SCID-Hu IC小鼠模型的建立及在rAd5HPV16L1-E7疫苗中的应用

目的研究脐血移植建立SCID-Hu IC小鼠嵌合动物模型以及在HPV16L1-E7重组腺病毒中的应用.方法 (1)采用尾静脉注射法给实验组注射脐血单个核细胞悬液,对照组注射RPMI 1640培养液;8周末检测血中人IgG抗体水平及人CD45、CD3、CD19;(2)将SCID-Hu IC小鼠和SCID小鼠,分别经腹腔注射和滴鼻途径给予rAd5HPV16L1-E7 重组病毒,于第4周末取血检测rAd5HPV16L1-E7重组病毒特异性IgG抗体、脾细胞培养液中IFN-γ的含量及T-淋巴细胞增殖.结果 (1)实验组中人IgG抗体阳性数为10/15,含量为(0.167±0.089);人CD45为(9.39±4.21),CD3为(3.25±3.99);CD19为(1.69±0.75),对照组未测出人IgG抗体以及CD45、CD3、CD19;(2)腹腔注射组和滴鼻组病毒特异性IgG抗体均为100%阳性;实验组培养液中IFN-γ的含量和T-淋巴细胞增殖实验病毒特异性蛋白刺激组均高于非刺激组(P<0.05);对照组SCID小鼠rAd5HPV16L1-E7 重组病毒特异性IgG抗体、T-淋巴细胞增殖和培养液中人IFN-γ均为阴性.结论 (1)采用人脐血移植SCID小鼠建立SCID-Hu IC小鼠嵌合动物模型是可行的;(2)人新鲜脐血移植重建的SCID-Hu IC小鼠具有对rAd5HPV16L1-E7 重组病毒的免疫应答能力,而未移植者(对照组)则对rAd5HPV16L1-E7重组病毒无免疫应答能力.     

尖锐湿疣患者HPV亚型与T细胞亚群的初步研究

目的　检测尖锐湿疣 (CA)患者人乳头瘤病毒 (HPV)亚型及 T细胞亚群。方法　用 S· P法检测 CA患者外周血CD3、CD2、CD8+T细胞。用 PCR法检测 CA患者皮损中的 HPV6/11,HPV16/18,HPV-DNA。结果　 61例患者疣体标本 HPV6/11阳性 40例 (A组 ) ;HPV16/18阳性 12例 (B组 ) ;HPV6/11阴性、HPV16/18阴性、HPV-DNA阳性 9例 (C组 ,简称 HPV其他型阳性 )。三组相比 ,CD3 +T细胞差异无显著性 (P>0 .10 )。CO4+T细胞 B组低于 A组 P<0 .0 2 5 )、低于 C组 (P<0 .0 2 5 ) ,差异有显著性。 CD8+T细胞 B组低于 A组 (P<0 .0 0 5 )、 C组低于 A组 (P<0 .0 0 5 ) ,有高度显著性差异。 CD4/CD8比值 ,B值低于 C组 (P<0 .0 2 5 ) ,有显著性差异。结论　 HPV16/18感染的 CA患者细胞免疫力明显降低。推测 HPV16/18亚型感染可能更易致尖锐湿疣复发     

宫颈鳞状细胞癌HPV16/18、p53、p21表达及意义

目的　探讨人乳头状瘤病毒 (HPV) 16型、18型及 p5 3、p2 1基因蛋白的表达情况以及与宫颈癌的关系。 方法　应用免疫组化二步法检测 5 0例宫颈鳞癌、4 0例正常宫颈黏膜中HPV16、HPV18、p5 3、p2 1的表达。肿瘤组分 <6 0岁和≥ 6 0岁 2个年龄组 ,观察HPV感染情况。结果　宫颈鳞癌中HPV16、HPV18、p5 3、p2 1表达分别为 4 8%、2 0 %、5 4 %和 5 0 % ,而正常宫颈黏膜中的表达分别为 10 %、0、0、10 % ,两者经统计学比较差异有显著性 (P <0 0 1) ;<6 0岁组和≥ 6 0岁组HPV16阳性率分别为 84 2 %和 2 5 8% ,年青组明显高于年老组 ,经统计学比较差异有显著性 (P <0 0 1)。结论　 (1)宫颈鳞癌的发生与HPV16、HPV18感染有密切关系 ,提示检测宫颈HPV16、HPV18感染情况对于宫颈鳞癌的随访和早期诊断有着重要的参考价值。 (2 )提示宫颈黏膜在HPV感染后 ,可能在p5 3、p2 1多种癌基因的共同作用导致宫颈鳞癌的发生发展。     

HPV 16 E6E7基因修饰树突状细胞疫苗诱导CTL致CaSki细胞凋亡体外实验研究

目的:采用人乳头状瘤-16(HPV-16)E6E7重组腺病毒(pAd-E6E7)转染树突状细胞(Dendritic cell,DC),观察基因修饰的DC疫苗诱导细胞毒性T淋巴细胞(Cytotoxic Tlymphocyte,CTL)致使CaSki细胞凋亡的效果。方法:将pAd-E6E7转染体外培养的小鼠未成熟树突状细胞制备DC疫苗,激光共聚焦显微镜观察转染的小鼠未成熟树突状细胞绿色荧光蛋白表达,流式细胞术检测转染前后小鼠树突状细胞表面标志物(CD40、CD86、MHCⅡ和CD11C)。DC疫苗诱导产生特异性细胞毒性T淋巴细胞,与CaSki细胞共培养后,采用DAPI、TUNEL及流式细胞术检测CaSki细胞凋亡情况。结果:pAd-E6E7成功转染体外培养的小鼠未成熟树突状细胞,体外转染效率约为40%～50%,成功制备了HPV16 E6E7基因修饰树突状细胞疫苗,诱导产生细胞毒性T淋巴细胞,经DAPI、TUNEL及流式细胞术检测证明CaSki出现凋亡。结论:以带有HPV16 E6E7基因的重组腺病毒载体转染DC制备基因修饰的DC疫苗,诱导CTL致使CaSki细胞出现凋亡。     

类风关滑膜浸润T细胞特性与致病机制研究

为研究类风湿关节炎时关节滑膜浸润性T细胞生物学特性与致病机制 ,对 10例RA患者滑膜液中淋巴细胞的免疫表型、细胞因子分泌格局与趋化因子受体表达进行了分析。用双色荧光标记法分别测定滑膜液中和外周血淋巴细胞表型与趋化因子受体表达。用ELISA方法检测滑膜液与外周血中IFN γ、IL 10、IL 4与IL 12的含量。结果是滑膜液中的CD4 + T淋巴细胞为 4 0 0 %± 11% ,CD8+ T细胞为 34 0 %± 6 % ,CD4 + 与CD8+ T细胞的比值为 1 2 ,显著低于外周血中CD4 /CD8的比值。滑膜液中CD3和CD2 5双阳性的活化T细胞占 16 %± 6 0 %。趋化因子受体CCR5表达较低 ,与外周血无明显差异。但CX CR3表达水平较高 ,为 16 %± 4 0 % ,远远高于外周血 (仅为 0 5 %± 0 3% )。IFN γ在滑膜液中含量很高 ,达 (36 6 7± 4 3 2 )pg/ml,而外周血中含量仅为 (2 0 1± 3 2 )pg/ml。IL 4含量未能测得 (<15pg/ml ) ,与外周血相似。IL 12含量为 (4 19 9±89 2 )pg/ml,远高于外周血中的含量 (6 5 32± 34 2 )pg/ml。IL 10含量为 (187 7± 34 5 )pg/ml,高于外周血中的含量 (85±12 7)pg/ml。在所测细胞因子中 ,关节滑膜液中IFN γ和IL 12的含量与外周血相比具有显著的统计学差异。表明RA关节滑膜液中有相当数量的T细胞浸润。这些T细胞     

乳腺浸润性导管癌中HPV18、HPV16感染的研究

目的 :了解乳腺浸润性导管癌中HPV18、HPV16的感染情况 ,分析其是否是乳腺癌发生的危险因素及与临床病理的相关性。方法 :根据HPV16、HPV18的DNA序列 ,合成相应特异的寡核苷酸片段 ,用加尾标记法制备地高辛标记探针 ,用原位杂交法检测 5 1例乳腺浸润性导管癌、10例相应正常乳腺上皮及 15例良性乳腺病变中HPV18、HPV16的感染 ,并分析其与患者发病年龄、肿块大小及淋巴结转移的相关性。结果 :浸润性导管癌中HPV18或 16的总阳性率达 70 6 % ,其中HPV18与HPV16的阳性率分别为 5 8 8%、4 5 1% ,均明显高于正常乳腺上皮的感染率 (30 0 %、10 0 % ;P <0 0 5 ) ;乳腺良性病变的HPV18、16阳性率分别是 6 0 0 %、6 0 % ,其中HPV18的阳性率亦显著高于正常乳腺上皮 (P <0 0 5 )。结论 :(1)HPV16和18可能是乳腺浸润性导管癌发生的致病因子 ,HPV18尚可能与乳腺良性病变的发生有关。 (2 )HPV的感染与患者年龄、肿块大小及淋巴结转移无相关性。     

HPV16 E5多表位肽免疫原性研究

目的 筛选人乳头瘤病毒16型（HPV16）E5蛋白多表位肽段，并评估其诱导小鼠产生特异性体液免疫和细胞免疫的免疫原性反应。方法 通过Uniprot获得HPV16 E5蛋白的氨基酸序列，采用EXPASY和SYFPEITHI等软件预测HPV16 E5蛋白的表位，筛选出富含B细胞和CTL表位的氨基酸肽段——HPV16 E5蛋白N端处aa28-46(LIRPLLLSVSTYTSLIILV)，进一步用该多表位肽免疫C57BL/6小鼠，评估其诱导产生特异性体液免疫和细胞免疫的反应。结果 该肽段可诱导小鼠产生较高水平的特异性IgG抗体，且抗体滴度随着免疫次数增加而升高，同时可诱导小鼠产生特异性的CTL反应，在效靶比为40∶1时，对靶细胞产生显著的特异性杀伤作用。结论 HPV16 E5蛋白多表位肽aa28-46(LIRPLLLSVSTYTSLIILV)具有良好的免疫原性，可为基于HPV16 E5蛋白的疫苗研究提供基础。     

卡介菌多糖核酸提高复发性生殖器疱疹患者CD8+ T细胞的细胞因子表达

目的　研究卡介菌多糖核酸 (BCG PSN)对复发性生殖器疱疹 (RGH)患者外周血CD8 T细胞IL 12、IFN γ、IL 4表达的影响。方法　应用流式细胞仪对 4 5例RGH患者和 15名健康志愿者外周血CD8 T细胞IL 12、IFN γ和IL 4的表达进行检测。结果　治疗前RGH患者外周血IFN γ和IL 12阳性CD8 T细胞百分率均显著下降 (P <0 .0 5 ) ,Tc1 Tc2平衡失调 (P <0 .0 5 )。治疗后BCG PSN组外周血IL 12和IFN γ阳性CD8 T细胞百分率显著升高 (P <0 .0 5 ) ,Tc1 Tc2恢复平衡。病例对照组IL 12、IFN γ及IL 4阳性CD8 T细胞百分率于治疗前后变化差异均无显著性 (P >0 .0 5 )。BCG PSN组与病例对照组治疗前后IL 12、IFN γ阳性CD8 T细胞百分率间差异均有显著性 (P <0 .0 5 )。BCG PSN组复发率较低 ,复发病情较轻。结论　RGH患者存在以Tc1水平低下为主的Tc1 Tc2比例失衡和IL 12表达水平低下 ;BCG PSN通过上调外周血CD8 T细胞IFN γ、IL 12的表达 ,纠正机体细胞因子失衡状态而减少疾病的复发     

人乳头状瘤病毒(HPV)16L1病毒样颗粒蛋白与HPV16L1基因联合免疫的体液免疫反应及其抗体的体外中和实验

目的用含有编码人乳头状瘤病毒(HPV)16L1和HPV16L1病毒样颗粒(VLP)蛋白联合免疫小鼠,与用HPV16L1重组表达质粒比较,观察VLP蛋白免疫对免疫小鼠产生抗体的增强作用,以及抗体的体外中和作用,寻找研制HPV16感染的预防性疫苗的有效途径。方法将c57BL／6小鼠,随机分为4组：Ⅰ组：pcDNA-L1(100μl/鼠),Ⅱ组：pcDNA—L1(70μl/鼠) HPV16K1 VLP,Ⅲ组：pcDNA3.1(100μl/鼠)空质粒,Ⅳ组：PBS缓冲液。质粒免疫3次,间隔3周。ELISA法检测其血清抗体,红细胞凝集实验和HPV16病毒样颗粒结合抑制实验体外检测抗体的中和活性。结果pcDNA-L1 HPV16L1 VLP较pcDNA—L1免疫组,3次免疫后的抗体水平均增高,尤其以第2次和第3次免疫后的抗体水平增加更显著。pcDNA—L1 HPV16L1 VLP联合免疫组血清的抑制活性高于pcDNA—L1免疫组,且．He[a细胞结合抑制实验染色呈阴性。结论HPV16L1 VLP联合免疫可以增加目的抗原的中和抗体产生,可能是HPV16有效预防性疫苗研制的更有希望的策略。     

HPV16、58型感染导致宫颈微环境中免疫因子变化及宫颈细胞中病毒整合的初步研究

目的：探讨人乳头状瘤病毒（Hapillomavirus,HPV）16和58型感染导致宫颈局部微环境中免疫因子白介素（Interleukin,IL）-12、IL-4,干扰素（Interferon,IFN）-γ浓度变化及宫颈细胞中HPV16/58病毒DNA整合与宫颈病变进展的关系。方法：收集2012年8月～2013年5月在广西医科大学附属肿瘤医院妇瘤科住院行HPV DNA分型检测,且组织病理结果诊断为宫颈癌患者的宫颈灌洗液共89例及宫颈脱落细胞140例,根据HPV分型结果将89例宫颈灌洗液分组为：①HPV16（＋）58例;②HPV58（＋）31例。应用酶联免疫法（Enzyme linked immunosorbent assay ,ELISA）法检测89例患者宫颈微环境中免疫因子IL-12、IL-4、IFN-γ的浓度;将140例宫颈脱落细胞根据HPV分型结果分组为：①HPV16（＋）106例;②HPV58（＋）34例。采用荧光定量PCR（ qRT-PCR）检测HPV16/58的E2和E6基因,根据E2/E6拷贝数比值判定HPV16/58 DNA的存在状态。结果：①HPV16（＋）组中IL-4浓度明显高于HPV58（＋）组（ P＜0.05）;IL-12、IFN-γ的浓度明显低于HPV58（＋）组（ P＜0.05）。②HPV16 DNA与HPV58 DNA的存在状态有着显著差异（ P＜0.05）,HPV16 DNA整合比率比HPV58 DNA整合比率明显增大。结论：HPV16和58型感染不但可以导致宫颈细胞外局部免疫微环境中的IL-12、IFN-γ的降低和（或） IL-4的升高,还可导致病毒在宫颈细胞内整合,通过宫颈细胞内外变化导致宫颈病变甚至宫颈癌的发生;HPV16较HPV58更容易造成宫颈组织发生癌变。     

Another human chimaera

Another human chimaera is described, which we are unable to classify because of limited family data. Evidence of chimaerism was confined to haemopoietic tissues. There were two red cell populations with differences in four blood group systems and, although the patient was female, chromosome analysis of cultured lymphocytes showed that 96% of cells had the male karyotype 46,XY. Possible mechanisms for the production of chimaeras are discussed.     

Further observations on the Birmingham chimaera.

The appropriate ABH-gene specified glycosyltransferases in the plasma of the Birmingham chimaera were estimated. These observatiions and the demonstration of A1Leb blood group specific glycosphingolipid in the plasma indicate that the minority population of red blood cells probably represents the true blood groups of the patient.     

Comparison of methods available for identification of Mycobacterium chimaera

ObjectivesMycobacterium chimaera is a recently described nontuberculous mycobacterium belonging to the Mycobacterium avium complex (MAC). Because this species is implicated in a worldwide outbreak due to contaminated heater–cooler unit water tanks during open-heart surgery, it has become mandatory for clinical microbiology laboratories to be able to differentiate M. chimaera from the other MAC species, especially M. intracellulare. Such identification has so far been restricted to specialized laboratories because it required the analysis of several gene sequences. The aim of this study was to evaluate commercial methods for identifying M. chimaera with regard to the reference gene sequencing ITS, the internal transcribed spacer 16–23S.MethodsForty-seven clinical and environmental isolates including 41 MAC were identified by (a) PCR sequencing of the ITS and hsp65 genes, (b) three molecular biology kits (INNO-LiPA Mycobacteria, GenoType Mycobacterium CM and GenoType NTM-DR) and (c) matrix-assisted desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using Microflex LT.ResultsThere was a high concordance for species determination between the reference ITS sequencing and the GenoType NTM-DR test (39/41, 95%), the INNO-LiPA Mycobacteria test (38/41, 93%) and the hsp65 sequencing (38/41, 93%). The GenoType Mycobacterium CM test did not distinguish M. chimaera from M. intracellulare. MALDI-TOF MS distinguished two M. chimaera–M. intracellulare groups separated from M. avium and from the other mycobacterial species on a score-oriented dendrogram, but it also failed to differentiate the two species.ConclusionsINNO-LiPA Mycobacteria and GenoType NTM-DR are efficient assays for M. chimaera identification in clinical microbiology laboratories.     

ACh receptor protein drives primary and memory autoantibody responses in chimeric human-SCID mice

The native antigen that drives the T-helper cells regulating production of muscle acetylcholine receptor (AChR) autoantibodies is unknown. Human T cell lines activated by autoantigens in vitro are of unproven relevance to B cell help. Here we report the functional interaction and unprecedented longevity of AChR-specific human T and B lymphocytes residing in SCID mice. Lymphoid cells from myasthenia gravis (MG) patients and healthy subjects were injected ip. Recombinant human AChR-alpha1-subunit-1-210 was injected after day 75. Human AChR-specific Ig was produced rapidly in MG-SCID mice challenged once. Only 1 of 32 control hu-SCID mice produced AChR-specific Ig. This required multiple immunizations, was initially cross-reactive with Torpedo AChR, and had a slow course. Thus, memory T and B lymphocytes specific for human AChR-alpha1-subunit are readily demonstrable in MG patients, interact to produce autoantibody of the same restricted specificity found in the donor's serum, and are long-lived without exogenous autoantigen challenge. In healthy subjects, AChR-specific lymphocytes are infrequent and exhibit naive response characteristics, including apparent affinity maturation of Ig specificity.     

Prosthetic Valve Endocarditis and Bloodstream Infection Due to Mycobacterium chimaera

Prosthetic valve endocarditis (PVE) due to fast-growing nontuberculous mycobacteria (NTM) has been reported anecdotally. Reports of PVE with slowly growing NTM, however, are lacking. We present here one case of PVE and one case of bloodstream infection caused by Mycobacterium chimaera. Randomly amplified polymorphic DNA (RAPD)-PCR indicated a relatedness of the two M. chimaera strains. Both patients had heart surgery 2 years apart from each other. A nosocomial link was not detected.     

Mycobacterium chimaera: Coming out of Nowhere to Dominate 2016 Infection Prevention Discussions

Mycobacterium chimaera is a bacterium of the 21st century. The organism was named in 2004 when molecular assays showed it to belong to the Mycobacterium avium complex. More specifically, it was found to be a close relative of Mycobacterium intracellulare. Merely a decade later, M. chimaera turned out to be the cause of infections associated with heater-cooler units used during open-heart surgery. There are still less than 100 confirmed cases worldwide, but the mycobacterial species has captured the attention of patients and caregivers alike because many of these infections are manifested months to years following surgery. No one currently knows the number of people who have been infected with M. chimaera or how many will ultimately be affected.     

A human dispermic chimaera first suspected from analyses of the blood group gene-specified glycosyltransferases

The red cells of a normal male blood donor, K.S., were first grouped as B but he was found to lack anti-A in his serum. Closer investigation revealed that his red cells had very weak A activity, demonstrable only by absorption and elution of anti-A. He is a non-secretor of ABH and a secretor of Lea. Blood group A-, B and H-gene specified glycosyltransferases were detected in his serum. In contrast to the finding of a B antigen of normal strength on his red cells, the B transferase in his serum was only about 30% of the normal level and, despite the very weak A activity of K.S's red cells, the A transferase level was about 50% of that found in the serum of group A individuals with normal strength of A antigen. Moreover, the A transferase on the basis of its pH optimum, Km values for donor and acceptor substrates, activation by divalent cations, isoelectric focusing profile and capacity to convert O to A-active cells, was characterized as the product of an A1 gene. A family study showed that K.S's wife is group A2 and that they have two sons, one group A2 and the other group B. The group B son is assumed to have inherited a B gene from the propositus but the level of B transferase in the son's serum is three times as high as that in his father's serum. The wife of the propositus and his group A2 son have normal A2 transferases in keeping with their A2 red cell status. The A2 son therefore appears to have inherited an A2 gene from his mother but neither the A1 nor the B gene shown to be carried by his father. The distribution of transferase activities in K.S's red cells differs from that in his serum. A level of B transferase within the normal range was found in his red cell membranes but a very low level of A transferase was detected. The discrepancies between the serum transferases and ABO red cell group, together with the pattern of inheritance within the family, led to a suspicion of chimaerism. This was confirmed by the finding of fibroblasts with the female 46XX karyotype in cultures of the propositus' skin. These results suggest that K.S. is a dispermic chimaera with two different cell lines of the genotypes BO and A1O or A1A1. The group A2 son is assumed to have inherited an O gene from his father. It seems probable that K.S.'s bone marrow and reproductive organs are comprised predominantly of the XY cell line which carried the blood group BO genotype whereas his skin and other tissues which contribute the A1 transferase to his plasma, are partly made up of the XX cell line which carries the blood group A1O or A1A1 genotype.     

Differential drug susceptibility patterns of Mycobacterium chimaera and other members of the Mycobacterium avium-intracellulare complex

Objectives

To determine MIC distributions for Mycobacterium chimaera, Mycobacterium intracellulare, Mycobacterium colombiense and Mycobacterium avium, and to derive tentative epidemiological cut-off (ECOFF) values.