibition of lung tumorigenesis.Whenever this project has been successfully done, it could provide the groundwork for theunderstanding molecular mechanism of human VEGF-A in the promotion of pulmonarytumorigenesis.應用基因轉殖科技產製癌症模式動物，已成為腫瘤生物學一項重要的研究工具。血管內皮成長因子(vascular endothelial growth factor, VEGF)之基因家族包括PIGF、VEGF-A、-B、-C、-D、-E共六個成員，其中VEGF-A分子具有促進血管增生(angiogenesis)，增加血管壁通透性，促進細胞存活以及促進細胞遷移之功能，並於許多試驗中得知於腫瘤生長過程中VEGF-A表現量會聚集增加，因而促進腫瘤生長與腫瘤轉移(metastasis)之能力。在肺癌的研究中發現最主要的肺腺癌多數衍生自細支氣管非纖維性的Clara細胞，而該細胞會釋泌一種特殊的分泌蛋白(Clara cell secretory protein; CCSP)，係一種保護性蛋白，具有抑制免疫反應，降低發炎反應之效能。基於上述特徵，本研究計畫採用小鼠CCSP 之啟動子銜接人類VEGF-A165 基因建構一個轉殖基因[CCSP-Vegf-A165-SV40 poly(A)]，經鼠胚基因顯微注射建立一套於鼠肺部細支氣管上皮專一性表現VEGF-A之基因轉殖小鼠動物模式，初步試驗結果顯示VEGF-A過量表現可直接誘發基因轉殖小鼠於12月齡時產生肺部腫瘤，此一新穎性研究發現提供一個良好肺癌動物模式，作為探討VEGF-A誘導肺腫瘤生成之機轉。本計畫擬分三年逐步完成：第一年之重點於不同VEGF-A表現量之基因轉殖小鼠品系的建立，觀察肺部腫瘤生成的速率與VEGF-A含量的關係、利用組織病理切片分析肺部腫瘤組織之癌症特性、並度量血管新生之效應。第二年之重點則分別投予尼古丁衍生物NNK致突變劑，給予雙重誘癌因子探討VEGF-A基因轉殖小鼠之肺癌加成的衍生速度，並且利用動物活體螢光影像分析系統進行即時之腫瘤影像分析；此外，將以小鼠43K cDNA基因晶片進行基因轉殖肺癌鼠之全基因體表現分析，以瞭解VEGF-A誘導肺腫瘤生成過程中所牽涉之訊息傳遞途徑。第三年之重點則著重於利用CpG islands甲基化基因晶片探討VEGF-A誘導肺癌產生過程中所更動之基因體甲基異常修飾的腫瘤抑制基因群，並測試此群抑癌基因對於肺部之保護功能，本研究亦將投予基因轉殖肺癌鼠VEGF-A抗體與抑制劑，觀察小鼠肺部腫瘤之抑制程度。透過本計畫之順利執行，預期將可釐清人類VEGF-A在誘發肺部腫瘤生成過程中所扮演的角色，對於未來肺腺癌的預防與醫療將有所助益

一種研究肺部腫瘤之動物模式的製造方法及其用途

本發明所揭一種研究肺部腫瘤之動物模式的製造方法及其用途，其係以基因工程製備一非人類的基因轉殖動物，使該非人類的基因轉殖動物能夠於其肺部專一性表現人類A165型血管內皮新生因子(Vascular endothelial growth factor A165,VEGF-A165)。藉由人類A165型血管內皮新生因子之表現，使該非人類的基因轉殖動物之肺部細胞會有發炎反應或是腫瘤之產生，藉此可用以作為肺腺癌作為分析肺腺癌調控及生成機制之動物模式

Production of Biologically Active B-Domain Deleted Human Factor VIII in the Milk of Transgenic Animals Driven by Mammary-Specific Expression Cassettes

血友病(Hemophilia)為遺傳性凝血功能異常之疾病，患者中約有80%為A 型血友病(Hemophilia A)，人類第八凝血因子(human factor VIII; hFVIII)係體內凝血過程不可或缺之重要成分，人體一旦缺乏或減少第八凝血因子，將會引發不同程度之凝血功能問題，而導致所謂的A 型血友病。目前血友病患係以血漿製劑或由正常人血液中純化之hFVIII 蛋白進行針劑補充療法，然而因費用昂貴，以及無法避免血源病毒感染（如HBV 或HIV）之潛在危險，因此利用生物科技發展出以非血源性的hFVIII 醫藥蛋白生產平台更顯迫切。又根據資料統計，A 型血友病好發於東方人，因此在東方國家第八凝血因子蛋白的短缺是醫療衛生上的一大問題，所以大量製造重組FVIII 醫藥蛋白必為當務之急。本研究室的先前研究成果以利用基因轉殖小鼠模式，成功在乳腺組織生產人類第八凝血因子，並於乳汁中檢測出含有7.0–50.2ug/ml 的FVIII 重組蛋白，其濃度為正常血液含量的35-200 倍之多，並且具有13.4U/ml 的凝血活性。在此一基礎下，本計畫將進一步以基因轉殖猪為動物模式（目前亦產製出No. 235 與No. 335 兩個品系），應用基因工程技術改造全長的FVIII 基因為B-區刪除的FVIII 基因(B-domain deleted FVIII; FVIII△B)，探討其提升重組蛋白產製的效果，三年期計畫之重點研究規劃，包括：第一年度將建構三種不同的FVIII轉殖基因，分別為全長FVIII cDNA、B-區刪除FVIII cDNA (FVIII△B)、及FVIII△B 加上His-Tag 等形式，並於基因轉殖小鼠系統中進行重組蛋白質之生產效能與純化效率分析；第二年度將針對已產製的FVIII△B 轉殖基因猪，建立不同的系譜族群，進行染色體基因定位與基因表現分析，並於猪乳樣中分析重組蛋白之含量；第三年則著重於基因轉殖猪乳之蛋白質體分析、FVIII△B 重組蛋白之純化與活性測定、並以A 型血友病基因刪除鼠為動物模式，進行活體之凝血功能確效分析。評估於基因轉殖乳汁中大量生產並純化人類第八凝血因子之可行性，以期降低昂貴FVIII 醫藥蛋白之生產成本，未來可開發成應用於A 型血友病治療之新式醫藥蛋白生產來源。Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency orabnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with wholeplasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and thecost of highly purified FVIII are the major factors that restrict prophylaxis in hemophiliatherapy. One of the challenges created by the biotechnology revolution is the development ofmethods for the economical production of highly purified proteins in large scales. Recentdevelopments indicate that manipulating milk composition using transgenesis has focusedmainly on the mammary gland as a bioreactor to produce pharmaceuticals. In our pilot study,a fusion gene containing bovine -lactalbumin and human FVIII full-length cDNA (7.2-kb)was constructed for microinjection into the pronuclei of newly fertilized mouse eggs. Totalnumber of 9 (4 females/ 5 males) mice produced were confirmed to be successfully integratedand stably germ-line transmitted with the LA-hFVIII fusion gene. Western-blot analysisagainst milk samples have further shown that the recombinant hFVIII was secreted into themilk of the transgenic mice. The concentrations of rFVIII ranged from 7.0 to 50.2 g/ml, over35- to 200-fold higher than that in normal human plasma. Up to 13.4 U/ml of rFVIII wasdetected in an assay in which rFVIII restored normal clotting activity to FVIII-deficienthuman plasma. In this 3 years proposal, to improve the large-scale production of rFVIIIpharmaceutical protein from transgenic milks, we will attempt to generate B-domain FVIII(FVIII △ B) gene constructs and mammary gland-specific expression in the milks oftransgenic pigs during lactation. A fast protein liquid chromatography (FPLC) will be appliedto large-scale purify recombinant FVIII△B protein and the in vivo clotting functional assaywill also be validated in FVIII-knockout mice. Whenever this project has been successfullydone, it could provide a novel strategy to large-scale production of a potential rFVIIIpharmaceutical drug use in Hemophilia A therapy