葡萄球菌肠毒素A诱导小鼠体内T细胞无能过程中CD28/CTLA-4的变化

目的在葡萄球菌肠毒素A(SEA)诱导小鼠体内T细胞无能的过程中,观察CD28和CTLA-4的变化规律及相互关系。方法单次(1×SEA组,n=4)或多次(2×SEA组,n=4;3×SEA组,n=4)向小鼠腹腔注射SEA,每次注射间隔3d,分别于末次注射后的第1、3、7、14天,将小鼠处死,制备小鼠脾淋巴细胞。通过流式细胞仪检测细胞膜表达CD28和CTLA-4、细胞内表达CTLA-4和IL-2的阳性率。结果1×SEA组,细胞膜CD28和细胞内IL-2的表达显著上升;而CTLA-4在细胞内和细胞膜表面的表达均降低且无明显变化;2×SEA组CD28和IL-2的表达稍有上升;CTLA-4在细胞内和细胞膜表面的表达均明显增加,胞内CTLA-4的增加尤为显著。3×SEA组,细胞膜CD28、细胞膜和细胞内CTLA-4的表达呈下降趋势,细胞内IL-2在第7天已经检测不到。结论SEA初次诱导小鼠脾淋巴细胞时,能显著促进T细胞的活化;经SEA多次(3次)刺激可诱导T细胞的无反应性。在无能的形成过程中,IL-2产生减少与CD28表达下调可能有着密切联系;CTLA-4的表达上调有利于无能的诱导,但可能不参与无能的维持。     

葡萄球菌肠毒素A诱导小鼠体内T细胞无能过程中CD28/CILA-4的变化

目的 在葡萄球菌肠毒素A(SEA)诱导小鼠体内T细胞无能的过程中,观察CD28和CTLA-4的变化规律及相互关系.方法 单次(1×SEA组,n=4)或多次(2×SEA组.n=4;3×SEA组,n=4)向小鼠腹腔注射SEA,每次注射间隔3d,分别于末次注射后的第1、3、7、14天,将小鼠处死,制备小鼠脾淋巴细胞.通过流式细胞仪检测细胞膜表达CD28和CTLA-4、细胞内表达CTLA-4和IL-2的阳性率.结果 1×SEA组,细胞膜CD28和细胞内IL-2的表达显著上升;而CTLA-4在细胞内和细胞膜表面的表达均降低且无明显变化;2×SEA组CD28和IL-2的表达稍有上升;CTLA-4在细胞内和细胞膜表面的表达均明显增加,胞内CTLA-4的增加尤为显著.3×SEA组,细胞膜CD28、细胞膜和细胞内CTLA-4的表达呈下降趋势,细胞内IL-2在第7天已经检测不到.结论 SEA初次诱导小鼠脾淋巴细胞时,能显著促进T细胞的活化;经SEA多次(3次)刺激可诱导T细胞的无反应性.在无能的形成过程中,IL-2产生减少与CD28表达下调可能有着密切联系;CTLA-4的表达上调有利于无能的诱导,但可能不参与无能的维持.     

高迁移率族蛋白B1对调节性树突状细胞的免疫效应及其作用机制

目的 观察高迁移率族蛋白B1(high mobility group box-1 protein,HMGB1)对分泌IL-10树突状细胞(dendritic cell,DC)亚群CD11clowCD45RBhigh DC功能的影响. 方法 采用磁珠分选技术获得Bab/c小鼠脾脏CD11clowCD45RBhighDC和CD4+T淋巴细胞,加不同浓度HMGB1处理(20,100,500 ng/ml,以不加HMGB1的细胞作为对照)CD11clowCD45RBhighDC细胞;流式细胞术检测CD11clow CD45RBhigh DC表面分子CD40、CD80、CD86、Ⅰ-a/e及Toll样受体(TLR)4的表达强度;应用ELISA检测CD11clowCD45RBhighDC培养液中IL-10的含量.将CD4+T淋巴细胞分为正常对照组(未作任何处理)、未刺激组(加入未经HMGB1处理的CD11clowCD45RBhighDC与CD4+T淋巴细胞混合培养)、高浓度HMGB1刺激组(加入经500 ng/ml HMGB1处理后的CD11clowCD45RBhighDC与CD4+T淋巴细胞混合培养)、高浓度HMGB1刺激+抗体1组(加入经500 ng/mlHMGB1处理后的CD11clowCD45RBhighDC、抗IL-10抗体与CD4+T淋巴细胞混合培养)和高浓度HMGB1刺激+抗体2组(加入经500 ng/ml HMGB1处理后的CD11clowCD45RBhighDC、IL-10的同型对照抗体与CD4+T淋巴细胞混合培养).流式细胞术检测CD4+T淋巴细胞培养液中IL-4和干扰素-γ(IFN-γ)的含量. 结果 与未加HMGB1刺激相比,HMGB1能显著增强CD11clowCD45RBhighDC表面分子CD86、TLR4的表达及IL-10的分泌,其中IL-10分泌呈HMGB1浓度依赖性.高浓度HMGB1刺激组IFN-γ水平为(279±17) pg/ml,显著低于未刺激组(963±11)pg/ml(P＜0.05);高浓度HMGB1刺激组IL-4水平为(372±14) pg/ml,明显高于未刺激组(213±10) pg/ml(P ＜0.05). 结论 HMGB1可促进CD11clowCD45RBhighDC IL-10的分泌,诱导CD4+T淋巴细胞向Th2型反应分化,通过激活CD11clowCD45RBhighDC介导机体免疫抑制活性.     

肝硬化大鼠模型肠黏膜相关淋巴组织T淋巴细胞表达的研究

目的研究肝硬化大鼠模型肠黏膜相关淋巴组织T淋巴细胞及其亚群的表达,了解肝硬化肠黏膜免疫屏障功能的变化。方法采用皮下注射40%四氯化碳建立大鼠肝硬化模型,分离肠上皮内、固有层、派伊尔结和肠系膜淋巴结淋巴细胞,分别标记小鼠抗大鼠CD3、CD4、CD8单克隆抗体,流式细胞仪检测T淋巴细胞及其亚群的表达情况。结果与正常组相比,肝硬化组肠上皮内CD3+T淋巴细胞比例有所减少,但无统计学差异(P>0.05);CD4+CD8-亚群细胞比例减少(P<0.05),CD4+CD8+亚群细胞比例增加(P<0.05);肝硬化组固有层、派伊尔结、肠系膜淋巴结CD3+T淋巴细胞比例均明显降低(P<0.05或P<0.01)。结论肝硬化时肠黏膜相关淋巴组织细胞免疫功能降低。     

雷公藤甲素对调节性树突细胞的免疫效应及其机制研究

目的 探讨雷公藤甲素(TPT)对分泌IL-10的树突细胞(D C)亚群CDllclowCD45RBhighDC功能的影响.方法 采用磁珠分选技术获得C57BL/6小鼠脾脏CD11clowCD45RBhighDC和CD4+T淋巴细胞.CD11clowCD45RBhighDC分别加入1、10、20ng/ml TPT后进行培养,以不加TPT的细胞作为对照,流式细胞仪检测细胞表面分子CD40、CD80、CD86、I-a/e的表达,ELISA法检测培养液中IL-10的含量.将CD4+T淋巴细胞分为正常对照组(未作任何处理)、未刺激组(与未经TPT处理的CD11clowCD45RBhighDC混合培养)、高浓度TPT刺激组(与经20ng/ml TPT处理后的CD11clowCD45RBhighDC混合培养)、高浓度TPT刺激+抗体1(抗IL-10抗体)组、高浓度TPT刺激+抗体2(IL-10的同型对照抗体)组,采用MTT法测定CD4+T淋巴细胞的增殖活性,流式细胞仪检测CD4+T淋巴细胞培养液中IL-4和IFN-γ的含量.结果 与对照组相比,TPT能显著降低CD11clowCD45RBhighDC表面分子CD40和I-a/e的表达,增强CD86的表达及IL-10的分泌,其中IL-10的分泌水平随TPT浓度的增加而增加.高浓度TPT刺激组和高浓度TPT刺激+抗体2组细胞增殖活性及IFN-γ分泌水平明显低于未刺激组,IL-4分泌水平明显高于未刺激组,而高浓度TPT刺激+抗体1组细胞增殖活性及IFN-γ分泌水平明显高于未刺激组,IL-4分泌水平明显低于未刺激组.结论 TPT可促进CD11clowCD45RBhighDCIL-10的分泌,诱导CD4+T淋巴细胞向Th2型分化,并可通过激活CD11clowCD45RBhighDC介导机体的免疫抑制活性.     

稳定转染小鼠IL-17全长基因的乳腺癌细胞株的建立及基因转染对4T1细胞的影响

目的建立稳定转染小鼠IL-17基因全长的小鼠乳腺癌4T1细胞株,并进行鉴定。方法脂质体法将携带小鼠IL-17基因全长的真核表达载体pcDNA3．1转染小鼠乳腺癌细胞4T1,经G418筛选出稳定表达IL-17的细胞株;镜下观察细胞形态,RT-PCR法、Western印迹和激光共聚焦法检测目的基因和蛋白的表达;收集转染和未转染IL．17的4T1细胞的培养上清,分别与巨噬细胞RAW264．7共培养,ELISA法检测RAw264．7细胞培养上清中IL-6水平;MTS法检测细胞的增殖情况,流式细胞技术检测细胞周期、凋亡以及细胞表面MHCI、MHC11、淋巴细胞功能相关抗原-1（LFA-1）等分子表达。结果获得1株稳定表达IL-17的4T1细胞,而且其培养上清可刺激小鼠巨噬细胞RAW264．7产生IL-6,证明该细胞可表达功能性IL-17。结论成功建立了稳定转染IL-17基因的小鼠乳腺癌细胞株。     

肾移植后诱导活化高表达CD25的CD4~+ CD25~(high)调节性T细胞的表型特点与免疫调节功能

目的 探讨肾移植受者活化并表达不同强度CD25的CD4细胞的表型特征和功能特点.方法依据CD25的表达水平,应用流式细胞仪将9例行初次亲属活体肾移植受者的外周血CD4~+ T细胞分为CD25~-、CD25高表达(CD25~(high))和CD25低表达(CD25~(low))3群,并比较3群细胞叉头翼状螺旋转录因子(FoxP3)、细胞毒性T淋巴细胞相关抗原-4(CTLA-4)表达强度.经免疫磁珠法和CD4~+ CD25~+调节性T细胞分选试剂盒分选得到受者外周血CD4~+ CD25~-、CD4~+ CD25~(high) T细胞,分别作为反应细胞,以Co~(60)γ射线照射灭活的供者外周血单个核细胞(PBMC)作为刺激细胞,建立体外单向混合淋巴细胞培养系统.采用半定量RT-PCR检测系统中IL-2 mRNA的表达.结果 在肾移植术后受者3群细胞中,FoxP3的表达以CD4~+ CD25~(high)细胞最高(93.7%±3.58%),其次是CD4~+CD25~(high)细胞(16.4%±6.8%),CD4~+ CD25~-细胞最低(1.8%±1.1%),三者间差异均有统计学意义(P<0.01);CTLA-4表达以CD4~- CD25~(high)细胞最高(80.8%±7.9%),其次是CD4~- CD25~(low)细胞(22.9%±6.5%),CD4~- CD25~-细胞最低(4.1%±2.4%),三者间差异均有统计学意义(P<0.01).在混合淋巴细胞培养中,CD4~- CD25~(high)细胞受移植抗原刺激后不表达IL-2 mRNA;按照1:2比例加入CD4~- CD25~(high)细胞后,CD4~-细胞表达的IL-2 mRNA平均被抑制了60%.结论 肾移植受者所有活化T细胞的表面均有CD25表达,高表达CD25的细胞群具有调节性T细胞的表型特点,为CD4~+ CD25~(high)曲调节性T细胞.     

洋参多糖提高放射性免疫功能低下小鼠免疫功能实验观察

目的 观察洋参多糖提高放射性免疫功能低下小鼠免疫功能的药效作用。方法 采用^60Coγ射线一次全身照射7Gy诱发放射性免疫功能低下小鼠动物模型。照射前后小鼠每日口服洋参多糖87．5mg/kg，连续服药10d。测定模型小鼠服药后脾NK细胞活性、淋巴细胞转化能力、T淋巴细胞亚群(CD4、CD8)、白介素2(IL-2)和足垫迟发超敏反应(DTH)等免疫功能变化。结果 洋参多糖可明显增加IL-2含量和T淋巴细胞亚群(CD4、CD8)数量，提高鼠脾NK细胞活性和淋巴细胞转化能力及促进模型鼠足垫迟发超敏反应。结论 洋参多糖可减轻辐射诱发的机体免疫功能改变，该化合物可能是一种治疗免疫功能低下的有效制剂。     

芳酰肼类小分子有机化合物J2的免疫抑制作用

目的:研究芳酰肼类小分子有机化合物J2的免疫抑制作用。方法:利用3H-TdR掺入法检测混合淋巴细胞培养,通过玫瑰花结计数评价细胞黏附实验,利用流式细胞术检测IL-2和IFN-γ,通过凝胶阻滞电泳分析(electrophoresis mob ility sh ift assay,EMSA)研究活化T细胞核因子(nuc lear factor of activated T cells,NF-AT)的表达。结果:J2对混合淋巴细胞培养中反应细胞的增殖具有抑制作用,其IC50为(67.75±4.47)μmol/L;J2可以抑制表达CD4的HEK293/CD4细胞和表达MHCⅡ类分子的Daud i细胞之间的细胞黏附;对反应细胞的IL-2和IFN-γ的产生具有抑制作用;并抑制NF-AT的表达。结论:小分子有机化合物J2具有一定的免疫抑制作用。     

卡拉胶寡糖对放射损伤小鼠T细胞功能和亚型的影响

目的探讨卡拉胶寡糖对受x射线照射小鼠T细胞功能和亚型的影响。方法采用直线加速器6MV X射线一次全身照射BALB／c小鼠，卡拉胶寡糖组照射前连续14d和照射后继续给药7d。检测小鼠脾脏淋巴细胞增殖反应，胸腺细胞表达CD69变化，外周血T细胞表达IL-2、IFN-γ和TNF-α变化及CD4^ ／CD8^ 比值。结果卡拉胶寡糖能有效提高脾脏T淋巴细胞的增殖能力，胸腺T细胞表达CD69显著升高，使受照射小鼠外周血T细胞产生IL-2和IFN—γ的能力显著增强，外周血T细胞表达TNF-α的能力降低，并能显著升高外周血T细胞CD4^ ／CD8^ 比值。结论卡拉胶寡糖对放射损伤小鼠的T细胞功能和亚型有明显调节作用。     

N-[C]methylpiperidine esters as acetylcholinesterase substrates: an in vivo structure–reactivity study

A series of simple esters incorporating the N-[¹¹C]methylpiperidine structure were examined as in vivo substrates for acetylcholinesterase in mouse brain. 4-N-[¹¹C]Methylpiperidinyl esters, including the acetate, propionate and isobutyrate esters, are good in vivo substrates for mammalian cholinesterases. Introduction of a methyl group at the 4-position of the 4-piperidinol esters, to form the ester of a teritary alcohol, effectively blocks enzymatic action. Methylation of 4- N-[¹¹C]methylpiperidinyl propionate at the 3-position gives a derivative with increased in vivo reactivity toward acetylcholinesterase. Esters of piperidinecarboxylic acids (nipecotic, isonipecotic and pipecolinic acid ethyl esters) are not hydrolyzed by acetylcholinesterase in vivo, nor do they act as in vivo inhibitors of the enzyme. This study has identified simple methods to both increase and decrease the in vivo reactivity of piperidinyl esters toward acetylcholinesterase.     

Can a cysteine challenge assay predict the in vivo behavior of Tc-labeled antibodies?

Recent investigations have shown that transchelation to cysteine is a principal mode of in vivo instability of ^99mTc-labeled antibodies. In this investigation, a cysteine challenge assay was used to measure the in vitro instability of ^99mTc directly labeled to two IgG antibodies (B72.3 and C110) via two established direct labeling methods employing mercaptoethanol and stannous ion for antibody reduction and by a novel method using glutathione for this purpose. For both antibodies, the greatest instability to cysteine occurred with stannous ion reduction. The stability of glutathione-reduced B72.3 was indistinguishable from mercaptoethanol-reduced B72.3 whereas glutathione-reduced C110 showed stability roughly intermediate between that of the other reducing agents for this antibody. Results obtained in normal mice were in the direction predicted by the assay: for both antibodies, urinary clearance of ^99mTc was fastest in mice receiving antibodies labeled via stannous ion reduction, presumably because of the increased transchelation of label to cysteine in vivo. Urinary clearance was slower and identical in mice receiving B72.3 labeled via glutathione or mercaptoethanol whereas clearance in the case of glutathione-reduced C110 was intermediate between that of the other two reducing agents. At both time points, higher radioactivity levels were observed in kidneys and lower levels in blood and most other tissues for both antibodies in the case of stannous ion reduction as expected for the label of greatest instability. In the B72.3 case, with only one exception, tissue and blood levels following administration of glutathione-reduced antibody were indistinguishable from that following administration of mercaptoethanol-reduced antibody. In the C110 case, significant differences in activity levels were observed in several tissues between glutathione- and mercaptoethanol-reduced antibodies. In conclusion, the relative in vivo behaviour of ^99mTc when administered to mice while labeled to two IgG antibodies were successfully predicted based on the results of an in vitro cysteine challenge assay.     

New iodinated progestins as potential ligands for progesterone receptor imaging in breast cancer. part 2: in vivo pharmacological characterization

On the basis of the observed high selective binding to both the human and rat progesterone receptor (PR) in vitro, three 17-iodovinyl-substituted nortestosterone derivatives, i.e., the Z-isomer of 17-iodovinyl-19-nortestosterone (Z-IVNT; Z-IPG1) and both the stereoisomers of 17-iodovinyl-18-methyl-11-methylene-19-nortestosterone (E-and Z-IPG2), were selected for radio-iodination and subsequently evaluated as potential radioligands for PR imaging in human breast cancer. Their target tissue uptake, retention, and uptake selectivity were studied in female rats. The distribution studies revealed that PR-mediated uptake in the uterus and ovaries could only be demonstrated for Z-[¹²³I]IPG2. The target tissue uptake selectivity was, however, low, with the highest uterus-to-nontarget tissue uptake ratios observed at 2–4 h postinjection (p.i.), being 4.4, 1.8, and 7.4 for the uterus-to-blood, -fat, and -muscle ratio, respectively. For Z-[¹²³I]IPG2, distribution was also studied in dimethylbenzanthracene (DMBA)-induced mammary tumour-bearing rats and in normal rabbits. Mammary tumour uptake of Z-[¹²³I]IPG2 in the mammary tumour-bearing rat was also found to be PR-specific. In rabbits, higher selective target tissue uptake of Z-[¹²³I]IPG2 was observed than in rats, resulting in uterus-to-blood, -fat, and -muscle ratios of 6.6, 2.2, and 21.3 at 2–4 h p.i., respectively. In conclusion, Z-[¹²³I]IPG2, which displayed high binding affinity for both the human and rat PR in vitro, showed specific PR-mediated target tissue uptake in rats and rabbits in vivo, the uptake selectivity being highest in the latter. Because the binding characteristics appeared to vary between species, a pilot study in breast cancer patients may be needed to decide whether Z-[¹²³I]IPG2 can be of potential use as PR imaging agent in breast cancer.     

Methodological aspects for in vitro characterization of receptor binding using C-labeled receptor ligands: A detailed study with the benzodiazepine receptor antagonist [C]Ro 15-1788

As a complement to in vivo studies with positron emission tomography (PET), it is desirable to perform in vitro characterization of newly developed ¹¹C tracers. In this report we describe the technique for determination of receptor-ligand kinetics utilizing ligands labeled with the short-lived radionuclide ¹¹C. The limitations and advantages are discussed. The benzodiazepine antagonist [¹¹C]Ro 15-1788 was used as a model substance, and the use of storage phosphor plates for quantification of radioactivity was validated. Storage phosphor plates showed an excellent linear range (˜10³) and acceptable resolution (˜ 0.5 mm). Receptor-ligand kinetics, including depletion, association and dissociation, saturation and displacement were evaluated with good results through the use of short-lived radiotracers and storage phosphor plates.     

In Vivo localization of diglycylcysteine-bearing synthetic peptides by nuclear imaging of oxotechnetate transchelation

A phenomenon of in vivo transchelation of oxotechnetate from a complex with glucoheptonic acid to synthetic peptides bearing oxotechnetate-binding motifs and a technique for in vivo visualization of these peptides are described. Using two model peptides bearing two tandem diglycylcysteine (GGC) motifs (P1) or three GGC motifs (P2), we demonstrated that: (i) these peptides efficiently transchelated oxo-[^99mTc]technetate from a complex with glucoheptonic acid in vitro (a complex with peptides was stable at least 24 h; radiochemical purity exceeded 95% by high performance liquid chromatography); (ii) injection of peptides into the rectus femoris muscle (at 0.5-1 μmol of SH groups) followed by an intravenous injection of ^99mTc-glucoheptonate (0.25-0.5 mCi per animal) yielded visualization of the injected muscle by nuclear imaging within 1 h after injection; (iii) the experimental/control (contralateral) thigh muscle ratio was 1.80 ± 0.05 for peptide P1 and 3.0 ± 0.1 for P2; (iv) the injection of a control peptide P2 with SH groups covalently modified with N-ethylmaleimide resulted in a ratio of 1.4 ± 0.2. These findings argue for specific association of oxo-[^99mTc]technetate with free thiols within the binding motif of injected peptides in vivo. In vivo transchelation of oxo-[^99mTc]technetate may be useful for the purpose of noninvasive imaging of gene expression, i.e., when the expression product bears GGC motifs.     

In Vivo evaluation of an In-labeled ST-peptide analog for specific-targeting of human colon cancers

In vitro competitive binding studies of In-DOTA-NCS-6-Ahx-Phe¹⁹-ST[1–19] vs. ¹²⁵I-Tyr⁵-6-Ahx-Phe¹⁹-ST[1–19] with guanylate cyclase -C (GC-C) receptors on human colon cancer LS-180 cells revealed an IC₅₀ value of 7.7 ± 0.1.6 nM. The in vitro cellular residualization studies of the ¹¹¹In-DOTA-NCS-ST peptide and GC-C receptor mediated stimulated cGMP production with LS-180 cells demonstrates that this peptide selectively binds to LS-180 cells in an agonistic fashion. In vivo biodistribution studies in LS-180 tumor bearing SCID mice demonstrates that the ¹¹¹In-DOTA-NCS-ST peptide targets the tumor with a specific uptake of 0.94 ± 0.31%ID/g at 1 hr p.i. and approximately 23% was retained by the tumor at 4 hrs p.i. The radioactivity cleared rapidly from the blood stream with 84.5 ± 3.4%ID at 1h p.i. found in the urine. High activity in urine and kidney, and minimal activity in liver and intestines, demonstrates preferential clearance of the radioactivity through the renal/urinary pathway. The specific in vitro and in vivo accumulation of the radioactivity by LS-180 human colonic cancer cells highlights the potential of radiometallated-DOTA-ST analogs as diagnostic/therapeutic radiopharmaceuticals.     

Syntheses of carbon-11 labeled piperidine esters as potential in vivo substrates for acetylcholinesterase

A series of carbon-11 labeled N-methylpiperidinyl esters were prepared as potential in vivo substrates for acetylcholinesterase (AChE). Target compounds were designed based on the structure of N-[¹¹C]methylpiperidin-4-yl propionate, an ester currently used to measure AChE enzymatic activity in the human brain, to examine the structure–activity relationship for in vivo enzymatic hydrolysis. Changes in steric bulk and in the ester order (“reverse” esters) were made. Addition of methyl groups was made to both the acid side chain (synthesis of N-[¹¹C]methylmethylpiperidin-4-yl isobutyrate) and to the piperidine ring (syntheses of N-[¹¹C]methyl-4-methylpiperidin-4-yl propionate, N-[¹¹C]methyl-4-methylpiperidin-4-yl acetate, and N-[¹¹C]methyl-3-methylpiperidin-4-yl propionate). Alterations of the order of the ester heteroatoms was accomplished through syntheses of the N-[¹¹C]methyl-2,3- and 4-piperidinecarboxylic acid ethyl esters. Finally, an additional piperidine-based ester (N-[¹¹C]methylpiperidin-2-yl)methyl propionate was also prepared. All carbon-11-labeled esters were prepared by N-[¹¹C]methylation reactions, using the desmethyl precursors and no-carrier-added [¹¹C]methyltriflate, and were obtained in decay-corrected yields (not optimized) of 10–40% and high specific activities.     

Evaluation of phenylmethanesulfonyl fluoride (PMSF) as a tracer candidate mapping acetylcholinesterase in vivo

The availability of phenylmethanesulfonyl fluoride (PMSF), an irreversible cholinesterase inhibitor, for a tracer mapping acetylcholinesterase (AchE) in vivo in brain and other organs was evaluated using [³⁵S]PMSF in mice and rats. [³⁵S]PMSF was well taken up into the brain, heart and muscle, and the radioactivities were trapped in these organs. Pretreatment with non-labeled PMSF decreased 33–40% of the trapped radioactivities in the brain and other organs in mice. However, regional distribution of [³⁵S]PMSF in rat brain did not correlate well with that of AchE activity, suggesting that the selectivity of PMSF toward AchE may be insufficient for use as an in vivo tracer mapping AchE.     

A new Tc-complex with a germanium-hydrazide (GeTH) ligand

A new tetrahydrazido-germanium (GeTH) ligand was synthesized, characterized and complexed with ^99mTc. The negatively charged ^99mTc-chelate was shown to form in high yields at neutral pH in the absence of other reducing agents and exhibits high in vitro and in vivo stability.     

Influence of acetylcholine on binding of 4-[i]iododexetimide to muscarinic brain receptors

The distribution of nicotinic and muscarinic cholinergic receptors in the human brain in vivo has been successfully characterized using radiolabeled tracers and emission tomography. The effect of acetylcholine release into the synaptic cleft on receptor binding of these tracers has not yet been investigated. The present study examined the influence of acetylcholine on binding of 4-[¹²⁵I]iododexetimide to muscarinic cholinergic receptors of porcine brain synaptosomes in vitro. 4-Iododexetimide is a subtype-unspecific muscarinic receptor antagonist with high affinity. Acetylcholine competed with 4-[¹²⁵I]iododexetimide in a dose-dependent manner. A concentration of 500 μM acetylcholine inhibited 50% of total specific 4-[¹²⁵I]iododexetimide binding to synaptosomes when both substances were given simultaneously. An 800 μM acetylcholine solution reduced total specific 4-[¹²⁵I]iododexetimide binding by about 35%, when acetylcholine was given 60 min after incubation of synaptosomes with 4-[¹²⁵I]iododexetimide. Variations in the synaptic acetylcholine concentration might influence muscarinic cholinergic receptor imaging in vivo using 4-[¹²³I]iododexetimide. Conversely, 4-[¹²³I]iododexetimide might be an appropriate molecule to investigate alterations of acetylcholine release into the synaptic cleft in vivo using single photon emission computed tomography.