中性粒细胞胞外诱捕网在痛风性疾病中的作用

痛风性疾病是单钠尿酸盐(MSU)晶体在关节、非关节组织及器官中沉积所引发炎症疾病的统称,以严重疼痛性关节炎的间歇性发作为特征。中性粒细胞胞外诱捕网(NETs)在机体先天免疫中发挥重要作用;MSU晶体可引发NETs的产生,通过不同的作用机制影响痛风性疾病的进展,既增加痛风性疾病的炎症反应,又参与痛风性疾病的炎症消退及痛风石的产生。本文主要从NETs的组成、形成方式、NETs参与痛风性疾病三个方面进行综述,以期发现痛风性疾病潜在的药物作用靶点并为痛风性疾病的靶向治疗提供新的研究思路。     

抗中性粒细胞胞外诱捕网的细菌毒力因子

中性粒细胞胞外诱捕网(Neutrophil extracellular traps,NETs)作为人体先天免疫系统的组成部分,具有捕杀病原体的作用。最近已经明确,几种细菌毒力因子有利于细菌逃避NETs的作用。本文讨论5种毒力因子,包括核酸酶、多糖荚膜、丙氨酰脂磷壁酸、唾液酸化聚糖分子模拟物、β-羟基丁酸。     

支气管哮喘患儿中性粒细胞胞外诱捕网水平与患者免疫功能及临床预后的关系

目的探讨支气管哮喘患儿中性粒细胞胞外诱捕网水平与患者免疫功能及临床预后的关系。方法选取本院2017年9月-2018年9月支气管哮喘患者80例作为观察组,并以同期健康体检的30例儿童作为对照组,采用pico Green dsDNA荧光染色定量检测支气管哮喘患者NETs水平,采用T淋巴细胞亚群评价患者免疫功能,对比不同NETs水平患者免疫功能及临床特点,采用Logistic回归分析探讨小儿支气管哮喘急性发作的风险因素。结果观察组白细胞、中性粒细胞、单核细胞、Cf-DNA/NETs水平均高于对照组(P <0. 05),而淋巴细胞水平低于对照组,差异均有统计学意义(P <0. 05)。Logistic回归分析显示,哮喘家族史、呼吸道感染、Cf-DNA/NETs水平上升、有害气体接触、被动吸烟均是支气管哮喘急性发作的独立危险因素(P <0. 05)。结论中性粒细胞胞外诱捕网水平与支气管哮喘患者免疫功能及预后密切相关,值得临床重点关注。     

哮喘儿童来源的中性粒细胞经一氧化氮供体诱导形成胞外诱捕网的相关研究

目的 探讨一氧化氮供体对哮喘儿童来源的中性粒细胞胞外诱捕网(NETs)的诱导作用,寻找新的可能用于儿童哮喘的监测指标和治疗靶点。方法 选取2017年9月-2017年11月就诊于上海市第五人民医院儿科的38例支气管哮喘患儿为研究对象,利用Percoll密度梯度离心法从哮喘儿童的外周血中分离出中性粒细胞,加入一氧化氮供体SNP进行体外孵育,荧光染色法检测细胞上清中的双链DNA(NETs的主要组分)做定量分析,同时制备细胞爬片并利用免疫荧光的方法观察NETs的主要结构,此外还利用一氧化氮合酶(NOS)的抑制剂对SNP的诱导进行干预实验。结果 成功从哮喘儿童外周静脉血中分离出中性粒细胞,回收率达95%以上,细胞活性大于80%;PMA、SNP组在诱导3 h后明显可见释放到胞外的丝网状NETs结构,而PBS、SNP+L-NAME、SNP+SMT组在诱导3 h后均未见明显的NETs结构;干预组(PMA 160 nmol/L、LPS 100 ng/ml、SNP 400 μmol/L)的NETs相对生成量均大于阴性对照PBS组(P<0.05);SNP+NOS阻滞剂组(SMT 10mmol/L、L-NAME 500 μmol/L、L-NMMA 1 mmol/L)的NETs相对生成量均小于单纯SNP组(P<0.05);NOS抑制剂能够减少但不能完全阻滞NETs的生成(P<0.05);SNP诱导的第1 h、2 h、3 h的NETs相对生成量呈持续上升趋势,而PBS组在第3 h生成量下降(P<0.05);随着L-NAME干预时间的延长,SNP诱导的NETs相对生成量持续减少(P<0.05)。结论 SNP能够诱导哮喘儿童NETs形成,同时NOS的抑制剂能够减少NETs的产生,为儿童哮喘的监测和治疗提供了新的思路。     

中性粒细胞胞外诱捕网的形成及其在生殖相关疾病中的作用

中性粒细胞活化后可形成中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs),参与人体内诸多疾病的病理生理过程,但其具体的发病机制尚未阐明.目前研究表明,NADPH氧化酶等细胞因子在其形成过程中发挥重要作用,并通过蛋白酪氨酸激酶(protein tyrosine kinase,P...     

中性粒细胞胞外诱捕网水平与糖尿病肾脏疾病患者肾功能相关性研究

目的 探讨中性粒细胞胞外诱捕网(neutrophil extracellular Traps,NETs)水平与糖尿病肾脏疾病(diabetic kidney disease,DKD)患者肾功能的相关性.方法 搜集年龄≤65岁、病程≤5年2型糖尿病(T2DM)患者167例,根据尿微量白蛋白/肌酐比值(UACR)分为正常蛋...     

肠源性感染患者中性粒细胞胞外诱捕网水平与肠屏障及细菌移位的关系

目的 探讨肠源性感染患者中性粒细胞胞外诱捕网(NETs)水平与肠屏障及细菌移位的关系。方法 选择绍兴市中心医院医供体总院老年医学科2018年1月-2021年1月肠源性感染患者86例为感染组,并根据其住院预后情况分为死亡组和存活组,随机选择未发生肠源性感染患者80例为非感染组。患者在入院时通过电子病历系统调取其临床资料,采用酶联免疫吸附法检测血浆NETs相关标记物cf-DNA/NETs、MPO-DNA/NETs以及血清中内毒素和D-乳酸水平;收集患者新鲜粪便标本,进行双歧杆菌、乳酸杆菌、大肠埃希菌和金黄色葡萄球菌培养。结果 肠源性感染组cf-DNA/NETs、MPO-DNA/NETs、内毒素、D-乳酸、大肠埃希菌和金黄色葡萄球菌水平高于非感染组,乳酸杆菌和双歧杆菌水平低于非感染组(P<0.05);死亡组cf-DNA/NETs、MPO-DNA/NETs、内毒素、D-乳酸、大肠埃希菌和金黄色葡萄球菌水平高于存活组,乳酸杆菌和双歧杆菌水平低于存活组(P<0.05);ROC分析结果显示,cf-DNA/NETs、MPO-DNA/NETs诊断肠源性感染时曲线下面积分别为0.835和0.8...     

心胸外科术后手术部位感染游离中性粒细胞胞外诱捕网DNA水平及其短期预后价值

目的 探讨心胸外科术后手术部位感染患者中性粒细胞胞外诱捕网(NETs)水平变化及其评估短期预后的临床价值。方法 选择江苏大学附属武进人民医院心胸外科2015年1月-2021年1月进行手术并在住院期间发生手术部位感染(SSI)患者72例为研究对象,通过电子病历收集患者临床资料,随访患者术后30 d预后情况,分析血浆cfDNA/NETs水平动态变化及其评估短期预后的临床价值。结果 72例纳入患者共培养出病原菌61株,其中革兰阴性菌33株,占54.10%,革兰阳性菌27株,占44.26%,真菌1株,占1.64%;根据患者30 d预后情况分为预后不良组19例和预后良好组53例。两组患者术前APACHEⅡ评分和COUNT评分,术后有创机械通气时间比较差异有统计学意义(P<0.05);两组患者术前、术后1 d、3 d cfDNA/NETs水平比较差异无统计学意义,预后不良组术后7 d cfDNA/NETs水平高于预后良好组(P<0.05); ROC分析结果显示,术后7 d cfDNA/NETs对心胸外科术后手术部位感染患者短期预后不良预测曲线下面积为0.841, 95%CI为0.727～0.924,特异度为81.34%,敏感度为67.97%。结论 对于心胸外科术后手术部位感染患者,临床医师可针对术前APACHEⅡ评分、术前COUNT评分、有创机械通气时间和血浆cfDNA/NETs水平进行针对性干预,以期改善预后。     

重症颅脑损伤并发肺部感染患者外周血中性粒细胞胞外诱捕网水平及意义

目的 方法 分析重症颅脑损伤并发肺部感染患者外周血中性粒细胞胞外诱捕网(NETs)水平及意义。方法 选择2018年1月-2021年1月医院收治的重症颅脑损伤患者150例,根据是否并发肺部感染分为肺部感染组(n=58)和非肺部感染组(n=92),采用荧光酶标仪检测两组研究对象外周血NETs水平,同时测定患者外周血白细胞计数(WBC)、中性粒细胞计数(NEU)、C-反应蛋白(CRP)、降钙素原(PCT),统计感染组患者颅脑损伤后3个月预后情况。结果 肺部感染组外周血NETs水平高于非肺部感染组(P<0.05);肺部感染组WBC、NEU、CRP、PCT水平高于非肺部感染组(P<0.05);感染程度越严重,肺部感染组患者外周血NETs水平、WBC、NEU、CRP、PCT水平越高(P<0.05);重症颅脑损伤并发肺部感染预后不良患者外周血NETs水平、WBC、NEU、CRP、PCT水平高于预后良好患者(P<0.05);外周血NETs诊断重症颅脑损伤并发肺部感染预后的曲线下面积(AUC)为0.864,敏感度为85.70%,特异度为73.00%。结论 重症颅脑损伤并发肺部感染患者外周血NETs水平显著升高,且外周血NETs水平与患者病情严重程度及预后有关,有可能作为评估重症颅脑损伤并发肺部感染病情严重程度的生物学标志物及为预后评估和治疗提供指导。     

中性粒细胞外陷阱与冠心病急性心肌梗死关联的临床流行病学研究

目的  探讨冠状动脉粥样硬化性心脏病(coronary atherosclerotic heart disease, CAHD)急性心肌梗死(acute myocardial infarction, AMI)发作及冠脉旁路移植术(coronary artery bypass graft, CABG)后周围血中性粒细胞胞外陷阱(neutrophil extracellular traps, NETs)水平变化及作用,为有效防控CAHD及AMI提供新依据。 方法  选择AMI患者和健康对照(healthy control, HC)各52例,ELISA法检测AMI发病时治疗前(pre-treatment, PRT)组及手术治疗后(post-operative treatment, POT)组周围血NETs、B因子活化片段a(fragment a of the factor B, Ba)、补体片段5a(fragment a of the 5th complement, C5a)及髓过氧化酶(myeloperoxidase, MPO)水平,检测结果的组间比较采用t检验,PRT组内观察指标间的相关性分析采用Pearson相关分析。 结果  PRT组和POT组各检测指标均高于HC组,PRT组心肌肌钙蛋白I(cardiac troponin I, cTnI)、NETs、Ba、C5a和MPO高于POT组[分别为(0.279±0.132)ng/ml vs.(0.016±0.008)ng/ml, P < 0.001;(0.466±0.143)OD值vs.(0.378±0.151)OD值, P=0.002;(72.812±30.144)pg/ml vs.(60.491±28.323)pg/ml, P=0.001;(327.112±203.228)ng/ml vs.(260.411±135.984)ng/ml, P=0.025;(75.782±33.596)ng/ml vs.(58.462±29.647)ng/ml, P=0.008];PRT组中NETs分别与Ba、C5a及MPO呈正相关(分别为r=0.394, P=0.001;r=0.324, P=0.030和r=0.406, P < 0.001),且Ba与C5a呈正相关(r=0.436, P < 0.001),但cTnI仅与NETs呈正相关(r=0.352, P=0.008)。POT组中,cTnI虽已恢复正常值范围,但仍高于HC组cTnI [(0.016±0.008)pg/ml vs.(0.012±0.007)pg/ml, P=0.016]。 结论  CAHD患者在AMI发作期和接受CABG手术后,持续存在高水平NETs及与其相关的高补体旁路活化状态,有助于病情进展和心肌梗死再发,干预NETs形成可能是防治CHAD进展和AMI发生的潜在途径之一。     

Antihypertensive peptides derived from egg proteins

There have been studies of antihypertensive peptides derived from food proteins, but very few described the production of bioactive peptides from egg proteins. The first 2 antihypertensive peptides isolated in egg were obtained by enzymatic hydrolysis of ovalbumin. They correspond to the sequences Phe-Arg-Ala-Asp-His-Pro-Phe-Leu (ovokinin) and Arg-Ala-Asp-His-Phe-Leu (ovokinin 2-7). Both exhibited endothelium-dependent vasodilatory activity. Ovokinin (2-7) had higher antihypertensive potency than ovokinin in spontaneously hypertensive rats (SHR). Modifications in the sequence of ovokinin (2-7) improved the bioavailability of this peptide. It was also demonstrated that different ovalbumin hydrolysates can inhibit angiotensin I-converting enzyme (ACE). We recently obtained an egg white hydrolysate that inhibited the enzyme in vitro. It was obtained by treating egg white with pepsin and it exhibited antihypertensive activity in SHR. Some ACE-inhibitory peptides obtained from this hydrolysate (Tyr-Arg-Glu-Glu-Arg-Tyr-Pro-Ile-Leu, Arg-Ala-Asp-His-Pro-Phe-Leu, and Ile-Val-Phe) also showed antihypertensive activity in these rats. The egg products mentioned could be used as functional food ingredients with potential therapeutic benefit in the prevention and treatment of hypertension.     

Evaluation of novel fusion proteins derived from extracellular matrix binding domains of LigB as vaccine candidates against leptospirosis in a hamster model

Leptospira binds to host extracellular matrix (ECM) through surface exposed outer membrane proteins called adhesin in order to initiate infection. Of various adhesins present on the surface of the spirochete, Leptospira-immunoglobulin like proteins (Lig proteins) and LipL32 are most abundant, widely distributed among pathogenic serovars and well characterized. Various fragments of Lig proteins (Ligcon4, Ligcon4-7.5, LigBcen2) and C-terminus fragment of LipL32 all of that bind to host ECM were fused, expressed and purified in soluble form as fusion proteins. Four week hamsters were immunized subcutaneously with various fusion proteins emulsified in EMULSIGEN-D adjuvant and subsequently boosted at 3 weeks. The protective efficacy of these novel fusion proteins was evaluated against subsequent challenge with highly virulent L. interrogans serovar Pomona (MLD50-100). Our results indicate that fusion protein based vaccine induced significant protection against acute infection with respect to PBS-adjuvant vaccinated controls as revealed by enhanced survival and reduced pulmonary hemorrhage. Moreover, the protection mediated by these novel proteins was higher than that of conserved region of Lig protein (Ligcon, established protective antigen) and correlated to the level of antibodies. LipL32 failed to impart significant protection, however fusing its immunogenic C-terminus domain to Lig fragments slightly delayed the morbidity of the infected animals. Our results demonstrate that this novel strategy could be promising in developing effective subunit vaccine to combat this zoonotic infection.     

Immunogenicity of a new, low-cost recombinant hepatitis B vaccine derived from Hansenula polymorpha in adults

BACKGROUND: Hepatitis B virus (HBV) infection is highly prevalent world over, especially in developing countries. A new recombinant hepatitis B virus (GeneVac-B; Serum Institute of India Ltd.) vaccine is developed using Hansenula polymorpha yeast. We decided to assess the immunogenicity, and reactogenicity of this vaccine in a large adult population. MATERIAL AND METHODS: Seven hundred eighty-eight adults subjects (age: 19-57 years, male:female ratio 35:1) received three 20 microg doses of a H. polymorpha-derived recombinant hepatitis B vaccine in months 0, 1, and 6. All the eligible subjects had negative baseline serum HBs Ag, and anti-HBs. The anti-HBs titer was obtained 1 month after the last dose of vaccine and was considered seroconverted if more than 1 mIU/ml, and seroprotective if more than 10 mIU/ml. RESULTS: The seroprotection rate was 96% and seroconversion rate was 97%. Seroconversion and seroprotection rates declined with increasing age. The minimum geometric mean titre of anti HBs was 443 mIU/ml (95% CI 407-482). Seroprotection was 96% in age group<40 years, while the same was 91% in >40 years group (Odd's ratio-2.9100, Z value-2.6183, highly significant). No other factor like smoking, tobacco-chewing, alcohol consumption, chronic diseases, and obesity, affected the immune response. No significant adverse reactions were reported in any of the subjects. CONCLUSIONS: Three standard doses of the H. polymorpha-derived recombinant HBV vaccine are highly immunogenic and safe in a predominantly male adult population. Young adults respond better with this vaccine. Because of its low cost, the vaccine may be a good choice in prevention of hepatitis B infection.     

Immunogenicity and efficacy of an in-house developed cell-culture derived veterinarian rabies vaccine

The efficiency of an inactivated tissue culture rabies vaccine produced on BHK-21 cells, according to an in-house developed process, was evaluated and compared to a commercial cell-tissue culture vaccine (Rabisin). Fifteen experimental dogs from local common breed were duly conditioned during a quarantine period, then vaccinated via the subcutaneous route with 1 ml of either the tissue culture vaccine developed in-house or the commercial vaccine Rabisin. The immune response of each dog was monitored for 162 days. Serum-neutralizing antibodies titers to rabies virus were determined by the rapid fluorescent focus inhibition test (RFFIT) which confirmed the strong response of dogs to both vaccines except one dog in the Rabisin group. The dogs were then challenged in the masseter muscle with a rabies street virus of canine origin. All vaccinated dogs except the single dog in the Rabisin group that failed to respond to the vaccine, survived the challenge. In contrast, 80% of animals in the control non-vaccinated group, developed rabies and died. A field vaccine trial was also conducted: 1,000 local dogs living in field conditions received one subcutaneous dose of the locally developed vaccine. Serum neutralizing antibody titers to rabies virus was determined by RFFIT at days 0, 60 and 360. Mean rabies neutralizing antibody titers were equal to 0.786, 3.73 and 1.55 IU/ml, respectively. The percentage of dogs with a neutralizing rabies antibody titer higher than the 0.5 IU/ml mandated WHO threshold, was 30%, 91.4% and 87.5% at day 0, 2 months and 1 year post-vaccination, respectively. These data demonstrate the efficiency of the in-house developed vaccine produced on BHK-21 cells in both experimental and field conditions and support its use in dog mass vaccination campaigns.     

Immunogenicity of peptides derived from a fibronectin-binding protein of S. aureus expressed on two different plant viruses

The D2 peptide derived from an S. aureus fibronectin-binding protein (FnBP) was expressed on the surface of the icosahedral cowpea mosaic virus (amino acids 1-30 of D2) or on the rod-shaped potato virus X (amino acids 1-38 of D2), termed CPMV-MAST1 and PVX-MAST8, respectively. Mice and rats were immunized subcutaneously with CPMV-MAST1 and mice with PVX-MAST8 in adjuvant and high titres of FnBP-specific antibody were obtained. The mouse IgG was predominantly of the IgG2a and IgG2b isotypes, which strongly bound complement component C1q, suggesting a TH1-bias in the peptide-specific responses. Sera from mice and rats immunized with CPMV-MAST1 and from mice immunized with PVX-MAST8 were shown to completely inhibit the binding of fibronectin to immobilised recombinant FnBP and rat sera against CPMV-MAST1 were able to block adherence of S. aureus to fibronectin. These studies demonstrate that the D2 peptide is highly immunogenic when expressed on 2 different plant viruses and highlight the potential of plant virus-based vaccines to protect against S. aureus infections.     

Immunogenicity of virus-like particles containing modified human immunodeficiency virus envelope proteins

Extensive glycosylation and variable loops of the HIV envelope protein (Env) are reported to shield some neutralizing epitopes. Here, we investigated the immunogenicity of mutated HIV Envs presented in virus-like particles (VLPs). We immunized mice with simian human immunodeficiency virus (SHIV) VLPs containing mutant HIV Env with reduced glycosylation (3G), variable loop-deleted mutations (dV1V2), or combinations of both types of mutations (3G-dV2-1G), and evaluated immune responses. Immune sera from mice that received VLPs with modified HIV Envs (3G or dV1V2) showed higher neutralizing activities against the homologous HIV 89.6 virus as well as heterologous viruses when compared with wild type SHIV VLP-immunized mice. Lymphocytes from immunized mice produced HIV Env-specific cytokines, with the 3G-dV2-1G mutant producing high levels of cytokines. Interestingly, both dendritic cells and B cells were found to interact with VLPs suggesting that VLPs are effective immunogens. Therefore, this study suggests that VLPs containing modified HIV Env have the potential to be developed as candidate vaccines capable of inducing cellular and humoral immune responses including neutralizing activities.     

Immunogenicity of the recombinant leptospiral putative outer membrane proteins as vaccine candidates

Leptospiral putative outer membrane proteins (OMPs) are likely to be essential components of more effective vaccines. Recently completed genomic sequences of Leptospira allowed us to target putative OMPs for the development of recombinant vaccines. We focused on 12 putative OMPs that had no homology with other organisms listed in the NCBI database except MceI and MceII of Leptospira, which are approximately 25% homologous to MceI of Mycobacterium tuberculosis. All putative OMPs were cloned, expressed and purified as glutathione-S-transferase (GST) fusion proteins. Primary screening for immunoprotective potential was performed in hamsters challenged with an LD50 inoculum of low passage serovar Pomona. Out of these 12 OMPs three fusion proteins viz. rLp1454, rLp1118 and rMceII were found to be protective in a hamster model of leptospirosis. The protective efficacy was evaluated on the basis of survival, histopathological lesions in vital organs and antibody responses against each antigen. All the recombinant proteins were able to enhance the survival and reduce the histopathological lesions. In contrast, control animals immunized with rGST demonstrated low survival and had significant lesions. Further, these three proteins were evaluated for synergistic protective efficacy as compared to LigA, which has already been established as a protective antigen. Our results indicate that rLp1454, rLp1118, and rMceII showed protection individually and synergistically against serovar Pomona infection, which may help us to develop a multicomponent vaccine for leptospirosis.