Homeostatic properties of Lactobacillus jensenii engineered as a live vaginal anti-HIV microbicide.
Homeostatic properties of Lactobacillus jensenii engineered as a live vaginal anti-HIV microbicide.
复制标题
DOI：
10.1186/1471-2180-13-4
复制
发表时间：
2013-01-08
期刊：
影响因子：
4.2
通讯作者：
Fichorova RN
中科院分区：
文献类型：
--
作者：
Yamamoto HS;Xu Q;Fichorova RN
研究阴道益生菌作为预防细菌性阴道病和HIV的二元策略。我们应用了一种创新的实验模型，使用原代和永生化的人宫颈和阴道上皮细胞来评估詹氏乳杆菌的功能特性，詹氏乳杆菌是健康阴道微生物组的主要组成部分，经工程改造可表达HIV-1进入抑制剂修饰的氰威蛋白-N（mCV-N）。在该模型中，细菌在24-72小时的时间段内定殖于上皮细胞。星形孢菌素和Toll样受体2/6配体巨噬细胞活化脂肽-2（MALP-2）分别作为细胞凋亡和促炎活化的阳性对照。以24小时间隔，在显微镜下评估定植上皮，收集上清液以测量可溶性免疫炎症介质和CV-N的产生，并裂解细胞以评估：1）通过切割的与总的半胱天冬酶-3测定的细胞凋亡; 2）通过荧光素酶报告基因测定的NF-κB活化;或3）在布鲁氏菌琼脂中的上皮相关集落形成单位（CFU）。野生型（WT）L. Jensenii 1153在不存在上皮损伤和凋亡的情况下始终定殖于宫颈和阴道细胞。表达mCV-N或对照质粒的生物工程衍生物显示出相同的稳定定殖模式，其在技术人员和细菌批次之间是可重复的（实验和上皮细胞类型内和实验和上皮细胞类型之间的CFU变异系数<10%）。MALP-2激活NF-κB，并导致宫颈阴道环境中具有临床意义的促炎介质（IL-1α、IL-1β、IL-6、TNF-α、IL-8、RANTES、MIP-3α和ICAM-1）水平成倍增加，通过多重电化学发光测定。同时，通过ELISA测量的保护性抗炎介质白细胞介素1受体拮抗剂（IL-1 RA）和分泌性白细胞蛋白酶抑制剂（SLPI）的水平保持恒定（IL-1 RA）或适度增加（SLPI）。与MALP-2相似，L. jensenii WT激活NF-κB;然而，与合成的TLR 2/6配体不同，活微生物在所有炎症相关蛋白的分泌水平上没有诱导显著变化。蛋白质印迹和HIV-1 gp 120结合试验分别证实了mCV-N的产生和功能。与WT L相比，生物工程乳酸杆菌表达的mCV-N在上皮细胞环境中保留了抗HIV活性，并且没有引起显著的免疫炎症变化。jensenii。这些结果突出了定殖模型的转化价值，并证明了L.詹氏衍生物
Vaginal probiotics are investigated as a binary strategy for prevention of bacterial vaginosis and HIV. We applied an innovative experimental model using primary and immortalized human cervical and vaginal epithelial cells to assess the functional properties of Lactobacillus jensenii, a predominant constituent of the healthy vaginal microbiome, engineered to express the HIV-1 entry inhibitor modified cyanovirin-N (mCV-N). In this model bacteria colonize the epithelial cells over a period of 24-72 h. Staurosporine and the Toll-like receptor 2/6 ligand macrophage-activating lipopeptide-2 (MALP-2) serve as positive controls for apoptosis and proinflammatory activation, respectively. In 24-hour intervals, the colonized epithelium is assessed microscopically, supernatants are collected for measurement of soluble immunoinflammatory mediators and production of CV-N, and cells are lysed for assessment of: 1) apoptosis by cleaved versus total caspase-3 assay; 2) NF-κB activation by a luciferase reporter assay; or 3) epithelia-associated colony forming units (CFU) in Brucella agar. Wild type (WT) L. jensenii 1153 consistently colonized cervical and vaginal cells in the absence of epithelial damage and apoptosis. The bioengineered derivatives expressing mCV-N or control plasmids showed the same stable colonization pattern, which was reproducible between technologists and bacterial batches (CFU coefficient of variation <10% within and between experiments and epithelial cell types). MALP-2 activated NF-κB and caused fold-increased levels of proinflammatory mediators with clinically established significance in the cervicovaginal environment (IL-1α, IL-1β, IL-6, TNF-α, IL-8, RANTES, MIP-3α, and ICAM-1), measured by a multiplex electrochemiluminescence assay. At the same time levels of protective anti-inflammatory mediators interleukin 1 receptor antagonist (IL-1RA) and secretory leukocyte protease inhibitor (SLPI), both measured by ELISA, remained constant (IL-1RA) or moderately increased (SLPI). Similarly to MALP-2, colonization by L. jensenii WT activated NF-κB; however, unlike the synthetic TLR2/6 ligand, the live microorganisms did not induce significant changes in the secreted levels across all inflammation-associated proteins. The mCV-N production and function were confirmed by western blot and a HIV-1 gp120 binding assay, respectively. The bioengineered lactobacilli expressed mCV-N with anti-HIV activity preserved in the epithelial cell context and caused no significant immunoinflammatory changes as compared to the WT L. jensenii. These results highlight the translational value of the colonization model and justify further clinical investigation of the homeostatic and anti-HIV effectiveness of the L. jensenii derivates.
登录
查看更多内容
新型的阴道菌群定殖模型为杀生作用机理提供了新的见解。
影响因子：
6.4
作者：
Fichorova RN;Yamamoto HS;Delaney ML;Onderdonk AB;Doncel GF
通讯作者：
Doncel GF
影响因子：
11.8
作者：
Cherpes, TL;Melan, MA;Hillier, SL
通讯作者：
Hillier, SL
影响因子：
6.4
作者：
Hawes, SE;Hillier, SL;Holmes, KK
通讯作者：
Holmes, KK
使用细胞因子的组合在潜在感染的CD4+ T细胞中诱导HIV-1复制。
DOI：
10.1084/jem.188.1.83
发表时间：
1998-07-06
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
通讯作者：
--
DOI：
10.1073/pnas.1934747100
发表时间：
2003-09-30
影响因子：
11.1
作者：
Chang, TLY;Chang, CH;Lee, PP
通讯作者：
Lee, PP