两种分泌型人谷氨酸脱羧酶65片段DNA疫苗的构建与鉴定

目的:构建和鉴定分泌型人谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)65片段DNA疫苗.方法:从人GAD65质粒中扩增出GAD190-315和GAD490-570两个片段的cDNA,分别与hIL-2信号肽cDNA基因拼接,将拼接后的片段克隆入pBudCE4.1真核表达载体,测序鉴定,并用Western印迹检测融合基因的表达.结果:核酸序列测定表明克隆的融合基因序列与报告序列一致,开放阅读框正确,Western印迹检测显示这两种人GAD65片段分泌表达.结论:两种分泌型人GAD65片段DNA疫苗均成功构建,为1型糖尿病的预防提供了实验基础.     

LPS触发的TLR4对NF-κB和IRF3信号通路的调控差异

目的探讨脂多糖(LPS)触发的Toll样受体4(TLR4)对NF-κB和干扰素调节因子3(IRF3)信号通路的调控差异。方法 LPS刺激小鼠原代腹腔巨噬细胞,用免疫荧光法检测TLR4及两种转录因子p65及IRF3的定位,用Western blot方法检测转录因子p65和IFR3的磷酸化水平。结果巨噬细胞经LPS刺激30 min内,细胞膜上的TLR4荧光强度增加(P0.01),胞质中TLR4荧光强度显著增加(P0.01),且与早期内体抗原1(early endosome antigen 1,EEA1)共定位;当LPS刺激90和180 min时,胞膜和胞质内的TLR4信号均显著下降(P0.01),与EEA1共定位的信号也明显减少(P0.01)。静息状态下,TLR4的下游信号通路分子p65和IRF3的荧光信号均出现在胞质中;LPS刺激后,两者的荧光信号在细胞核中逐渐增加(P0.05),但p65荧光信号比IRF3增强得更早,且更持久。p65磷酸化的修饰也明显早于IRF3,且持续时间更长。结论 TLR4活化后的定位变化导致其下游IRF3信号通路的传递时间明显延后,且比NF-κB信号通路短暂。     

匹诺塞林缓解大鼠肝细胞系BRL-3A低氧/复氧损伤

目的探讨匹诺塞林(PIN)对低氧/复氧(H/R)诱导的大鼠肝细胞损伤的保护作用及其可能机制。方法将大鼠肝细胞(BRL-3A细胞系)分为正常对照组、PIN实验组、低氧复氧损伤模型组和PIN预处理组。CCK-8检测细胞存活率;Annexin V-FITC/PI双染法流式细胞计量术检测细胞凋亡;检测细胞培养液中谷丙转氨酶(ALT)活性;ELISA检测TNF-α和IL-1β含量;Western blot检测细胞中TLR4、IκB-α和NF-κB P65蛋白水平;RT-q PCR检测细胞中TLR4、IκB-α和NF-κB P65mRNA表达。结果在H/R条件下细胞存活率明显降低(P0.01),细胞凋亡率增高(P0.001),ALT活性升高(P0.01),IL-1β和TNF-α含量增多(P0.01),TLR4和NF-κB P65蛋白与mRNA表达水平显著提高(P0.01)而IκB-α降低(P0.05);经PIN预处理后,细胞存活率显著提高(P0.01),细胞凋亡率显著减小(P0.001),ALT活性降低(P0.01),IL-1β和TNF-α含量降低(P0.01),TLR4和NF-κB P65蛋白与mRNA表达水平明显降低(P0.01)而IκB-α升高(P0.05)。结论 PIN对H/R诱导的BRL-3A肝细胞的损伤具有保护作用,且该作用可能是通过TLR4/NF-κB信号通路实现的。     

HSP65-PRA identification of non-tuberculosis mycobacteria from 4892 samples suspicious for mycobacterial infections

Various molecular methods have been used for the rapid identification of mycobacterial species. In this survey, evaluation of antibiotic resistance and PCR-restriction fragment length polymorphism analysis (PRA) of the hsp65 gene was carried out for identification of non-tuberculosis mycobacteria (NTM) isolates from different clinical specimens. Forty-eight different mycobacterial isolates were selected and followed by the conventional and PRA of hsp65 for species identification. The antibiotic susceptibility test was carried out according to standard methods. A 439 bp PCR product of hsp65 in all selected isolates was amplified and digested with the BstEII and HaeIII restriction enzymes. The restriction fragment length polymorphism (RFLP) patterns were analyzed for species identification. Using PRA for 48 mycobacterial selected isolates, including 15 M. tuberculosis, one M. bovis and all 32 isolates of NTM, revealed 11 different species among the NTM isolates. The most frequent NTM isolates were M. kansasii, M. gordonae III, M. marinum, M. chelonae, M. scrofluaceum and M. gastri. In most cases, the PRA results were perfectly in accordance with the classical biochemical method. Combination of resistance to rifampin and isoniazid was present among M. kansasi, M. gordoniae III, M. scrofluaceum, M. chelonae, M. marinum, M. gastri, M. gordoniae II and M. trivale isolates. A high incidence of co-resistance to six, five, four and three anti-TB drugs was observed in 18.5%, 9.1%, 6.6% and 11.7% of all NTM isolates, respectively. Our results showed that PRA, in comparison with classical methods, is rapid and accurate enough for the identification of mycobacterial species from LJ medium. Additionally, we found that in Iran we have a highly diverse population of NTM isolates among patients suspected of having TB.     

Suppression of NF-κB p65 nuclear translocation and tumor necrosis factor-α by Pongamia pinnata seed extract in adjuvant-induced arthritis

Abstract

Pongamia pinnata is a plant known for its therapeutic usage in Indian traditional medicine. Despite the controversy regarding toxic flavonoid and erucic acid content, the seed of this plant is consumed in tribal medicine and its oil is used in Ayurveda to treat psoriasis and arthritis. This study explored the potential anti-arthritic effects of a P. pinnata seed (hexane) extract (PSE) at non-lethal doses in an adjuvant-induced arthritic rat model; possible mechanisms of any observed effects were also explored. After establishing the lethal doses arising from oral exposure to the extract, the material was administered per os daily at two doses (0.3?g/kg/day; 0.5?g/kg/day) to arthritic rats. Other rats received indomethacin or vehicle (control). Treatments were performed for a total of 14 days. One day after the final exposure, the rats were euthanized to permit harvest of various cells, blood, and tissues for analyses. Paw diameter and tissue myeloperoxidase activity in the paws were evaluated as indices for edema and neutrophil infiltration into the tissue. The severity of arthritis in the experimental rats was assessed via measures of urinary hydroxyproline (HP) and glucosamine, and of serum pro-inflammatory TNFα and anti-inflammatory IL-10. The extent of NF-κB p65 nuclear translocation in peritoneal macrophages harvested from naïve rats and then treated in vitro was also assessed. The results indicated that exposure to PSE significantly decreased paw diameter, tissue myeloperoxidase level, and levels of urinary HP and glucosamine, as well as of serum TNFα and IL-10 in adjuvant-injected (arthritic) rats. In vitro PSE treatment also resulted in a marked inhibition of NF-κB p65 nuclear translocation in primary cultures of peritoneal macrophages. Thus, PSE appears to be able to prevent experimental arthritis, in part, by helping to maintain the balance between pro- and anti-inflammatory cytokines and by inhibiting NF-κB activation.     

Characteristics of in‐vitro phenotypes of glutamic acid decarboxylase 65 autoantibodies in high‐titre individuals

Previous studies have indicated phenotypical differences in glutamic acid decarboxylase 65 autoantibodies (GADA) found in type 1 diabetes (T1D) patients, individuals at risk of developing T1D and stiff‐person syndrome (SPS) patients. In a Phase II trial using aluminium‐formulated GAD₆₅ (GAD‐alum) as an immunomodulator in T1D, several patients responded with high GADA titres after treatment, raising concerns as to whether GAD‐alum could induce GADA with SPS‐associated phenotypes. This study aimed to analyse GADA levels, immunoglobulin (Ig)G1–4 subclass frequencies, b78‐ and b96·11‐defined epitope distribution and GAD₆₅ enzyme activity in sera from four cohorts with very high GADA titres: T1D patients (n = 7), GAD‐alum‐treated T1D patients (n = 9), T1D high‐risk individuals (n = 6) and SPS patients (n = 12). SPS patients showed significantly higher GADA levels and inhibited the in‐vitro GAD₆₅ enzyme activity more strongly compared to the other groups. A higher binding frequency to the b78‐defined epitope was found in the SPS group compared to T1D and GAD‐alum individuals, whereas no differences were detected for the b96·11‐defined epitope. GADA IgG1–4 subclass levels did not differ between the groups, but SPS patients had higher IgG2 and lower IgG4 distribution more frequently. In conclusion, the in‐vitro GADA phenotypes from SPS patients differed from the T1D‐ and high‐risk groups, and GAD‐alum treatment did not induce SPS‐associated phenotypes. However, occasional overlap between the groups exists, and caution is indicated when drawing conclusions to health or disease status.     

重组BCG疫苗和结核杆菌HSP65 DNA疫苗免疫原性的比较研究

目的　研究重组BCG(recombinantBCG ,rBCG)疫苗和人结核杆菌热休克蛋白 (heatshockprotein ,HSP) 6 5DNA疫苗对小鼠免疫应答的影响 ,评价两种疫苗的免疫原性。方法　用rBCG疫苗和HSP6 5DNA疫苗免疫BALB/c小鼠 ,免疫 8周时 ,采血、取腹腔巨噬细胞和脾脏进行实验。以NO释放量检测巨噬细胞吞噬活性 ,以淋巴细胞刺激指数 (SI)反映细胞增殖能力 ,以ELISA试剂盒检测血清及脾淋巴细胞培养上清的IL 2和IFN γ的含量。结果　rBCG疫苗以 10 6CFU/鼠的剂量经皮下免疫小鼠后 ,其脾淋巴细胞的增殖能力明显高于对照组 ,BCG组和HSP6 5DNA疫苗组 (P <0 .0 5 ) ;腹腔巨噬细胞一氧化氮 (NO)的含量和血清IL 2的含量明显高于对照组 (P <0 .0 1) ;血清IFN γ的含量明显高于HSP6 5DNA疫苗组 (P <0 .0 5 ) ;其余测定参数均有升高趋势 ,但差异无显著性 (P >0 .0 5 )。而HSP6 5DNA疫苗以 5 0 μg/鼠的剂量经肌注免疫小鼠后 ,其血清IL 2的含量明显高于对照组 (P <0 .0 1) ;血清IFN γ的含量明显低于rBCG组 (P <0 .0 5 ) ;其余测定参数与其他各组相比差异均无显著性(P >0 .0 5 )。结论　本实验结果显示 ,rBCG疫苗具有良好的免疫原性 ,能明显增强BALB/c小鼠的免疫应答反应。HSP6 5DNA疫苗亦能刺激机体的免疫应答 ,但其反应强度似乎不     

Activation of nuclear factor-kappa B correlates with tumor necrosis factor-alpha in oral lichen planus: a clinicopathologic study in atrophic-erosive and reticular form

Backgroud:  Nuclear factor-kappa B (NF-κB) is believed to be involved in the pathogenesis of various inflammatory diseases, including oral lichen planus (OLP). The objective of the present study was to investigate the possible relationship between NF-κB activation and expression of tumor necrosis factor-alpha (TNF-α) in OLP and their expression pattern in relation to several clinical features.
Methods:  Thirty OLP cases were divided into atrophic-erosive form (14 cases) and reticular form (16 cases) according to their clinical manifestations. The expression of NF-κB p65 and TNF-α of both two groups were investigated by immunohistochemical staining, and the percentage of positive cells was calculated in each case. Biopsies of 10 normal oral mucosa (NOM) also underwent the same procedure as controls.
Results:  Nuclear factor-kappa B p65 nuclear staining was found in nuclei of basal and suprabasal epithelial keratinocytes in OLP, however, no positive staining was found in NOM. Positive TNF-α staining was detected in cytoplasm of basal epithelial keratinocytes in OLP, and only scattered staining was detected in NOM. Expression of NF-κB p65 and TNF-α were significantly different with respect to clinical forms and lesion sites ( P  < 0.05), except for genders ( P  > 0.05) in 30 OLP cases. NF-κB nuclear staining positively correlated ( r  = 0.676, P  < 0.01) with TNF-α overexpression in OLP.
Conclusions:  Nuclear factor-kappa B activation and its correlation with overexpression of TNF-α may play an important role in pathogenesis of OLP. There might be a positive regulatory loop between NF-κB and TNF-α, which may contribute to inflammation in OLP; NF-κB may also protect epithelial keratinocytes from excessive apoptosis.     

牙龈鳞癌中p65 和VEGF 的表达及意义

 目的 研究p65和VEGF在牙龈癌组织中的表达及意义。方法 应用免疫组化SP法检测46例牙龈鳞癌组织中p65和VEGF的表达，并与临床病理指标结合进行分析。结果 牙龈鳞癌组织中p65与VEGF阳性率分别为63．04％与56．52％。p65的表达与牙龈鳞癌的病理分级、淋巴结转移无相关性，与临床分期明显相关（P〈O．05）。VEGF的表达与牙龈鳞癌的病理分级、淋巴结转移密切相关（P〈O．05）。p65与VEGF在牙龈鳞癌的表达呈正相关。结论 p65和VEGF参与牙龈鳞癌的发生发展过程，可作为评估牙龈鳞癌生物学行为的一项指标，用于识别高危转移和不良预后者。