重组人可溶性CD40配体对树突状细胞体外分化、成熟及功能的影响

用Ficoll密度离心及贴壁法获得外周血单个核细胞 (PBMC ) ,PBMC经细胞因子组合诱导分化成树突状细胞 (DC ) :GM CSF (10 0ng/ml)与IL 4 (5 0ng/ml)诱导 5d后 ,分别加入TNF α (10ng/ml)或rhsCD4 0L (2 μg/ml)继续培养 4d ;倒置显微镜下观察DC形态 ,免疫荧光标记和流式细胞术分析DC表型 (CD1a、CD80、CD83、HLA DR、CD14、CD16、CD19)及摄取FITC Dextran抗原的能力 ;3 H TdR掺入法检测DC刺激自体混合淋巴细胞体外增殖反应 (MLR )能力 ;ELISA法分析DC培养上清中IL 12的水平 ;Trans well细胞趋化实验检测DC对自体外周T淋巴细胞的趋化能力。发现经rhsCD4 0L刺激的DC表面分子 (CD1a、CD80、CD83、HLA DR )的表达水平高于经典的细胞因子组合组 (GM CSF +IL 4 +TNF α ) ,同时rhsCD4 0L刺激后的DC摄取FITC Dextran的能力下降而刺激自体MLR和分泌IL 12的能力明显提高 ;而且rhsCD4 0L诱导的DC表面趋化因子受体CXCR4的表达水平及对自体外周T淋巴细胞的趋化能力均强于TNF α或FL激发的DC。rhsCD4 0L在体外不仅具有显著的诱导DC分化 ,促进DC成熟的功能 ,而且经rhsCD4 0L作用的DC能更有效地激发T淋巴细胞     

钙离子载体在诱导人外周血单核细胞分化为树突状细胞中的作用

目的研究钙离子载体(CI)能否诱导人外周血单核细胞(PBMC)分化为树突状细胞(DC)，并初步探讨其信号转导途径。方法分离健康献血者的PBMC，在体外用CI(A23187)或人重组粒细胞／巨噬细胞集落刺激因子(rhGM—CSF)和CI培养40h，或rhGM-CSF和白细胞介素4(IL-4)培养7d，部分PBMC预先用W-7或CsA或K35926处理30min后，再加入上述细胞因子或CI。相差显微镜下观察细胞形态，流式细胞仪检测细胞表面CD14、CD80、CD86、CD83等分子的表达，MTT比色法检测其对同种异体混合T淋巴细胞的刺激增殖作用。结果健康献血者的PBMC经CI培养40h，或rhGM—CSF与CI培养40h，或rhGM-CSF与IL-4培养7d，均可获得DC的典型形态和表面分子的表达，包括CD14表达下调、共刺激分子(CD80、CD86)表达上调，以及较强刺激同种异体混合T淋巴细胞增殖的作用；CI诱导的DC其CD14分子的下调及CD83分子的上调更明显，刺激混合T淋巴细胞的增殖能力更强；rhGM-CSF可协同CI诱导PBMC向DC的分化。经rhGM—CSF及CI处理的PBMC，其形态、表面标志物及对T细胞的刺激增殖能力，均受到W-7或CsA或KT5926不同程度的抑制；而rhGM-CSF及IL-4所诱导的PBMC其形态、表面分子的表达以及刺激T细胞增殖的作用却不受上述抑制剂的影响。结论CI可快速诱导PBMC向DC分化，其分化过程可能受控于Ca^2 /钙调蛋白及其下游的多个细胞信号转导途径的调节。     

用含有特定细胞因子的无血清培养基培养慢性乙型肝炎病人的树突状细胞

目的　体外扩增慢性乙肝病人的树突状细胞 (dendriticcell,DC) ,从细胞表型和功能上鉴定和研究。方法　用含GM CSF和IL 4的无血清培养基AIM V体外培养慢性乙肝病人外周血单个核细胞 ,获得树突状细胞。流式细胞仪检测细胞表型 ,IL 1 2ELISA试剂盒检测DC分泌IL 1 2的水平 ,并观察加入细胞因子TNF α后对DC培养的影响。结果　慢性乙肝病人的外周血单个核细胞用AIM V培养及细胞因子诱导后 ,经贴壁法纯化 1 0 0mL可获得 0 .5× 1 0 7～ 1 .5× 1 0 7成熟的具有典型形态的DC ,加入TNF α后 ,CD83阳性占 60 .80 % ,明显高于未加组 (P <0 .0 5) ,IL 1 2分泌较未加TNF α组增高近 1 0倍。结论　①慢性乙肝病人的DC可用AIM V无血清培养基及特定的细胞因子诱导在体外大量获得 ,TNF α是诱导DC成熟的重要的细胞因子。②典型的细胞形态和CD1 4 -、HLA DRhigh+、CD86high+的细胞表面分子特征可作为临床上快速鉴定培养DC的标志。     

单核细胞源性树突状细胞表达淋巴细胞趋化因子mRNA的初步研究

目的:体外诱导、培养单核细胞源性树突状细胞(DC),研究其淋巴细胞趋化因子(lymphotactin,Lptn)mRNA表达的动态变化。方法:采用密度梯度离心的方法分离人外周血中的单个核细胞(PBMC),用重组人粒细胞-巨噬细胞集落群体刺激因子(rhGM-CSF)、重组人白细胞介素-4(rhIL-4)刺激贴壁的单核细胞,诱导培养DC,第6天用重组人肿瘤坏死因子-α(rhTNF-α)诱导DC成熟。用流式细胞术检测成熟和未成熟的DC表面分子CD1a和CD83;在电镜下观察成熟DC的形态;以RT-PCR法扩增其LptncDNA并克隆至pGM-TEasyT载体中,测序;以RT-PCR结合凝胶成像分析系统,半定量分析培养3、5及7dDC的LptnmRNA表达的强度。结果:电镜观察培养7d的细胞具有典型的DC形态,流式细胞术检测DC表面分子CD83呈高水平表达。用RT-PCR法克隆的cDNA序列与GenBank中U23772(登陆号)提供的序列一致。培养3d的DC不表达LptnmRNA,培养7d的DC较培养5d的DCLptnmRNA表达增强。结论:单核细胞源性DC能表达LptnmRNA,随着DC的成熟,LptnmRNA的表达增强。     

补体C5b-9复合物促进树突状细胞成熟

探讨补体C5b-9复合物对树突状细胞成熟及免疫学功能的影响。rhGM-CSF+rhIL-4体外诱导单核细胞分化为不成熟树突状细胞,体外于不成熟树突状细胞表面用补体蛋白纯品组装CSb-9,37℃,5%CO_2温育96 h,流式分析细胞表型及抗原捕获能力;与同种异体CD4~+和CD8~+T细胞共培养检测其免疫刺激功能;ELISA检测细胞因子分泌。结果显示,亚溶解型C5b-9处理DC表面标志CD83、HLA、CD80、CD86、B7-H1、B7-H3、B7-H4以及BTLA等表达上调;DC分泌IL-12及TNF-α上调,抗原捕获能力降低;C5b-9处理DC刺激CD4~+T细胞活化及分泌IFN-γ、IL-2能力增强。结论:补体C5b-9复合物可以促进树突状细胞成熟,衔接天然免疫和特异性免疫。     

IL-27促进外周血单个核细胞来源树突状细胞分化成熟

目的:研究IL-27体外对人外周血单个核细胞(PBMC)的来源树突状细胞(DC)分化、成熟及其免疫活性的影响。方法:采集健康成人外周血,密度梯度离心法获得PB-MC,给予GM-CSF、IL-4诱导DC,第5天后根据不同处理因素将DC分为3组:IL-27组、TNF-α组和阴性对照组。倒置显微镜和透射电镜下观察DC形态;流式细胞术(FCM)检测DC表面分子CD1a、CD80、CD83和CD86的表达情况;MTT法检测DC刺激T细胞增殖的能力。结果:IL-27刺激后PB-MC来源DC呈现典型的成熟DC形态学特征。IL-27组DC表面CD1a、CD80、CD83和CD86表达水平较对阴性对照组均明显上调(P<0.05),与TNF-α组DC相比无显著差异。IL-27组DC诱导T细胞增殖的能力较对照组DC明显增高(P<0.05)。结论:IL-27可刺激PBMC来源DC的成熟。     

树突状细胞负载肺癌细胞抗原的方法研究

目的:建立自体热休克凋亡肺癌细胞抗原制备、树突状细胞(DC)体外诱导、以及抗原负载方法, 为制备DC肿瘤疫苗提供技术基础.方法:采用酶消化法从手术切除的肺癌新鲜组织获得单细胞悬液, 热休克后用白桦脂酸诱导其凋亡制备成细胞抗原;采用血细胞分离机采集外周血单个核细胞(PBMC), 贴壁法获得单核细胞, 经GM-CSF与IL- 4体外诱导成未成熟树突状细胞(imDC);负载细胞抗原后制备成DC肿瘤疫苗, 并对DC疫苗的形态、数量及表型特征进行分析.结果:(1)肿瘤细胞抗原得率:(13.68±1.30)×106/g组织;平均凋亡率:(93.79±2.31)%;(2)imDC平均得率为(9.37±0.83)×106/(118.77±12.56)×106个PBMC, 活率>95%;imDC表型分析:CD11c CD14-、 CD11c HLA-DR 、 CD11c CD80 、 CD11c CD83 、 CD11c CD86 表达率分别为(87.45±3.42)%、 (87.16±1.08)%、 (2.80±1.52)%、 (5.64±1.56)%、 (5.11±1.09)%;(3)DC疫苗平均得率为(5.75±0.69)×106/(9.37±0.83)×106个imDC, 活率>95%, DC疫苗表型:CD11c CD14-、 CD11c HLA-DR 、 CD11c CD80 、 CD11c CD83 、 CD11c CD86 表达率分别为(92.73±1.21)%、 (89.35±2.31)%、 (86.80±1.23)%、 (69.53±7.21)%、 (94.92±1.48)%. 结论:该方法稳定、安全、可靠, 可制备出具有成熟DC表型的肿瘤疫苗.     

小鼠骨髓来源树突状细胞的分离与扩增培养

目的 :探讨树突状细胞 (DC)分离纯化及其体外扩增的方法。方法 :无菌制备C57BL/6小鼠骨髓 ;依次用红细胞裂解液去除红细胞 ,通过半粘附法去除T、B细胞 ,又在粒细胞巨噬细胞集落刺激因子 (GM CSF)和白细胞介素 4 (IL 4 )协同诱导下培育 ,DC前体分化发育成DC并扩增。在第 7天用脂多糖 (LPS)和肿瘤坏死因子 a (TNF a)刺激 4 8h ,检测细胞因子白介素 12 (IL 12 )浓度及细胞表面标志CD11c、CD80、CD86和MHCⅡ。结果 :DC细胞数增加 ,其形态在光镜下多为特征性星形 ,也有梭形和多角形 ;至培养第 9天DC细胞表面标志CD11c、CD80、CD86、MHCⅡ阳性率分别为 86 .32± 12 .14 %、76 .4 2± 8.4 5%、77.12± 9.0 5%、6 8.4 5± 6 .84 % ,IL 12浓度较未用LPS和TNF a组明显增加 (P <0 .0 1)。结论 :①结果所得细胞的形态和功能符合DC ;②用LPS和TNF a刺激可以获得成熟DC。     

IL-34 对类风湿关节炎髓系树突状细胞表型的影响

目的:探讨IL-34 对类风湿关节炎(RA)患者单核细胞向髓系树突状细胞(DC)分化的诱导作用,及其在分化过程中对细胞表型的影响。方法:采集RA 患者外周血,Ficoll 密度梯度离心法获得PBMC,培养4 h 后将贴壁细胞分别用GM-CSF+IL-4、IL-4、IL-4+IL-34 刺激3 d,流式细胞术检测CD83、CD86 和CD14 的表达水平;再将GM-CSF+IL-4 刺激3 d 后的细胞加入TNF-α和(或)IL-34 培养4 d,流式细胞术检测CD83、CD86 和(或)CD14 的表达水平。结果:(1)IL-34+IL-4 诱导后CD83、CD86 表达水平较IL鄄4 单独作用明显上调(P<0.01),CD14 表达无差异(P>0.05);IL-34+IL-4 诱导CD86、CD14 表达水平较GM-CSF+IL-4 刺激组下降(P<0.05),CD83 表达无差异(P>0.05)(2)GM-CSF+IL-4+IL-34 诱导CD83、CD86 表达水平较GM-CSF+IL-4+TNF-α组低(P<0.05),但与GM-CSF+IL-4 刺激组对比CD83、CD86 表达水平差异无统计学意义(P>0.05)。(3)GM-CSF+IL-4+TNF-α+IL-34 诱导DC CD83 表达水平较GM-CSF+IL-4+TNF-α刺激组低(P<0.05),但两组CD86、CD14 表达差异无统计学意义(P>0.05)。结论:IL-34 在不成熟DC 诱导过程中发挥一定作用,效应略弱于GM-CSF;IL-34 对成熟DC诱导作用不显著,但参与了不成熟DC 的维持。     

不同方法分离外周血单个核细胞对树突状细胞增殖的影响

目的 :探讨不同方法分离的外周血单个核细胞 (PBMC)对树突状细胞 (dentriticcells,DC)增殖产量的影响。方法 :取肝素抗凝血 ,分别用羟乙基淀粉 (hydroxyethy1starch ,HES)离心沉淀法、Ficoll密度梯度离心法分离单个核细胞 ,直接进行贴壁计数及DC的诱导培养。结果 :HES离心沉淀法、Ficoll密度梯度离心法分离的外周血单个核细胞 ,其贴壁细胞经FACS检测 ,CD1 4 细胞分别占贴壁总数的 6 5 0 6 %、6 3 80 % ,每 4 0ml外周血扩增的DC产量分别为 (0 .88～ 3.1 7)× 1 0 6和 (0 .79～ 3.0 2 )× 1 0 6。结论 :羟乙基淀粉法分离的PBMC更适用于DC的诱导培养。     

Pepsinogen secretion from dispersed glands from rabbit stomach

In dispersed glands from rabbit stomach, pepsinogen secretion was stimulated by carbamylcholine, the C-terminal octapeptide of cholecystokinin (CCK-8), and structurally related peptides physalaemin and A23187 but not by bombesin or histamine. Reducing the incubation temperature from 37 degrees to 4 degrees C or adding carbonyl cyanide m-chlorophenylhydrazone reduced basal as well as stimulated pepsinogen secretion. Dibutyryl cGMP inhibited the stimulation of pepsinogen secretion caused by cholecystokinin but not that caused by carbamylcholine; atropine inhibited the stimulation of pepsinogen secretion caused by carbamylcholine but not that caused by cholecystokinin. Each cholecystokinin-related peptide had the same efficacy for stimulating pepsinogen secretion. In terms of the concentration that caused half-maximal stimulation of pepsinogen secretion, the relative potencies of cholecystokinin-related peptides were caerulein greater than CCK-8 greater than [desSO3]CCK-8 greater than gastrin. Removing extracellular calcium did not alter basal or cholecystokinin-stimulated pepsinogen secretion. These results illustrate that dispersed glands from rabbit stomach constitute a suitable preparation for examining the actions of various agents on pepsinogen secretion in vitro.     

谷氨酸在分离的小鼠皮层突触连接体的释放

目的 分析兴奋性神经递质谷氨酸在体外分离的小鼠皮层突触连接体的释放。 方法 解剖分离小鼠的大脑皮层,组织经匀浆后使用3层147 μm的尼龙网筛进行过滤,离心后重悬沉淀为突触连接体。谷氨酸释放检测实验根据谷氨酸脱氢酶酶促反应原理进行,利用多功能读数仪读取产物 烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的荧光值。采用谷氨酸标准品绘制标准曲线,使用制备的突触连接体进行谷氨酸释放检测,进行3次独立重复实验。结果 谷氨酸标准品浓度与荧光值具有良好的线性关系。新鲜制备的突触连接体在加入去极化剂KCl后,释放谷氨酸显著增加并逐渐达到平台期,加入Triton X-100裂解突触连接体后释放谷氨酸明显增加。冻融1次的突触连接体失去活性,与新鲜制备的突触连接体的谷氨酸释放曲线不同。 结论 对小鼠皮层突触连接体的提取方法进行了改良,检测和分析了兴奋性神经递质谷氨酸在分离制备的小鼠皮层突触连接体的释放。     

“Far from the heart far from the eye”: Evidence from the Capgras delusion

Introduction. Capgras syndrome is characterised by the belief that a significant other has been replaced by an identical-looking impostor. These patients have no difficulties with visual recognition but fail to show a skin conductance response (SCR) to the objects of the delusion. A case of Capgras delusion (YY), specifically characterised by the absence of brain lesions, constituted a good opportunity to test the relationship between SCR hyporesponsiveness and eye movement patterns to familiar and unfamiliar faces.

Methods. Visual scan path and SCR were recorded for YY and 8 controls during the presentation of family members’ photographs matched with unfamiliar faces of the same sex, age, and physical likeness. Eye movement patterns were explored by selecting three specific areas of interest (AOI) involving the eyes, the mouth, and the face regions.

Results. In contrast with controls, YY showed a reduction in number and sum of fixation durations to the eyes (p<.01) and no differential SCRs (p>.05) to familiar vs. nonfamiliar faces. SCR and fixation duration to family members’ eyes were significantly correlated (r=.77) in both YY and controls.

Conclusions. Eye region exploration seems to be related to the autonomic reactivity elicited by the affective valence of familiar faces.     

The separation of renal activity from lung and from the venous effluent from perfused lungs

1. Antidiuretic and salt-retaining activity has been separated from cat and ox lung, and from the venous effluent of blood-perfused cat lungs, by acetone extraction, gel filtration, partition and thin-layer chromatography.2. The chromatographic properties of the renally active substance from lung and from pulmonary venous blood are similar.3. The renal actions of these substances, demonstrable in vivo and in the isolated organ, are characterized by reduction in the excretion of water and Na. Renal blood flow, glomerular filtration rate and the urinary Na/K are not significantly affected.4. Concentrations of the lung substance which equate with angiotensin II-val(5)-amide in reduction of urinary Na in rats during water diuresis and under alcohol sedation do not raise the mean arterial pressure of the anaesthetized rat nor contract the superfused rat colon. At these dose levels angiotensin II is markedly vasopressor and contracts the superfused rat colon.     

Differentiating betweenBesnoitia besnoiti from cattle andSarcocystis hoarensis from rodents

To provide a biological basis for studies designed to establish the mode of transmission of the veterinary pathogenBesnoitia besnoiti, we compared salient features of this pathogen in cattle with those ofSarcocystis hoarensis in rodents. The cysts and cystozoites of these organisms can readily be distinguished morphologically. In contrast toS. hoarensis which is well adapted to rodents,B. besnoiti fails to mature in jirds or mice and generally is lethal in jirds. Serological reagents discriminately detect these pathogens.B. besnoiti, therefore, can unambiguously be differentiated fromS. hoarensis either by morphological or serological methods or on the basis of experimental comparisons of virulence in laboratory rodents.     

Clinicians are from Mars and pathologists are from Venus

CONTEXT: Text reports convey critical medical information from pathologists, radiologists, and subspecialty consultants. These reports must be clear and comprehensible to avoid medical errors. Pathologists have paid much attention to report completeness but have ignored the corresponding issue of report comprehension. This situation presents an increasingly serious potential problem. As laboratories are consolidated and as reports are disseminated in new ways (eg, via the World Wide Web), the target audience becomes more diverse and less likely to have any contact with pathologists beyond the written reports themselves. OBJECTIVE: To compare clinician comprehension with pathologist intent in written pathology reports. METHODS: Typical surgical pathology reports relevant to surgeons and covering a range of specimen complexity were taken from our hospital files. Questionnaires based on these cases were administered open-book-examination style to surgical attending physicians and trainees during surgical conferences at an academic medical center. MAIN OUTCOME MEASURES: Scores from questionnaires. RESULTS: Surgeons misunderstood pathologists' reports 30% of the time. Surgical experience reduced but did not eliminate the problem. Streamlined report formatting exacerbated the problem. CONCLUSIONS: A communication gap exists between pathologists and surgeons. Familiarity with report format and clinical experience help reduce this gap. Paradoxically, stylistic improvements to report formatting can interfere with comprehension and increase the number of misunderstandings. Further investigation is required to reduce the number of misunderstandings and, thus, medical errors.     

Identification of trypanosomes isolated from reduviidae from North Chile

Examination of 54Triatoma infestans from a village near the European Southern Observatory La Silla in Chile and of 9Triatoma spinolai from the territory of the observatory showed that 10T. infestans were infected with trypanosomatids. Mice were infected with in vitro cultures initiated with five different trypanosomatid isolates and treated with the immunosuppressive drug cyclophosphamide to increase the parasitemia of the flagellates. Evidence of the presence ofT. cruzi was provided by a comparative biometrical analysis of blood trypomastigotes and the occurrence of intracellular amastigotes. Three methods for further identification were used: examination of kDNA ultrastructure, disc electrophoresis of soluble proteins and theAaptos papillata II lectin induced agglutination. We obtained the following results for all isolates: (1) presence of a central band of the kDNA; (2)T. cruzi specific double bands of the protein patterns; (3) positive reaction withAaptos papillata II. No differences between the five isolates from Chile andT. cruzi orT. cruzi-like strains from other countries could be observed. Based on these results an infection of the bugs withT. rangeli andT. conorhini could be excluded.Supported by BMZ, Research Program FIOCRUZ, Rio de Janeiro/BNI, Hamburg     

Aftereffects from jogging

After running on a treadmill, runners who attempted to jog in place on solid ground inadvertently jogged forwards. One-legged hopping on the treadmill produced an aftereffect in the same leg, but not in the other leg. This non-transfer suggests a peripheral neural site. Judgments of velocity and slope were affected; running on a backward-moving treadmill made a stationary test treadmill seem to move forwards, and running on an uphill-sloping treadmill made a horizontal test treadmill seem to slope downhill. These aftereffects suggest an automatic gain control process.