Mechanisms underlying Actinobacillus pleuropneumoniae exotoxin ApxI induced expression of IL-1β, IL-8 and TNF-α in porcine alveolar macrophages

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes fibrino-hemorrhagic necrotizing pleuropneumonia in pigs. Production of proinflammatory mediators in the lungs is an important feature of A. pleuropneumoniae infection. However, bacterial components other than lipopolysaccharide involved in this process remain unidentified. The goals of this study were to determine the role of A. pleuropneumoniae exotoxin ApxI in cytokine induction and to delineate the underlying mechanisms. Using real-time quantitative PCR analysis, we found native ApxI stimulated porcine alveolar macrophages (PAMs) to transcribe mRNAs of IL-1β, IL-8 and TNF-α in a concentration- and time-dependent manner. Heat-inactivation or pre-incubation of ApxI with a neutralizing antiserum attenuated ApxI bioactivity to induce cytokine gene expression. The secretion of IL-1β, IL-8 and TNF-α protein from PAMs stimulated with ApxI was also confirmed by quantitative ELISA. In delineating the underlying signaling pathways contributing to cytokine expression, we observed mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK) were activated upon ApxI stimulation. Administration of an inhibitor specific to p38 or JNK resulted in varying degrees of attenuation on ApxI-induced cytokine expression, suggesting the differential regulatory roles of p38 and JNK in IL-1β, IL-8 and TNF-α production. Further, pre-incubation of PAMs with a CD18-blocking antibody prior to ApxI stimulation significantly reduced the activation of p38 and JNK, and subsequent expression of IL-1β, IL-8 or TNF-α gene, indicating a pivotal role of β2 integrins in the ApxI-mediated effect. Collectively, this study demonstrated ApxI induces gene expression of IL-1β, IL-8 and TNF-α in PAMs that involves β2 integrins and downstream MAPKs

B5, a thioredoxin reductase inhibitor, induces apoptosis in human cervical cancer cells by suppressing the thioredoxin system, disrupting mitochondrion-dependent pathways and triggering autophagy

published_or_final_versio

The Role of Age in Predicting the Outcome of Caustic Ingestion in Adults: A Retrospective Analysis

<p>Abstract</p> <p>Background</p> <p>Although the outcomes of caustic ingestion differ between children and adults, it is unclear whether such outcomes differ among adults as a function of their age. This retrospective study was performed to ascertain whether the clinical outcomes of caustic ingestion differ significantly between elderly and non-elderly adults.</p> <p>Methods</p> <p>Medical records of patients hospitalized for caustic ingestion between June 1999 and July 2009 were reviewed retrospectively. Three hundred eighty nine patients between the ages of 17 and 107 years were divided into two groups: non-elderly (< 65 years) and elderly (≥ 65 years). Mucosal damage was graded using esophagogastroduodenoscopy (EGD). Parameters examined in this study included gender, intent of ingestion, substance ingested, systemic and gastrointestinal complications, psychological and systemic comorbidities, severity of mucosal injury, and time to expiration.</p> <p>Results</p> <p>The incidence of psychological comorbidities was higher for the non-elderly group. By contrast, the incidence of systemic comorbidities, the grade of severity of mucosal damage, and the incidence of systemic complications were higher for the elderly group. The percentages of ICU admissions and deaths in the ICU were higher and the cumulative survival rate was lower for the elderly group. Elderly subjects, those with systemic complications had the greatest mortality risk due to caustic ingestion.</p> <p>Conclusions</p> <p>Caustic ingestion by subjects ≥65 years of age is associated with poorer clinical outcomes as compared to subjects < 65 years of age; elderly subjects with systemic complications have the poorest clinical outcomes. The severity of gastrointestinal tract injury appears to have no impact on the survival of elderly subjects.</p

Reactive Sputtering Process Study for Vanadium Oxynitride Films

In this study, vanadium oxynitride thin films were deposited by reactive magnetron sputtering using pure vanadium targets, Ar as a plasma carrier, and a mix of N2 and O2 as reactive gases. Various ratios of mass flow rates between two reactive gases were maintained as a constant during the process. To obtain crystalline phases of oxynitrides, rapid thermal annealing in Ar atmosphere at 600 &deg;C and 700 &deg;C for 5 min was conducted after the deposition. This study aims to define the range of the process parameters of magnetron sputtering to deposit vanadium oxynitride thin films. The assessment for the characterization of films utilizes the surface profiler, scanning electron microscope, X-ray diffraction, X-ray photoelectron spectroscopy, four-point probe, Hall analyzer, and UV-visible-NIR spectrometer. Experimental results reveal that the annealed films can be oxynitrides when the oxygen flow rate is below 0.25 sccm, and the ratio of oxygen/nitrogen is no more than ~1/10. The annealed vanadium oxynitride films, in terms of their properties, are closer to vanadium nitrides than to oxides, due to the intended low supply of oxygen during deposition. For instance, the film is more metallic than semi-conductive with dark appurtenance and high optical absorbance across the spectrum between 200 and 900 nm. For practical purposes, the deposition conditions of O2:N2 = 1/20, O2 &lt; 0.25 sccm, and 600 &deg;C annealing are recommended to obtain vanadium oxynitride films with relatively lower resistivity (10&minus;2 &Omega; cm) and optical transmittance (&lt;15%) through films

Effects of Actinobacillus pleuropneumoniae exotoxin on different cells

胸膜肺炎放線桿菌Actinobacillus pleuropneumoniae (APP)外毒素Apx為APP強毒力因子之一，該毒素具有溶血性及細胞毒性，於APP感染豬隻可發現毒素所造成嚴重的肺臟病變。本研究主要目的為探討不同種類或不同來源細胞對於Apx之敏感性。首先以APP血清型第10型所分泌毒素ApxI氣管內接種豬隻，利用H&E染色、免疫化學染色、TUNEL、及Hoechst染色等方法探討ApxI對豬肺臟組織所造成之影響。結果顯示，豬隻經ApxI毒素接種後，肺臟組織中可見嗜中性球浸潤及肺臟間質組織增厚，伴隨輕度炎症反應；並於TUNEL標幟後發現較高比例之細胞呈凋亡，而凋亡細胞中吞噬及非吞噬細胞各佔54%與46%。接種經熱不活化ApxI毒素的肺臟組織則可見輕度炎症反應，而生理食鹽水處理組組則無明顯組織病變，且TUNEL染色結果發現熱不活化毒素與生理食鹽水處理組細胞凋亡比例無顯著差異，且細胞凋亡數明顯較ApxI毒素處理組低。為進一步瞭解不同種類細胞對於不同血清型別之Apx毒素之敏感性，本研究利用不同來源之細胞(株)包括豬肺臟巨噬細胞(PAM)、BL-3、BL-3.1、RAW 264.7、PK-15、Vero細胞，比較這些細胞對APP血清型第1及2型外毒素的敏感性。結果發現，BL-3、BL-3.1與PAM對於APP血清型第1及2型菌株所產生的毒素具最高之敏感性，三者間差異不明顯；小鼠巨噬細胞株RAW 264.7對APP血清型第1型分泌毒素有微弱感受性，而腎臟上皮細胞PK-15與Vero則對Apx毒素不具敏感性。此外，本研究利用自殺載體及同源重組互換原理，成功構築APP血清型第10型之apxIA突變菌株，經PCR及基因定序確定其具有正確之基因型，亦利用生長曲線、Biolog生化代謝、溶血試驗、細胞毒性試驗、細胞凋亡試驗及西方轉漬法進行表現型分析。結果顯示，apxIA基因突變菌株與親代株有相似的生長曲線及生化代謝性狀；此外，由於該菌株失去產生ApxI毒素能力，因此突變菌株之培養上清液不具溶血性、細胞毒性，不會誘發PAM凋亡。Actinobacillus pleuropneumoniae (APP) exotoxin (Apx) is one of the strong virulent factors of APP. Apx possesses hemolytic and cytotoxic activities which cause serious lung damage in APP infected pigs. The aim of this study was to evaluate the sensitivity of different cell types or cell lines originated from different species toward Apx. APP serotype 10-derived exotoxin ApxI was used to intratracheally inoculate piglets; lung sections were subjected to H&E, immunohistochemical, TUNEL, and Hoechst stains in order to assess the effects of ApxI on porcine lung tissue. Lung sections of ApxI treated group had mild neutrophil infiltration and interstitial hyperplaisa. TUNEL staining revealed higher percentage of apoptotic cells in ApxI treated group of which 54% were phagocytic cells. Piglets inoculated with heat-inactivated ApxI showed mild inflammation in lungs while no obvious histopathological changes observed in saline treated piglets. Both groups had lower percentage of apoptotic cells in lung sections as compared to ApxI treated group. To further understand the sensitivities of cell lines from different species toward Apx of different serotypes, porcine alveolar macrophage (PAM), BL-3, BL-3.1, RAW 264.7, PK-15, and Vero cells were used for comparison. Results showed that BL-3, BL-3.1, and PAM had the highest sensitivity toward exotoxins derived from APP serotype 1 (APP1) and 2 (APP2), and no significant difference was found between these cells. RAW 264.7 cells were mildly sensitive toward APP1 exotoxin, and PK-15 and Vero cells were not sensitive toward exotoxins of APP. Further, an APP serotype 10 apxIA mutant was successfully constructed through homologous recombination of APP genomic DNA with a suicide vector carrying apxIA gene inserted with a kanamycin-resistant determinant. The mutant with desired mutation was selected and confirmed by antibiotic resistance, PCR, and DNA sequence analysis. Growth curves, Biolog metabolic profiles, hemolytic assay, cytotoxic assay, TUNEL assay, and Western blot analysis were used to verify the phenotypes of mutant strain. The apxIA mutant had similar growth rate and exhibited similar metabolic profiles compared to the parental strain. In addition, apxIA mutant strain lost its ability to produce ApxI, therefore no hemolytic, cytotoxic, or apoptosis inducing activity was observed in the bacterial culture supernatant.頁數 中文摘要 ----------------------------------------------------------------------------------------- i 英文摘要 ----------------------------------------------------------------------------------------- ii 目次 ----------------------------------------------------------------------------------------- iv 表次 ----------------------------------------------------------------------------------------- vii 圖次 ----------------------------------------------------------------------------------------- viii 第一章 前言 --------------------------------------------------------------------------- 1 第二章 文獻探討 ------------------------------------------------------------------------- 2 第一節 胸膜肺炎放線桿菌(APP)簡介 --------------------------------- 2 第二節 APP血清型分型 ------------------------------------------------- 2 第三節 APP毒力因子 ----------------------------------------------------- 3 一、LPS ------------------------------------------------------------ 3 二、CPS ------------------------------------------------------------ 3 三、攜鐵蛋白 ------------------------------------------------------- 4 第四節 APP外毒素&not;Apx -------------------------------------------------- 4 第五節 Apx特性 ------------------------------------------------------------ 4 第六節 Apx生物活性 ------------------------------------------------------- 5 一、溶血性Hemolytic activity ---------------------------------- 5 二、細胞毒性Cytotoxic effect --------------------------------- 6 三、Apx誘發細胞凋亡(apoptosis) ----------------------------- 6 第七節 RTX toxins --------------------------------------------------------- 7 第八節 現今APP疫苗發展 ----------------------------------------------- 8 第三章 實驗材料及方法 ----------------------------------------------------------------- 10 第一節 菌株、質體與細胞株 --------------------------------------------- 10 第二節 APP粗製毒素製備 ----------------------------------------------- 10 第三節 粗製Apx溶血力價測定 ----------------------------------------- 10 第四節 細胞製備與培養 -------------------------------------------------- 11 一、豬肺泡巨噬細胞(PAM)灌洗取得、保存與培養 ----- 11 二、細胞株保存與繼代 ----------------------------------------- 12 第五節 粗製Apx細胞毒性力價測定 ------------------------------------ 13 第六節 血清中和抗體力價測定 ---------------------------------------- 13 第七節 豬隻氣管內攻毒試驗 -------------------------------------------- 14 第八節 細胞凋亡檢測 ----------------------------------------------------- 14 一、PAM ---------------------------------------------------------- 14 二、肺臟組織切片處理 ----------------------------------------- 15 三、免疫化學染色 ---------------------------------------------- 15 四、TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining-------------------------------------------- 15 五、Hoechst staining --------------------------------------------- 16 第九節 構築APP血清型第10型apxIA突變株 ---------------------- 16 一、APP基因體DNA抽取 ------------------------------------- 16 二、細菌質體DNA抽取 --------------------------------------- 16 三、熱休克轉型法(Heat Shock Transformation) ------------- 16 四、E. coli電穿孔轉型法 --------------------------------------- 17 五、APP電穿孔轉型法 ------------------------------------------ 17 六、建構突變株 --------------------------------------------------- 18 七、apxIA突變株於小鼠半致死劑量 (50% Lethal Dose, LD50)-------------------------------------------------------------------- 21 第四章 結果 ---------------------------------------------------------------------------- 23 第一節 APP粗製毒素溶血力價以及細胞毒性力價判定(Hemolytic unit, HU; Cytotoxic unit, CU)---------------------------------------- 23 第二節 不同細胞株與PAM對Apx敏感性測試 -------------------- 23 第三節 Apx造成細胞凋亡現象 – TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining----------------------- 24 一、In vivo study: Apx造成豬隻肺臟細胞凋亡現象 ------- 24 第四節 以APP血清型第10型構築apxIA突變株 --------------------- 25 一、自殺載體構築及鑑定 --------------------------------------- 25 二、APP血清型第10型apxIA基因突變候選株篩選及基因鑑定----------------------------------------------------------------------- 25 三、ApxIA336表現型確定 ------------------------------------- 26 四、APP10及ApxIA336於小鼠半致死劑量 ----------------- 27 第五章 討論 -------------------------------------------------------------------------------- 28 參考文獻 ------------------------------------------------------------------------------------------- 6