Transgenic Expression of Decoy Receptor 3 Protects Islets from Spontaneous and Chemical-induced Autoimmune Destruction in Nonobese Diabetic Mice

Decoy receptor 3 (DCR3) halts both Fas ligand– and LIGHT-induced cell deaths, which are required for pancreatic β cell damage in autoimmune diabetes. To directly investigate the therapeutic potential of DCR3 in preventing this disease, we generated transgenic nonobese diabetic mice, which overexpressed DCR3 in β cells. Transgenic DCR3 protected mice from autoimmune and cyclophosphamide-induced diabetes in a dose-dependent manner and significantly reduced the severity of insulitis. Local expression of the transgene did not alter the diabetogenic properties of systemic lymphocytes or the development of T helper 1 or T regulatory cells. The transgenic islets had a higher transplantation success rate and survived for longer than wild-type islets. We have demonstrated for the first time that the immune-evasion function of DCR3 inhibits autoimmunity and that genetic manipulation of grafts may improve the success and survival of islet transplants

A novel trivalent non-Fc anti-CD3 Collabody preferentially induces Th1 cell apoptosis in vitro and long-lasting remission in recent-onset diabetic NOD mice

Specific anti-CD3 treatment is deemed to be a promising therapy for allograft rejection and type 1 diabetes (T1D). Fc receptor (FcR) reduced-binding antibodies, by avoiding adverse effects of Fc and FcR interaction, have good therapeutic potential. We generated a trivalent anti-mouse-CD3 Collabody, h145CSA, by using a triplex-forming collagen-like peptide (Gly-Pro-Pro)10 to drive the trimerization of the Fab fragments. Exposure to h145CSA, but not its bivalent counterparts 145-2C11 and h145chIgGAA (FcR reduced-binding format), upregulates FasL expression on Th1 cells and causes Th1 cell apoptosis. Administration of h145CSA invokes minimal mitogenic effects in mice. The ability of multiple dosing of h145CSA to induce splenic CD4+ T-cell depletion is comparable to bivalent antibodies but is characterized by more rapid CD4+ T-cell recovery kinetics. h145CSA is more potent than h145chIgGAA in inducing long-lasting remission in recent-onset diabetic NOD mice. Its therapeutic effect is accompanied by a significantly lower percentage of CD4+IFNγ+ T cells and a higher Treg/Th1 ratio in pancreatic and mesenteric lymph nodes. The results of our study demonstrate that trivalent non-Fc anti-CD3 Collabody has the potential to be used in the treatment of T1D

Isolation of Hepatoma binding ligands from phage display random peptide libraries

Abstract Phage display technology, consisting of the expression of target sequences on the surface of phage particles and the subsequent affinity selection, is one of the most powerful tools in studying molecular recognition. The purpose of this study was to select peptide ligands with affinity to 3 hepatocellular carcinoma (HCC) cell lines from phage display random peptide libraries, so that the peptide ligands can be applied to the targeting therapy on hepatoma patients. A 39-mer random peptide library was used for sequential selections against 3 HCC cell lines. Without any amplification of the phages during the selection, 4 clones were obtained. Based on the results of subsequent phage ELISA analysis, one clone exhibiting high affinity to the HCC cell lines was subcloned into pET21c, resulting in the production of His-tagged pep-3 peptide in E coli BL21(DE3). Using pep-3-His peptide as the probe followed by treatment with anti-His-tag monoclonal antibodies, a reactive molecule was visualized. Unfortunately pep-3-His not only reacts with HCC cells but also nasopharygenal carcinoma, hypopharygenal carcinoma, hybridoma and human fibroblast. The result indicates that pep-3 although cannot be used for clinical treatment on hepatoma patients, maybe the pep-3 phage can transfect these cells efficiently since its receptors are abundantly existing on their surfaces. Through the process, a method was developed, which directly detect the peptide binding activity without phage particle. This may serve as a model system to detect the affinity between other selected peptides and their targets. I have also searched for the HCC-binding ligands form phage display C7C random peptide library. Clones with consensus HPQ amino acids were obtained in the control experiment using streptavidin as the selector. Although no obvious consensus sequence could be obtained from 15 randomly picked clones after sequential selection with three HCC cell lines, based on the result of streptavidin panning, it is most likely that the clones recovered from panning against three HCC cell lines sequentially should have some degree of affinity to three cell lines. However more experimental data are needed to prove this.中文摘要 噬菌體展示技術包含了將目標序列展示在噬菌體表面的基因庫構築工作以及後續的篩選過程兩大部分。其最大的特色就是在一個噬菌體顆粒上同時帶有目標基因以及蛋白產物，並且可以透過感染E. coli的過程不斷的複製。利用噬菌體展示系統表現逢機合成的寡核酸序列，就可以製備出具有各種結構的月生 月太 庫，稱為噬菌體逢機月生 月太 庫。藉由噬菌體逢機月生 月太 庫能夠展現各式各樣不同結構的特性，我們對肝癌細胞株進行了一系列的親和篩選，希望能夠找到對其具有專一性的多月生 月太 片段，應用在肝癌的標的療法上。首先以39個逢機胺基酸的月生 月太 庫作為來源，進行連續性親和篩選得到了六個疑似為選殖株的菌落，其中四個帶有插入子（insert）。經過ELISA分析，挑選對三株肝癌細胞株具有較高親和力的一個選殖株，將其插入子以His-tag融合蛋白的方式表現，再藉著anti-His-tag單株抗體的偵測觀察該段月生 月太 的結合情形。結果發現這段月生 月太 是一種細胞表面蛋白的配體，其受體存在於我們所測試的肝癌細胞株、鼻咽癌細胞株、融合瘤細胞株以及人類纖維母細胞上。由於不是肝癌細胞專屬的配體，所以無法應用在肝癌的臨床治療上，但是其結合會結合到所有的細胞上的特色，也許能夠應用在以噬菌體轉化哺乳動物細胞的研究。另一方面，我也使用了7個逢機胺基酸的月生 月太 庫，針對相同的目標進行階段性親和篩選。根據對照組實驗的結果，從streptavidin篩選出來的選殖株，隨機挑選了7株進行DNA定序分析，都具有已知的保守（conserve）胺基酸序列HPQ，顯示階段性篩選相當成功。雖然從肝癌細胞株所篩選到的選殖株，在隨機挑選15株進行DNA定序後，仍然沒有發現明顯的保守序列，但是基於對照組的結果，相信這些選殖株也對肝癌細胞具有某種程度的親和性，然而此一推論仍然需要進一步的實驗來證明。目錄 中文摘要…………………………………………………………………1 英文摘要…………………………………………………………………2 前言………………………………………………………………………3 實驗材料…………………………………………………………………9 實驗方法………………………………………………………………..11 結果與討論……………………………………………………………..31 圖表…………………………………………………………………..…42 參考文獻……………………………………………………………..…56 附錄一………………………………………………………………..…61 附錄二………………………………………………………………..…6

Estimation of Tire Mileage and Wear Using Measurement Data

Tire mileage and wear provide important information for vehicle applications. There are more and more studies discussing intelligent tires, but few focus on the role of tire mileage and wear. The conventional tire pressure monitoring system (TPMS) is one of the intelligent tire applications, but there has been no significant advancement in recent years in this regard. In order to increase the additional functions of intelligent tire applications, we propose a method that estimates the mileage and wear information of tires. The proposed method uses a three-axis sensor and a Hall sensor to implement the function. The proposed method also has a low power design to reduce the power consumption of the Hall sensor. The experimental results show the trend of tire wear status, rendering this method effective. This method also requires more accurate mileage information to support tire wear estimation. This experiment found that the correct rate of the proposed mileage estimation method is 99.4% and provides sufficient and correct mileage information for tire wear methods. If this method is used in autonomous vehicle applications, the autonomous control strategy algorithm has more conditions to plan the control strategy. The strategy system processes more meticulous control that increases the safety of autonomous vehicles

Nephrogenic Systemic Fibrosis Associated with Gadolinium Use

Nephrogenic systemic fibrosis (NSF) is an idiopathic, progressive, systemic fibrosis that occurs in patients with renal diseases. Recently, gadolinium-containing contrast (Gd-contrast) has become a suspected causal factor for NFS. This report discusses two female patients with end-stage renal disease, aged 70 and 51 years, respectively, who developed histologically proven NSF after exposure to Gd-contrast. Clinically, both patients were characterized by fibrosis and induration of skin and muscle mainly in the limbs with joint contracture. In the first case, NSF developed gradually after undergoing evaluation by Gd-contrast magnetic resonance imaging (MRI) and subsequent surgery for her urothelial carcinoma. In the second patient, NSF developed after undergoing evaluation by Gd-contrast MRI for her right shoulder bursitis with calcification, and the conditions of NSF continued to worsen after the surgical treatment of this right shoulder lesion. Although the role of Gd-contrast in NSF is still not well known, the correlation in our cases strongly suggests that it should be used with cautioned in patients with end-stage renal disease. Both of our patients underwent surgery before or during the development of NSF, indicating that the surgical procedure may be a contributing factor

Effect of Freshly Isolated Bone Marrow Mononuclear Cells and Cultured Bone Marrow Stromal Cells in Graft Cell Repopulation and Tendon-Bone Healing after Allograft Anterior Cruciate Ligament Reconstruction

Graft cell repopulation and tendon-bone tunnel healing are important after allograft anterior cruciate ligament reconstruction (ACLR). Freshly isolated bone marrow mononuclear cells (BMMNCs) have the advantage of short isolation time during surgery and may enhance tissue regeneration. Thus, we hypothesized that the effect of intra-articular BMMNCs in post-allograft ACLR treatment is comparable to that of cultured bone marrow stromal cells (BMSCs). A rabbit model of hamstring allograft ACLR was used in this study. Animals were randomly assigned to the BMMNC, BMSC, and control groups. Fresh BMMNCs isolated from the iliac crest during surgery and cultured BMSCs at passage four were used in this study. A total of 1 × 107 BMMNCs or BMSCs in 100 µL phosphate-buffered saline were injected into the knee joint immediately after ACLR. The control group was not injected with cells. At two and six weeks post operation, we assessed graft cell repopulation with histological and cell tracking staining (PKH26), and tendon-bone healing with histological micro-computed tomography and immunohistochemical analyses for collagen I and monocyte chemoattractant protein-1 (MCP1). At two weeks post operation, there was no significant difference in the total cell population within the allograft among the three groups. However, the control group showed significantly higher cell population within the allograft than that of BM cell groups at six weeks. Histological examination of proximal tibia revealed that the intra-articular delivered cells infiltrated into the tendon-bone interface. Compared to the control group, the BM cell groups showed broader gaps with interfacial fibrocartilage healing, similar collagen I level, and higher MCP1 expression in the early stage. Micro-CT did not reveal any significant difference among the three groups. BMMNCs and BMSCs had comparable effects on cell repopulation and interfacial allograft-bone healing. Intra-articular BM cells delivery had limited benefits on graft cell repopulation and caused higher inflammation than that in the control group in the early stage, with fibrocartilage formation in the tendon-bone interface after allograft ACLR

Craniofacial and olfactory sensory changes after long-term unilateral nasal obstruction—an animal study using MMP-3-LUC transgenic rats

Abstract Nasal obstruction exerts considerable physiological effects on the respiratory system and craniofacial morphology during the developmental stage. This study used MMP-3-LUC transgenic rats for in vivo tracking of long-term expression in the rat nasal region after unilateral nasal obstruction. Skeletal changes of the craniofacial, nasal, and sinus regions were measured through micro-computed tomography examination and analysis with 3D image processing and calculation. Matrix metalloproteinase-3 and olfactory marker protein expression were also investigated through immunohistochemistry (IHC). Unilateral nasal obstruction significantly reduced the MMP-3 signal in the nasal region of MMP-3-LUC transgenic rats, which was mainly expressed in the respiratory epithelium. Long-term obstruction also caused morphological changes of the craniofacial hard tissue, such as nasal septal deviation, longer inter-jaw distance, and increased maxillary molar dental height. It also caused compensatory growth in olfactory nerve bundles and the olfactory epithelium, as confirmed by IHC. In our study, long-term unilateral nasal obstruction caused nasal septal deviation toward the unobstructed side, hyper divergent facial development including longer molar dental height, and reduced MMP-3 production. However, further investigation is necessary to explore the mechanism in depth