Enhancing dendritic cell vaccine potency by combining a BAK/BAX siRNA-mediated antiapoptotic strategy to prolong dendritic cell life with an intracellular strategy to target antigen to lysosomal compartments

Dendritic cell (DC)-based vaccines have become important in immunotherapeutics as a measure for generating antitumor immune responses. We have previously demonstrated that linkage of the antigen gene to a lysosomal targeting signal, a sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1), enhances the potency of DC-based vaccines. DCs have a limited life span, hindering their long-term ability to prime antigen-specific T cells. In this study, we attempted to further improve the potency of a DC vaccine that targets human papilloma virus 16 (HPV16) E7 to a lysosomal compartment (DC-Sig/E7/LAMP-1) by combining a strategy to prolong DC life. We show that small interfering RNA-targeting Bak and Bax proteins can be used to allow transfected DCs to resist being killed by T cells. This is done by downregulating these proapoptotic proteins, which have been known as so-called gate keepers in mitochondria-mediated apoptosis. DCs expressing intact E7 or Sig/E7/LAMP-1 became resistant to attack by CD8+ T cells after transfection with BAK/BAX siRNA, leading to enhanced E7-specific T cell activation in vitro and in vivo. More importantly, vaccination with E7-presenting DCs transfected with BAK/BAX siRNA generated a strong therapeutic effect against an E7-expressing tumor in vaccinated mice, compared with DCs transfected with control siRNA. Our data indicate that a combination of strategies to enhance intracellular Ag processing and to prolong DC life may offer a promising strategy for improving DC vaccine potency.     

Alpha lipoic acid attenuates microvascular endothelial cell hyperpermeability by inhibiting the intrinsic apoptotic signaling

BACKGROUND: This study examined whether alpha lipoic acid (ALA), an antioxidant with anti-apoptotic properties, synthesized in mitochondria of endothelial cells, would inhibit intrinsic apoptotic signaling and microvascular endothelial cell hyperpermeability. METHODS: Rat lung microvascular endothelial cells were transfected with BAK (BH3) peptide (5 microg/mL) or active caspase-3 (5 microg/mL) and were pretreated with ALA (10 and 100 micromol/L). Hyperpermeability was determined using fluorescein isothiocyanate albumin-flux across the cells grown as monolayer. Reactive oxygen species (ROS) formation was determined using 123 dihydrorhodamine and mitochondrial membrane potential using JC-1. Cytochrome c levels and caspase-3 activity were determined using an enzyme-linked immunosorbent assay and a fluorometric assay, respectively. RESULTS: ALA (100 micromol/L) pretreatment attenuated BAK (BH3)-induced hyperpermeability and ROS formation. ALA restored BAK (BH3)-induced collapse in mitochondrial membrane potential and decreased BAK (BH3)-induced cytochrome c release and caspase-3 activity. CONCLUSIONS: These findings suggest that ALA attenuates BAK-induced monolayer hyperpermeability through the inhibition of ROS formation and intrinsic apoptotic signaling.     

Zur Frage der Transformation von Atem- in Blutalkoholkonzentrationen Experimentelle Untersuchungen mit einem geeichten Atemalkoholtestgerät Alcotest 7110 Evidential MK III

Mit dem Alcotest 7110 Evidential MK III, durch die PTB in Braunschweig zugelassen und geeicht, steht ein hochwertiges Ger?tesystem zur Messung der Alkoholkonzentration in der Ausatemluft zur Verfügung. Der Umrechnungsfaktor von Atem- in Blutalkoholkonzentrationen wurde von Schoknecht [17] im Gutachten des Bundesgesundheitsamtes 1992 experimentell als BAK/AAK-Quotient mit 2,098 ±0,11 bestimmt und auf 2,1 gerundet. Der Mittelwert von 455 zeitgleichen Wertepaaren der vorliegenden Arbeit lag bei 2,2. Der Maximalwert betrug 3,29, der Minimalwert 0,74. Die Streubreite stimmte im Wesentlichen mit den theoretischen Berechnungen von Wehner et al. [19] überein. Die gefundenen Quotienten w?ren theoretisch als Umrechnungsfaktoren von AAK in BAK denkbar. Aufgrund der sehr gro?en Variabilit?t sollte aber grunds?tzlich in forensischen Gutachten eine Transformation von AAK in BAK und umgekehrt nicht erfolgen. Demzufolge erscheint auch eine Rückrechnung mit „transformierten” BAK-Werten oder die Berücksichtigung von angegebenem Nachtrunk nicht m?glich. Insbesondere ist ein strafrechtsrelevanter AAK-Grenzwert auch nicht durch einfache Multiplikation zu errechnen, sondern bedarf einer breiten experimentellen psycho-physiologischen und verkehrsmedizinischen Grundlagenforschung.      

柔红霉素对培养人视网膜色素上皮细胞BAK和BCL-XL表达的影响

目的 观察BAK和BCL-XL在正常培养和经柔红霉素处理后的人视网膜色素上皮(retinal pigment epithelium,RPE)细胞中的表达。方法 人RPE细胞培养液中加入终浓度为200μg·L~(-1)柔红霉素作用12h,更换新鲜培养液继续培养24h后,运用抗BAK和BCL-XL的多克隆抗体进行免疫细胞化学染色,观察2种基因在RPE细胞中表达的变化。结果 BAK和BCL-XL在正常RPE细胞中均存在表达,但前者的染色强度大于后者(P<0.05),2者光密度分别为56.4±5.7和32.4±4.7,经柔红霉素处理后,RPE细胞BAK的染色强度增加(P<0.05),而BCL-XL的染色强度不变(P>0.05),其光密度值分别为79.2±6.5和34.7±3.9。结论 BAK和BCL-XL在人RPE细胞中存在表达。柔红霉素可以促进BAK的表达,这可能是其诱导RPE细胞凋亡的机理之一。     

SHARPIN regulates mitochondria-dependent apoptosis in keratinocytes

Background

The chronic proliferative dermatitis mutation (CPDM) in mice, due to Sharpin deficiency (Sharpin^cpdm), is a multisystem disorder characterized by peripheral blood eosinophilia and eosinophil infiltration of affected tissues including the skin, bone marrow, spleen, lung, heart, and other organs. The epidermis has numerous apoptotic keratinocytes which increase with age, coalesce, form vesicles, and rupture causing ulceration.

Objective

To clarify the molecular pathways involved in the keratinocyte apoptosis caused by loss of function of SHARPIN in mice.

Method

10-week-old Sharpin^cpdm and wildtype mice were used for experiments. Ultrastructural changes of skin were evaluated by transmission electron microscopy. Cross points of mitochondrial pathway were analyzed by in vitro and in vivo cellular and molecular assays.

Results

77.5% skin cells in Sharpin^cpdm mice were functionally apoptotic and dead cells, compared to only 18.1% unhealthy skin cells in wildtype mice, indicated by annexin-V/propidium iodide FACS analysis. Mitochondria in keratinocytes were disrupted containing prominent electron dense inclusions and membrane potential depolarization, accompanied by a shift in protein expression between the anti-apoptotic BCL2 and pro-apoptotic BAX proteins. Enzymatic activities of caspases 9 and 3, but not 8, were markedly increased in Sharpin^cpdm keratinocytes. Caspase-3 was cleaved in most cells in skin of 10-week-old mutant mice.

Conclusion

The present results indicated that keratinocyte apoptosis in Sharpin^cpdm mice was regulated by an intrinsic caspase-dependent mitochondria pathway.     

后路椎体间融合治疗腰椎滑脱症对腰椎生理前凸的影响

目的探讨后路椎弓根螺钉内固定加椎体间BAK融合手术治疗低度(Ⅰ～Ⅱ度)腰椎滑脱症的临床与放射学结果的变化。方法行椎弓根螺钉内固定加椎体间融合器BAK融合者共26例。峡部不连性18例,退变性8例。L4滑脱14例、L5滑脱12例。测量所有患者的术前、术后、最后一次随访的站立位侧位X线片,测量项目包括腰椎曲线指数(irldex of lumbar spinal curvaturc,LCI)、L4、5、L5S1节段前凸角、全腰椎前凸角。结果排除1例未融合数据,术后融合节段的节段前凸角明显改善,且经随访后无明显丢失。术前、术后、随访时全腰椎前凸角与LCI的差别无显著性意义。结论后路椎弓根螺钉内固定加椎体间BAK融合治疗腰椎滑脱症可以有效改善并维持腰椎的正常生理曲线。     

重组hIFN-α-2b-BCG对人外周血免疫细胞因子表达调节及抗膀胱肿瘤作用的实验研究

目的：探讨重组卡介苗（bacilli Calmette-Guérin BCG）对人外周血单个核细胞（peripheral blood monocytes,PBMC）IFN-γ、IL-12和TNF-α细胞因子表达、免疫增强及抗肿瘤效应。方法：重组BCG、野生型BCG和外源性人IFN-α-2b＋野生BCG,分别与PBMC共同培养,以单纯PBMC为对照,在不同时段收集培养液上清,经ELISA法检测上清液中IFN-γ、IL-12和TNF-α的表达水平;将BCG激活杀伤细胞（bacilli Calmette-Guérin activated killercell,BAK细胞）与人膀胱肿瘤细胞EJ细胞共同培养,采用乳酸脱氢酶释放试验（LDH）检测活化免疫细胞的抗肿瘤效果。结果：重组BCG诱导PBMC产生细胞因子的表达明显高于同浓度的野生型BCG组和野生型BCG＋外源性人IFN-α-2b组（P〈0.05）;重组BCG诱导的PBMC抗癌效应均高于对照组诱导的抗癌效果（P〈0.05）。结论：重组BCG诱导人PBMC高表达IFN-γ、IL-12和TNF-α,并通过增强人PBMC细胞因子的表达来提高抗膀胱肿瘤作用。     

人参二醇单体对大鼠心肌作用的单钙通道分析及ESR谱研究

应用细胞封接式膜片钳方法，在Ｗｉｓｔａｒ大鼠的单个心室肌细胞上，观察了人参二醇组皂甙Ｒｃ，Ｒｄ２００μｇ／ｍｌ对钙通道单通道活动的影响，并与钙通道阻滞剂异搏定３７．５μｇ／ｍｌ、钙通道激动剂ＢＡＹ 中８６４４μｍｍｏｌ／Ｌ对照，证明Ｒｃ对钙通道有阻滞作用；而Ｒｄ则无此作用。用电子自旋共振法测定了培养心肌细胞的自由基含量，Ｒｃ３０μｇ／ｍｌ能显著抑制黄嘌呤０．４２ｍｍｏｌ／Ｌ－黄嘌呤氧化酶５．３ｎｍ     

Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway

1. Download : Download high-res image (208KB)
2. Download : Download full-size image

     

颈椎椎体融合器的临床应用（附135例报告）

目的：观察比较3种颈椎椎体间融合器前路减压后椎体间融合的固定及融合效果。方法：采用BAK、CHTF和InterFix3种颈椎椎体间融合器行颈椎病及颈椎间盘突出症前路减压后椎体间融合135例,其中BAK 65例,CHTF42例,InterFix技术28例。术后颈椎X线片及CT检查,观察手术椎节的稳定性和融合情况。结果：随访8－31月,施术节段稳定,3种颈椎椎体间融合器固定融合率无显著差异。结论：颈椎椎体间固定融合技术使施术椎节立即稳定,避免了自体植骨引起的并发症,可作为替代传统颈前路椎体间植骨融合术的方法之一。