Replication of white spot syndrome virus in ovarian primary cultures from the kuruma shrimp, Marsupenaeus japonicus

Propagation of white spot syndrome virus (WSSV) was investigated in primary ovarian cultures from the kuruma shrimp Marsupenaeus japonicus. A WSSV strain, purified by sucrose density gradient centrifugation, was inoculated into 10-day-old primary ovarian cultures. WSSV infection induced marked cytopathic effect (CPE) on primary ovarian cells. Initially, virus-infected cells began to shrink 72 h post-inoculation, followed by the rounding of most cells which detached finally from flask surface. Electron microscopic observations clearly showed that the replication of WSSV occurred in nuclei of ovarian cells. Immunoblot analysis with antibodies against the WSSV envelope protein VP28 provided the evidence that the levels of WSSV antigens in culture supernatant gradually increased during the period between 24 and 120 h after virus inoculation. The results suggest that the use of primary ovarian cultures of the kuruma shrimp will facilitate characterization of the WSSV infection.     

HMGB1-Like Dorsal Switch Protein 1 Triggers a Damage Signal in Mosquito Gut to Activate Dual Oxidase via Eicosanoids

Several mosquitoes transmit human pathogens by blood feeding, with the gut being the main entrance for the pathogens. Thus, the gut epithelium defends the pathogens by eliciting potent immune responses. However, it was unclear how the mosquito gut discriminates pathogens among various microflora in the lumen. This study proposed a hypothesis that a damage signal might be specifically induced by pathogens in the gut. The Asian tiger mosquito, Aedes albopictus, encodes dorsal switch protein 1 (Aa-DSP1) as a putative damage-associated molecular pattern (DAMP). Aa-DSP1 was localized in the nucleus of the midgut epithelium in naïve larvae. Upon infection by a pathogenic bacterium, Serratia marcescens, Aa-DSP1 was released to hemocoel and activated phospholipase A₂ (PLA₂). The activated PLA₂ increased the level of prostaglandin E₂ (PGE₂) in the gut and subsequently increased Ca²⁺ signal to produce reactive oxygen species (ROS) via dual oxidase (Duox). Inhibition of Aa-DSP1 via RNA interference or specific inhibitor treatment failed to increase PGE₂/Ca²⁺ signal upon the bacterial infection. Thus, the inhibitors specifically targeting eicosanoid biosynthesis significantly prevented the upregulation of ROS production in the gut and enhanced mosquito mortality after the bacterial infection. However, such inhibitory effects were rescued by adding PGE₂. These suggest that Aa-DSP1 plays an important role in immune response of the mosquito gut as a DAMP during pathogen infection by triggering a signaling pathway, DSP1/PLA₂/Ca²⁺/Duox.     

Suppression of NSAID-induced small intestinal inflammation by orally administered redox nanoparticles

Patients regularly taking non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin (IND) have a risk of small intestinal injuries. In this study, we have developed an oral nanotherapeutics by using a redox nanoparticle (RNP^O), which is prepared by self-assembly of an amphiphilic block copolymer that possesses nitroxide radicals as side chains of hydrophobic segment via ether linkage, to reduce inflammation in mice with IND-induced small intestinal injury. The localization and accumulation of RNP^O in the small intestine were determined using fluorescent-labeled RNP^O and electron spin resonance. After oral administration, the accumulation of RNP^O in both the jejunum and ileum tissues was about 40 times higher than those of low-molecular-weight nitroxide radical compounds, and RNP^O was not absorbed into the bloodstream via the mesentery, thereby avoiding the adverse effects of nitroxide radicals in the entire body. RNP^O remarkably suppressed inflammatory mediators such as myeloperoxidase, superoxide anion, and malondialdehyde in the small intestines of IND-treated mice. Compared to low-molecular-weight nitroxide radical compounds, RNP^O also significantly increased the survival rate of mice treated daily with IND. On the basis of these results, RNP^O is promising as a nanotherapeutics for treatment of inflammation in the small intestine of patients receiving NSAIDs.     

Characterization of a Kunitz-type protease inhibitor (MjKuPI) reveals the involvement of MjKuPI positive hemocytes in the immune responses of kuruma shrimp Marsupenaeus japonicus

Serine proteases and their inhibitors play vital roles in biological processes. Serine protease inhibitors, including Kunitz-type protease inhibitors play important roles not only in physiological process (i.e. blood clotting and fibrinolysis) but also in immune responses. In this study, we characterized a Kunitz-type protease inhibitor, designated MjKuPI, from kuruma shrimp Marsupenaeus japonicus. An expression profile showed that MjKuPI was mainly expressed in hemocytes. Immunostaining revealed that some hemocytes expressed MjKuPI (MjKuPI⁺ hemocytes) and others did not (MjKuPI⁻ hemocytes). Injection of shrimp with Vibrio penaeicida and white spot syndrome virus (WSSV) upregulated the mRNA level of MjKuPI, and a flow cytometry analysis revealed that the proportion of MjKuPI⁺ hemocytes increased significantly 24 h after injection. Together, these results suggest that MjKuPI and MjKuPI⁺ hemocytes have a role in the innate immune system of kuruma shrimp.     

血管NAD(P)H氧化酶在血管重塑中的作用

NAD(P)H oxidase was initially found in phagocytes and it participates in the generation of reactive oxygen species(ROS). Recent researches have showed that NAD(P)H oxidase also expresses in other tissues including blood vessels and it plays a critical role in vascular remodeling through ROS which are important signaling molecules in vascular cells. This article reviews the biochemical characterization, activation paradigms, structure, and function of this enzyme.     

黄嘌呤氧化酶在激素诱导成骨细胞凋亡过程中的作用及机制

目的探究黄嘌呤氧化酶在糖皮质激素诱导小鼠胚胎成骨细胞前体细胞(MC3T3-E1)细胞凋亡过程中的作用及机制。方法将细胞分为空白组、模型组、5 mol/L组、10 mol/L组和15 mol/L组,接种完成后培养24 h,确认细胞贴壁后换液。空白组加入10%FBS培养基2 mL,模型组加入1 10-6 mol/L DEX培养基2 mL, 5 mol/L组加入5 mol/L别嘌醇DEX培养基2 mL,10 mol/L组加入10 mol/L别嘌醇DEX培养基2 mL,15 mol/L组加入15 mol/L别嘌醇DEX培养基2 mL。培养72 h后检测Caspase-3、STAT-1、Bax、Bcl-2等蛋白表达情况,检测细胞凋亡情况以及活性氧簇含量。检测细胞内黄嘌呤氧化酶活性、丙二醛(MDA)含量及线粒体膜电位。结果模型组中Caspase-3、STAT-1和Bax蛋白表达量明显升高,Bcl-2蛋白表达量降低,细胞凋亡比例增高,细胞内活性氧簇含量增高,细胞内黄嘌呤氧化酶活性、丙二醛含量增高,而线粒体膜电位降低,与空白组、5 mol/L组、10mol/L组和15 mol/L组相比,差异有统计学意义(P 0.05)。结论黄嘌呤氧化酶通过产生过量的活性氧簇从而导致成骨细胞氧化应激损伤,通过激活STAT-1和Bax蛋白来诱导成骨细胞凋亡,同时诱导线粒体膜电位稳态崩溃触发内源性线粒体凋亡途径。别嘌醇可以较好地抑制黄嘌呤氧化酶的活性,减少活性氧簇的产生,通过减少Caspase-3、STAT-1和Bax蛋白表达,稳定线粒体膜电位,从而减少成骨细胞凋亡。     

The role and regulation of catalase in respiratory tract opportunistic bacterial pathogens

Respiratory tract bacterial pathogens are the etiologic agents of a variety of illnesses. The ability of these bacteria to cause disease is imparted through survival within the host and avoidance of pathogen clearance by the immune system. Respiratory tract pathogens are continually bombarded by reactive oxygen species (ROS), which may be produced by competing bacteria, normal metabolic function, or host immunological responses. In order to survive and proliferate, bacteria have adapted defense mechanisms to circumvent the effects of ROS. Bacteria employ the use of anti-oxidant enzymes, catalases and catalase-peroxidases, to relieve the effects of the oxidative stressors to which they are continually exposed. The decomposition of ROS has been shown to provide favorable conditions in which respiratory tract opportunistic bacterial pathogens such as Haemophilus influenzae, Mycobacterium tuberculosis, Legionella pneumophila, and Neisseria meningitidis are able to withstand exposure to highly reactive molecules and yet survive. Bacteria possessing mutations in the catalase gene have a decreased survival rate, yet may be able to compensate for the lack of catalatic activity if peroxidatic activity is present. An incomplete knowledge of the mechanisms by which catalase and catalase-peroxidases are regulated still persists, however, in some bacterial species, a regulatory factor known as OxyR has been shown to either up-regulate or down-regulate catalase gene expression. Yet, more research is still needed to increase the knowledge base in relation to this enzyme class. As with this review, we focus on major respiratory tract opportunistic bacterial pathogens in order to elucidate the function and regulation of catalases. The importance of the research could lead to the development of novel treatments against respiratory bacterial infections.     

Hyperinflammation in chronic granulomatous disease and anti-inflammatory role of the phagocyte NADPH oxidase

Chronic granulomatous disease (CGD) is an immunodeficiency caused by the lack of the superoxide-producing phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. However, CGD patients not only suffer from recurrent infections, but also present with inflammatory, non-infectious conditions. Among the latter, granulomas figure prominently, which gave the name to the disease, and colitis, which is frequent and leads to a substantial morbidity. In this paper, we systematically review the inflammatory lesions in different organs of CGD patients and compare them to observations in CGD mouse models. In addition to the more classical inflammatory lesions, CGD patients and their relatives have increased frequency of autoimmune diseases, and CGD mice are arthritis-prone. Possible mechanisms involved in CGD hyperinflammation include decreased degradation of phagocytosed material, redox-dependent termination of proinflammatory mediators and/or signaling, as well as redox-dependent cross-talk between phagocytes and lymphocytes (e.g. defective tryptophan catabolism). As a conclusion from this review, we propose the existence of ROS high and ROS low inflammatory responses, which are triggered as a function of the level of reactive oxygen species and have specific characteristics in terms of physiology and pathophysiology.     

Reactive Oxygen Species in the Immune System

Reactive oxygen species (ROS) are a group of highly reactive chemicals containing oxygen produced either exogenously or endogenously. ROS are related to a wide variety of human disorders, such as chronic inflammation, age-related diseases and cancers. Besides, ROS are also essential for various biological functions, including cell survival, cell growth, proliferation and differentiation, and immune response. At present there are a number of excellent publications including some reviews about functions of these molecules either in normal cell biology or in pathophysiology. In this work, we reviewed available information and recent advances about ROS in the main immune cell types and gave summary about functions of these highly reactive molecules both in innate immunity as conservative defense mechanisms and in essential immune cells involved in adaptive immunity, and particularly in immune suppression.     

Oxygen and reactive oxygen species in articular cartilage: modulators of ionic homeostasis

Articular cartilage is an avascular tissue dependent on diffusion mainly from synovial fluid to service its metabolic requirements. Levels of oxygen (O₂) in the tissue are low, with estimates of between 1 and 6%. Metabolism is largely, if not entirely, glycolytic, with little capacity for oxidative phosphorylation. Notwithstanding, the tissue requires O₂ and consumes it, albeit at low rates. Changes in O₂ tension also have profound effects on chondrocytes affecting phenotype, gene expression, and morphology, as well as response to, and production of, cytokines. Although chondrocytes can survive prolonged anoxia, low O₂ levels have significant metabolic effects, inhibiting glycolysis (the negative Pasteur effect), and also notably matrix production. Why this tissue should respond so markedly to reduction in O₂ tension remains a paradox. Ion homeostasis in articular chondrocytes is also markedly affected by the extracellular matrix in which the cells reside. Recent work has shown that ion homeostasis also responds to changes in O₂ tension, in such a way as to produce significant effects on cell function. For this purpose, O₂ probably acts via alteration in levels of reactive oxygen species. We discuss the possibility that O₂ consumption by this tissue is required to maintain levels of ROS, which are then used physiologically as an intracellular signalling device. This postulate may go some way towards explaining why the tissue is dependent on O₂ and why its removal has such marked effects. Understanding the role of oxygen has implications for disease states in which O₂ or ROS levels may be perturbed.     

阿托伐他汀对大鼠缺血再灌注脑组织中NADPH氧化酶源性超氧阴离子的抑制作用

目的：脑组织在缺血再灌注的早期，超氧阴离子的大量生成加重了脑组织损伤，本实验研究阿托伐他汀对缺血再灌注脑组织保护作用的可能机制。方法:成年雄性Sprague-Dawley大鼠经线栓法阻断大脑中动脉建立脑缺血再灌注模型，再灌注前经腹腔给予阿托伐他汀（立普妥）治疗。脑梗死灶体积用四唑氮蓝染色后测量；NADPH氧化酶酶活性和超氧阴离子水平使用光泽精增强化学发光法定量测定；NADPH氧化酶膜亚基gp91phox、膜易位亚基p47phox和小GTP酶Rac-1蛋白的表达用蛋白质印迹分析。结果:缺血半暗区的NADPH氧化酶活性和超氧阴离子水平增高，于再灌注2 h达到高峰，但缺血中心区的NADPH氧化酶活性和超氧阴离子水平无明显增高。阿托伐他汀预治疗能抑制再灌注2 h后缺血半暗区的NADPH氧化酶活性和超氧阴离子增高，减少膜亚基gp91phox蛋白的表达和预防细胞质亚基p47phox蛋白易位至细胞膜。结论:阿托伐他汀对缺血再灌注脑组织NADPH氧化酶源性超氧阴离子的抑制作用，是其脑保护作用机制之一。     

芪苈强心对心肌梗死后大鼠心肌细胞凋亡的影响

目的: 探讨芪苈强心对心肌梗死大鼠心肌细胞凋亡的影响。方法: 结扎冠脉前降支制作大鼠心肌梗死模型,随机分为假手术组(sham, n=5)、心肌梗死组(MI, n=16)和芪苈强心组(4 g·kg^-1·d^-1, n=15),28 d后检测心肌梗死面积,TUNEL法检测心肌细胞凋亡指数(AI),免疫组化法检测Fas蛋白表达,Western blotting检测黄嘌呤氧化酶(XO)和caspase-3蛋白表达,比色法测定XO和清除活性氧活性。结果: MI组非梗死区心肌细胞AI升高,Fas、XO和caspase-3蛋白表达增强,XO活性增加,清除活性氧活性降低(P<0.01)。芪苈强心组与MI组比较,非梗死区心肌细胞AI降低,Fas和caspase-3蛋白表达减少,XO活性降低,清除活性氧活性升高(P<0.01),但梗死面积和XO蛋白表达在两组间无显著差异(P>0.05)。结论: 芪苈强心能够抑制非梗死区心肌细胞凋亡,其作用机制可能与减少活性氧簇生成及降低Fas和caspase-3的表达有关。     

Commensal and probiotic bacteria may prevent NEC by maturing intestinal host defenses

Necrotizing enterocolitis (NEC) is a devastating disease of prematurity with significant morbidity and mortality. Immaturity of intestinal host defenses predisposes the premature infant gut to injury. An abnormal bacterial colonization pattern with a deficiency of commensal bacteria may lead to a further breakdown of these host defense mechanisms, predisposing the infant to NEC. The presence of probiotic and commensal bacteria within the gut has been shown to mature the intestinal defense system through a variety of mechanisms. We have shown that commensal and probiotic bacteria can promote intestinal host defenses by reducing apoptotic signaling, blocking inflammatory signaling, and maturing barrier function in immature intestinal epithelia. Future studies aimed at elucidating the mechanisms by which probiotic and commensal bacteria exert their effects will be critical to developing effective preventive therapies for NEC.     

二烯丙基三硫诱导人髓样白血病HL-60细胞活性氧产生及其细胞毒性作用

目的：探讨二烯丙基三硫(DATS)诱导人白血病HL-60细胞产生活性氧(ROS)及其细胞毒性作用。方法: 用浓度为50、100、150、200 μmol/L DATS处理HL-60细胞1、3、6、12、24 h后，流式细胞计数法检测HL-60细胞内的ROS水平，NBT还原实验分析NADPH氧化酶的活性，分别用NADPH氧化酶特异性阻断剂apocynin与抗氧化剂NAC（N-acetyl-L-cystein）预处理30 min后，观察DATS对HL-60细胞产生活性氧的影响，分光光度法检测细胞质膜氧化产物丙二醛（MDA）与细胞蛋白氧化产物羰基化蛋白（protein carbonyl）。结果: DATS能诱导人白血病HL-60细胞产生ROS，当DATS处理细胞1-3 h 时，HL-60细胞产生ROS随DATS浓度的升高和处理时间的延长而升高，当浓度为150 μmol/L DATS处理细胞3h时，ROS的荧光强度达到最高峰，其后维持在一个较高水平。NADPH氧化酶活性与ROS的产生一致。HL-60细胞的脂质过氧化和羰基化蛋白浓度与DATS诱导ROS产生的趋势基本一致，都在3 h、150 μmol/L处理点达到最高值。应用NADPH氧化酶特异性阻断剂apocynin或抗氧化剂NAC后，能显著降低HL-60细胞ROS的产生和细胞损伤。结论: NADPH氧化酶是DATS诱导 HL-60 细胞产生ROS的主要酶系，ROS能氧化细胞质膜和蛋白质。     

胰淀素对离体大鼠胰岛细胞内活性氧生成的影响

目的：观察高浓度胰淀素对胰岛细胞内活性氧（ROS）生成的影响。方法：以体外培养胰岛单层细胞为模型,应用ROS特异性荧光染料2′,7′-二氯二氢荧光素双醋酸盐（H₂DCF）进行标记，以粘附细胞仪570型测定10 μmol/L胰淀素作用2 h后胰岛细胞内DCF荧光强度变化。结果：10 μmol/L胰淀素作用2 h，胰岛细胞内DCF荧光强度在16.7 mmol/L葡萄糖刺激后，迅速急骤升高进入平台期，升高速度为28×10^-3/s，而正常细胞DCF荧光强度无明显变化。光镜下可见部分细胞胞体缩小，胞质发生空泡变性，少数细胞核固缩、裂解，胞膜突起。结论：10 μmol/L胰淀素作用后的胰岛细胞内ROS生成增多，细胞形态受损，这些活性氧基的生成可以反映胰淀素对胰岛细胞损害程度，推测活性氧可作为胰岛细胞毒性损伤指标之一。     

Role of Nox2 in elimination of microorganisms

NADPH oxidase of the phagocytic cells (Nox2) transfers electrons from cytosolic NADPH to molecular oxygen in the extracellular or intraphagosomal space. The produced superoxide anion (O*2) provides the source for formation of all toxic oxygen derivatives, but continuous O*2 generation depends on adequate charge compensation. The vital role of Nox2 in efficient elimination of microorganisms is clearly indicated by human pathology as insufficient activity of the enzyme results in severe, recurrent bacterial infections, the typical symptoms of chronic granulomatous disease. The goals of this contribution are to provide critical review of the Nox2-dependent cellular processes that potentially contribute to bacterial killing and degradation and to indicate possible targets of pharmacological interventions.