CD4+ T Cell-Derived IL-2 Signals during Early Priming Advances Primary CD8+ T Cell Responses

Stimulating naïve CD8+ T cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. In this study, we show that the activation and differentiation of CD8+ T cells require IL-2 provided by activated CD4+ T cells at the initial priming stage within 0–2.5 hours after stimulation. This critical IL-2 signal from CD4+ cells is mediated through the IL-2Rβγ of CD8+ cells, which is independent of IL-2Rα. The activation of IL-2 signaling advances the restriction point of the cell cycle, and thereby expedites the entry of antigen-stimulated CD8+ T-cell into the S phase. Besides promoting cell proliferation, IL-2 stimulation increases the amount of IFNγ and granzyme B produced by CD8+ T cells. Furthermore, IL-2 at priming enhances the ability of P14 effector cells generated by antigen activation to eradicate B16.gp33 tumors in vivo. Therefore, our studies demonstrate that a full CD8+ T-cell response is elicited by a critical temporal function of IL-2 released from CD4+ T cells, providing mechanistic insights into the regulation of CD8+ T cell activation and differentiation

Measurement of event-shape observables in Z→ℓ+ℓ− events in pp collisions at √ s=7 TeV with the ATLAS detector at the LHC

Event-shape observables measured using charged particles in inclusive $Z$-boson events are presented, using the electron and muon decay modes of the $Z$ bosons. The measurements are based on an integrated luminosity of $1.1 {\rm fb}^{-1}$ of proton--proton collisions recorded by the ATLAS detector at the LHC at a centre-of-mass energy $\sqrt{s}=7$ TeV. Charged-particle distributions, excluding the lepton--antilepton pair from the $Z$-boson decay, are measured in different ranges of transverse momentum of the $Z$ boson. Distributions include multiplicity, scalar sum of transverse momenta, beam thrust, transverse thrust, spherocity, and $\mathcal{F}$-parameter, which are in particular sensitive to properties of the underlying event at small values of the $Z$-boson transverse momentum. The Sherpa event generator shows larger deviations from the measured observables than Pythia8 and Herwig7. Typically, all three Monte Carlo generators provide predictions that are in better agreement with the data at high $Z$-boson transverse momenta than at low $Z$-boson transverse momenta and for the observables that are less sensitive to the number of charged particles in the event.Comment: 36 pages plus author list + cover page (54 pages total), 14 figures, 4 tables, submitted to EPJC, All figures including auxiliary figures are available at http://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/STDM-2014-0

Helicobacter Pylori Infection and Caga Protein Translocation in Human Primary Gastric Epithelial Cell Culture

Background: Increasing evidence has shown that Helicobacter pylori CagA protein translocation into gastric epithelial cells plays an important role in the development of gastric inflammation and malignancy. Translocated CagA undergoes tyrosine phosphorylation in gastric adenocarcinoma cell line cells, and CagA involves disruption of cellular apical–junction complex in Madin–Darby canine kidney cells. Methods: To elucidate whether these events take place in normal human gastric epithelium, we infected human primary gastric epithelial cells with H. pylori. Results: Our results demonstrate that CagA protein was translocated into primary gastric epithelial cells and tyrosine phosphorylated . The translocated CagA induces cytoskeletal rearrangement and the disruption of tight junctions in primary gastric epithelial cells. Conclusions: This study provides direct evidence of the modulation of gastric epithelial cells by CagA protein translocation, and advances our understanding of the pathogenesis of H. pylori infection

CagA Translocation and Gastric Epithelial Tight Junction Destruction: Possible Carcinogenesis of Helicobacter pylor

雖然世界衛生組織基於流行病學的研究成果，認為幽門螺旋桿菌與人類胃部惡性腫瘤生成有關，特別是與人類胃腺癌形成有著相當密切的關係，已經將幽門螺旋桿菌列為第一類人類致癌因子，但是確實的致病機轉仍尚未被清楚的分析瞭解。由於幽門螺旋桿菌CagA蛋白可以被轉位進入胃上皮細胞，並且在酪氨酸磷酸化模體發生磷酸化，這現象被認為與致癌機轉有關。 所以我們研究CagA酪氨酸磷酸化、酪氨酸磷酸化模體變異性是否與增加致癌風險有關，另外我們建立初次培養人類胃上皮細胞感染幽門螺旋桿菌的模式，希望能比胃癌上皮細胞或非胃上皮細胞的模式，提供更接近人類活體感染的研究方式。 我們研究發現由胃腺癌病人的胃中所分離培養得來的幽門螺旋桿菌菌株發生CagA蛋白酪氨酸磷酸化的比例，比起自一般性胃炎病人的胃中分離得到的菌株，顯著要高了許多（27/29:15/29）。而CagA酪氨酸磷酸化模體變異性與CagA蛋白酪氨酸磷酸化並非絕對相關。所以發生CagA蛋白酪氨酸磷酸化現象與增加致癌風險有關，或者是功能完整的第四型分泌系統與致癌機轉有關。我們也建立初次培養人類胃上皮細胞感染幽門螺旋桿菌的模式，並且發現CagA蛋白酪氨酸磷酸化產生，與細胞形態變化有關；也發現CagA蛋白轉位至胃上皮細胞可以造成細胞緊密接合的破壞。 經由研究顯示能夠被轉位進入胃上皮細胞，並且被磷酸化的CagA蛋白會增加致癌風險，另外由CagA轉位造成細胞緊密接合的破壞而產生的可能致癌機轉，以初次培養上皮細胞的模式來看，是極有可能發生人類活體中。本研究提供對CagA轉位及被磷酸化的致癌機轉更進一步的瞭解。Background Tyrosine phosphorylation of Helicobacter pylori cytotoxin-associated protein (CagA) of in gastric epithelial cells is reported. Phosphorylated CagA trigger downstream cellular signaling that alters cell morphology and proliferation and induce pro-inflammatory process. Translocated CagA involves epithelial-to-mesenchymal transition and disruption of cellular apical-junction complex in MDCK cells. These events may play important role in the development of malignancy. To clarify these issues, our aims in this study are: 1) to examine the occurrence of CagA tyrosine phosphorylation in H. pylori strains isolated from differnet patients with gastric adenocarcinoma and gastritis, 2) to analyze the relationship between the diversity of tyrosine phosphorylation motifs and the presence of CagA tyrosine phosphorylation, 3) to establish primary human gastric epithelial cell for better understanding effects of CagA protein in non-cancer epithelial cells and 4) to observe cellular junction change in primary human gastric epithelial cell but not non-gastric epithelial cells . Methods Fifty-eight clinical isolates of H. pylori from patients with gastric adenocarcinoma (29 cases) and gastritis (29 cases) were studied for CagA tyrosine phosphorylation by Western blotting. Sequence diversity of tyrosine phosphorylation motifs was analysed among positive- or negative-CagA tyrosine phosphorylation isolates. To elucidate whether events of CagA translocation and phosphorylation take place in normal human gastric epithelium, we infected human primary gastric epithelial cells with H. pylori. Tight junction proteins: ZO-1 and occludin changes were observed by confocal microscopy. Results Positive CagA tyrosine phosphorylation was found in 93.1% (27 of 29) of strains from gastric adenocarcinoma patients and 51.7% (15 of 29) of strains from gastritis patients, (p<0.001). Intact motifs were found in H. pylori isolates with CagA tyrosine phosphorylation. Of the 16 negative CagA tyrosine phosphorylation isolates, intact tyrosine phosphorylation motifs were found in 15 isolates. Our results also demonstrate that CagA protein was translocated into primary gastric epithelial cells and tyrosine phosphorylated. The translocated CagA induces cytoskeleton rearrangement and the disruption of tight junctions in primary gastric epithelial cells. Conclusions CagA tyrosine phosphorylation, which is significantly greater in strains from gastric adenocarcinoma patients, may play a role in gastric carcinogenesis, and could be a better marker of more virulent strains than the cag pathogenicity island in Asia, where the cag pathogenicity island is present in nearly all H. pylori strains. Sequence diversity of tyrosine phosphorylation motifs on CagA was not related to the presence of tyrosine phosphorylation. The absence of tyrosine phosphorylation motif might result in negative tyrosine phosphorylation phenotypes, but such motifs are not the sole factors associated with CagA tyrosine phosphorylation. The establishment of primary culture gastric epithelial cells also provides direct evidence of the modulation of gastric epithelial cells by CagA translocation, and advances our understanding of the carcinogenesis of H. pylori infection.第一章 緒論 ……………………………………………… 1 (1.1) 幽門螺旋桿菌感染與人類上消化道疾病 …………… 2 (1.2) 幽門螺旋桿菌的致病因子 …………………………… 3 (1.2.1) 鞭毛(flagella) …………………………… 4 (1.2.2) 尿素酶(urease) …………………………… 4 (1.2.3) 黏著因子(adhesin) ……………………… 5 (1.2.4) 發炎反應因子與細胞毒素………………… 5 (1.2.5) 第四型分泌系統(Type IV secretion system) …………………………………… 6 (1.3) 幽門螺旋桿菌CagA蛋白及第四型分泌系統與致病機轉………………………………………………………… 6 (1.3.1) cagA基因在臨床疾病上的重要性 ………… 7 (1.3.2) CagA蛋白磷酸化對胃上皮細胞的影響 …… 7 (1.3.3) 幽門螺旋桿菌第四型分泌系統對胃上皮細胞的影響…………………………………… 8 (1.3.4) CagA蛋白的酪氨酸磷酸化 ………………… 8 (1.3.5) CagA蛋白的酪氨酸磷酸化模體 …………… 9 (1.3.6) CagA蛋白的轉位與上皮細胞緊密間隙變化 9 (1.4) 幽門螺旋桿菌感染與胃癌形成的相關機制…………… 10 (1.4.1) 自由基（Free radicals）的影響 ………… 10 (1.4.2) 細胞凋亡（Apoptosis）與細胞增生（proliferation）的影響……………………… 11 (1.4.3) 細胞分化及小腸化生（Intestinal metaplasia） 11 (1.5) 本研究的目標及重要性………………………………… 12 (1.6) 本研究的可能結果……………………………………… 13 第二章 材料與方法 …………………………………………… 15 (2.1) 材料菌株 ………………………………………………… 16 (2.1.1) 菌株：幽門螺旋桿菌 16 (2.1.2) 菌株：大腸桿菌 17 (2.1.3) 細胞株 18 (2.1.4) 質體 19 (2.1.5) 培養基 20 (2.1.6) 引子 21 (2.1.7) 抗體 23 (2.2) 幽門螺旋桿菌cagE與cagA基因的剔除（knock out） 25 (2.3) 幽門螺旋桿菌cagA基因的補回（complementation）… 27 (2.4) 酪氨酸磷酸化模體DNA定序…………………………… 28 (2.5) 人類胃上皮細胞之培養………………………………… 29 (2.6) 細胞株培養……………………………………………… 30 (2.7) 初次培養人類胃上皮細胞之確認……………………… 31 (2.8) 以幽門螺旋桿菌感染胃上皮細胞……………………… 32 (2.9) 共軛焦顯微鏡及免疫螢光染色(immunofluorescence） 33 (2.10) 免疫（西方）墨點法檢測……………………………… 34 (2.11) 資料統計分…………………………………………… 35 第三章 結果 …………………………………………………… 36 (3.1) 幽門螺旋桿菌cagA及cagE基因剔除………………… 37 (3.2) 檢測CagA蛋白及酪氨酸磷酸化CagA蛋白 …………… 38 (3.3) 研究個體的特性分析與CagA蛋白酪氨酸磷酸化…… 40 (3.4) 分析酪氨酸磷酸化模體之基因序列變異……………… 43 (3.5) 建立初次培養的人類為上皮細胞……………………… 45 (3.6) 初次培養人類胃上皮細胞感染幽門螺旋桿菌，CagA轉位與磷酸化……………………………………………… 46 (3.7) 幽門螺旋桿菌感染後初次培養人類胃上皮細胞的型態變化……………………………………………………… 47 (3.8) 幽門螺旋桿菌CagA引發初次培養胃上皮細胞緊密間隙破壞 ………………………………………………… 48 (3.9) 幽門螺旋桿菌cagA基因補回（complementation）的影響 …………………………………………………… 49 第四章 討論 …………………………………………………… 50 (4.1) 幽門螺旋桿菌CagA蛋白酪氨酸磷酸化與病人年齡… 51 (4.2) 幽門螺旋桿菌CagA蛋白酪氨酸磷酸化與cag-PAI的 相似意義………………………………………………… 52 (4.2.1) CagA蛋白酪氨酸磷酸化與TPMs變異 52 (4.2.2) CagA蛋白TPMs變異與疾病生成的關係 53 (4.2.3) CagA蛋白的酪氨酸磷酸化與蛋白轉位 54 (4.3) 胃上皮細胞培養與幽門螺旋桿菌致病機制…………… 56 (4.4) CagA蛋白轉位進入初次培養的胃上皮細胞………… 57 (4.5) CagA蛋白轉位進入初次培養的胃上皮細胞與緊密接合損壞 ………………………………………………… 58 (4.6) 胃癌細胞株與初次培養的胃上皮細胞的差異………… 60 英文總結 ………………………………………………………… 61 參考文獻 ………………………………………………………… 64 圖表 ……………………………………………………………… 78 附錄 ……………………………………………………………… 10

Caga Tyrosine Phosphorylation in Gastric Epithelial Cells Caused by Helicobacter Pylori in Patients with Gastric Adenocarcinoma

Background. Tyrosine phosphorylation of Helicobacter pylori cytotoxin- associated protein of in gastric epithelial cells is reported. The goals of this study are first to examine the occurrence of CagA tyrosine phosphorylation in H. pylori strains isolated from patients with gastric adenocarcinoma and gastritis, and second to clarify the relationship between the diversity of tyrosine phosphorylation motifs and the presence of CagA tyrosine phosphorylation. Methods. Fifty-eight clinical isolates of H. Pylori from patients with gastric adenocarcinoma (29 cases) and gastritis (29 cases) were studied for CagA tyrosine phosphorylation by Western blotting. Sequence diversity of tyrosine phosphorylation motifs was analysed among positive- or negative-CagA tyrosine phosphorylation isolates. Results. Positive CagA tyrosine phosphorylation was found in 93. 1% ( 27 of 29) of strains from gastric adenocarcinoma patients and 51.7% ( 15 of 29) of strains from gastritis patients (p<0 .001). Intact motifs were found in H. pylori isolates with CagA tyrosine phosphorylation. Of the 16 negative CagA tyrosine phosphorylation isolates, intact tyrosine phosphorylation motifs were found in 15 isolates. Conclusions. CagA tyrosine phosphorylation, which is significantly greater in strains from gastric adenocarcinoma patients, may play a role in gastric carcinogenesis , and could be a better marker of more virulent strains than the cag pathogenicity island in Asia, where the cag pathogenicity island is present in nearly all H. pylori strains. Sequence diversity of tyrosine phosphorylation motifs on CagA was not related to the presence of tyrosine phosphorylation. The absence of tyrosine phosphorylation motif might result in negative tyrosine phosphorylation phenotypes, but such motifs are not the sole factors associated with CagA tyrosine phosphorylation

A repeating pattern based Query-by-Humming fuzzy system for polyphonic melody retrieval

Query-by-Humming involves retrieving music with a melody that matches the hummed query. An improved Query-by-Humming system for extracting pitch contour information based on a fuzzy inference model is introduced. In addition, an improved content-based music repeating pattern extraction model is introduced. Our bar-indexing method can extract the melody, identify repeating patterns and handle polyphonic MIDI files. To verify the effectiveness of the system, 15 volunteers recorded queries that were fed as input to the system and the longest common subsequence (LCS) was used to identify the most related top N matches. The system achieves 70% accuracy among the top 5 items retrieved

Abdominal obesity and hypertension are correlated with health-related quality of life in Taiwanese adults with metabolic syndrome

ObjectiveMetabolic syndrome (MetS) gains more attention due to high prevalence of obesity, diabetes and hypertension among adults. Although obesity, diabetes and hypertension can certainly compromise health-related quality of life (HRQoL), the correlations of sociodemographic factors, quality of life and MetS remains unclear. This study aims to investigate the association between HRQoL and MetS in an Asian community of the sociodemographic characteristics.Research design and methodsWe performed a cross-sectional study by recruiting 2588 Taiwanese patients aged ≥30 years between August 2015 and August 2017. Sociodemographic data and anthropometric variables were obtained from medical records and physical examination. Meanwhile, HRQoL was assessed by 36-Item Short-Form Health Survey questionnaires.ResultsThe overall prevalence of MetS was 32.8%. Multivariate analysis revealed that age ≥65 years (OR=1.987, p&lt;0.001), body mass index (BMI) ≥24 kg/m2 (OR=7.958, p&lt;0.001), low educational level (OR=1.429, p=0.014), bad self-perceived health status (OR=1.315, p=0.01), and betel nut usage (OR=1.457, p=0.048) were associated with the development of MetS. For patients with MetS, the physical and mental health domains of HRQoL are negatively correlated with abdominal obesity and hypertension, respectively.ConclusionsAdult MetS in Taiwan was associated with certain sociodemographic factors including older age, high BMI, low educational level, bad self-perceived health status, and betel nut use. Abdominal obesity and hypertension was correlated with HRQoL in patients with MetS

The objective structured clinical examination as an assessment strategy for clinical competence in novice nursing practitioners in Taiwan

Abstract Background The conventional written tests and professional assessment have limitation in fair judgement of clinical competence. Because the examiners may not have total objectivity and may lack standardization throughout the assessment process. We sought to design a valid method of competence assessment in medical and nursing specialties. This work was aimed to develop an Objective Structured Clinical Exam (OSCE) to evaluate novice nursing practitioners’ clinical competency, work stress, professional confidence, and career satisfaction. Methods A Quasi-experimental study (pre-post). Fifty-five novice nursing practitioners received the OSCE three-months following their graduation, which consisted of four stations: history taking, physical examination, problem-directed management, interpersonal communication, and the required techniques of related procedures. The examiners had to complete an assessment checklist, and the participants had to complete a pre-post questionnaire (modified from a Nursing Competency Questionnaire, a Stress scale, and Satisfaction with Learning scale). Results Among the novice nursing practitioners, 41 of them (74.5 %) passed the exam with a mean score of 61.38 ± 8.34. There was a significantly higher passing rate among nurses who were working in medical-surgical wards (85.7 %) and the intensive care unit-emergency department (77.8 %) compared to novice nursing practitioners working in other units. All the novice nursing practitioners at Station A had poor performance in assessing patients with a fever. OSCE performance was more associated with educational attainment and work unit, rather than the gender. Finally, the participants showed statistically significant increases in their clinical competency, confidence in their professional competence, satisfaction with the clinical practice, and decreased work stress after the OSCE. Conclusions We found that the OSCE process had a positive educational effect, in providing a meaningful and accurate assessment of the competence of novice nursing practitioners. An appropriate OSCE program is vital for novice nursing practitioners, educators, and administrators. The effective application of OSCEs can help novice nursing practitioners gain confidence in their clinical skills

Regulation of cell cycle by IL-2 signals at priming.

<p>(A) In the presence (•) or absence (▪) of rhIL-2 (5 ng/mL) at priming, the anti-CD3/CD28-stimulated CD8⁺ T cells were counted day by day (left). Folds increase was determined day by day (right). (B) In the presence or absence of rhIL-2 at priming, CFSE-labeled naïve CD8⁺ T cells were stimulated by anti-CD3/CD28 antibodies for indicated time, 24, 48 and 72 hours, and cell divisions were analyzed by flow cytometry (black filled). Gray filled: CFSE-labeled naïve CD8⁺ T cells. (C) Naïve CD8⁺ T cells were stimulated by anti-CD3/CD28 antibodies for the indicated time, 0, 24, 36 and 48 hours, and analyzed by flow cytometry for the BrdU incorporation to determine the percentage of cell entering S phase (Solid line). Cells stimulated by anti-CD3/CD28 antibodies also with rmIL-2 administration at priming were shown as a dotted line. Dashed line shows cells additionally treated by rmIL-2 and anti-mouse IL-2 at priming. R and R' stands for restriction point. (D) Total cell extracts of 1.5×10⁶ CD8⁺ T cells at 18 hours after stimulation were collected for further Immunoblotting with antibodies against cyclins A, D1 and E, p19, p27 and actin, respectively. IL-2 (5 ng/mL) was added at priming. The data represent three independent experiments. (D  =  day)</p