Influence of Size, Dosage, and Surface Structure on Clearance and Tissue Distribution of Intravenous Microspheres

Intravenously injected polystyrene microspheres with functional amino groups (AP-MSs, 0.2,1.0, and 4.0 urn in diameter) were cleared from the blood very rapidly. The calculated half-lives for 0.2-, 1.0-, and 4.0-μm AP-MSs were about 55,60, and 50s; no significant differences were found with 10⁶,10⁷, and 10⁸ microspheres/rat. Loading experiments showed that the liver, spleen, lung, kidney, and heart had a very high capacity to take up AP-MSs. The AP-MSs were distributed mainly to the liver, lung, and spleen, whereas other organs contained less than 1 % of injected AP-MSs. In terms of numbers of AP-MSs per gram of tissue, the highest contents were found in spleen, liver, and lung for 0.2-, 1.0-, and 4.0-um AP-MSs, respectively. There was indication of redistribution of particles from one organ to another during the first 6 h after injection. Chondroitin sulfate A (Chon) and hyaluronic acid (Hya) adsorbed or covalently linked to AP-MSs increased uptake in the liver, with Chon AP-MSs (adsorbed or linked) showing the best effect: about 25% increase compared with unadsorbed 1-μm AP-MSs. Experiments with separated cells in vitro demonstrated that 1 um AP-MSs, intravenously injected, associated only with Kupffer cells. When the microspheres were adsorbed with Chon, there was also association with liver endothelial cells. This finding indicates that conjugation of microspheres with ligands for endothelial receptors may be a useful method for directing microspheres to specific organs, even if the receptors are not by themselves phagocytic     

A sensitive assay for detecting low-affinity interactions at the cell surface reveals no additional ligands for the adhesion pair rat CD2 and CD48

The ligand for the T cell antigen CD2 is CD48 in rodents, but CD58 in humans. The extracelluar parts of these three antigens are structurally related in that all contain two immunoglobulin superfamily (IgSF) domains. There have been reports of alternative ligands for CD2 in the human, but not so far in rodents. We describe the analysis of ligands for rat CD2 and CD48 using fluorescent beads capable of displaying a high ligand density and detecting low-affinity interactions like that of CD2 with CD48 (Kd = 60 ? 90 μM). Monovalent chimeric proteins containing the two IgSF domains of rat CD48 or CD2 and domains 3 and 4 of rat CD4 (CD4d3+4) were anchored to fluorescent covaspheres^TM via a CD4 monoclonal antibody (mAb) with the CD48 or CD2 domains available for ligand binding. Multivalent CD48-CD4d3 + 4 covaspheres^TM gave strong specific binding to rat CD2 expressed on the surface of transfected Jurkat cells. CD48-CD4d3+4 was compared with CD48-IgG and CD48-IgM as tools for detecting binding at the cell surface. Soluble divalent CD48-IgG and decavalent CD48-IgM bound to soluble CD2 with a K_off of around 10^?3 s^?1 as determined using a BIAcore^TM biosensor. However, binding to cells by CD48-IgG and CD48-IgM was only detectable when they were immobilized on covaspheres^TM and represented no increase in sensitivity over CD48-CD4 covaspheres^TM when tested for binding to cells expressing high and low levels of CD2. CD48-CD4d3 + 4 covaspheres^TM only bound to rat cells expressing CD2. In the reverse orientation, binding of CD2-CD4d3 + 4 covaspheres^TM was dependent on expression of CD48. Pre-incubation of cells with CD2 or CD48 mAb abolished all binding of CD48-CD4d3 + 4 or CD2-CD4d3 + 4, respectively. The data provide no evidence for an alternative ligand for rat CD2 or CD48.     

Comparative biodistribution of indium- and yttrium-labeled B3 monoclonal antibody conjugated to either 2-(p-SCN-Bz)-6-methyl-DTPA (1 B4M-DTPA) or 2-(p-SCN-Bz)-1,4,7,10-tetraazacyclododecane tetraacetic acid (2B-DOTA)

The biodistribution of indium-111/yttrium-88-labeled B3 monoclonal antibody, a murine IgG1k, was evaluated in non-tumor-bearing mice. B3 was conjugated to either 2-(p-SCN-Bz)-6-methyl-DTPA (1B4M) or 2-(p-SCN-Bz)-1,4,7,10 tetraazacyclododecane tetra-acetic acid (2B-DOTA) and labeled with ¹¹¹In at 1.4–2.4 mCi/mg and ⁸⁸Y at 0.1–0.3 mCi/mg. Non-tumor-bearing nude mice were co-injected i.v. with 5–10 Ci/4–10 g of ¹¹¹In/⁸⁸Y-labeled B3 conjugates and sacrificed at 6 h and daily up to 168 h post-injection. Mice injected with ¹¹¹In/⁸⁸Y (IB4M)-B3 showed a similar biodistribution of the two radiolabels in all tissues except the bones, where significantly higher accretion of ⁸⁸Y than ¹¹¹In was observed, with 2.8% ± 0.2% vs 1.3% ± 0.16% ID/g in the femur at 168 h, respectively (P<0.0001). In contrast, mice receiving the ¹¹¹In/⁸⁸Y-(DOTA)-B3 conjugate showed significantly higher accumulation of ¹¹¹In than ⁸⁸Y in most tissues, including the bones, with 2.0% ± 0.1% vs 1.2% ± 0.09% ID/g in the femur at 168 h, respectively (P<0.0001). Whereas the ratios of the areas underneath the curve (%ID × h/g) in the blood, liver, kidney and bone were 0.96, 1.12, 1.13, and 0.74 for ¹¹¹In/⁸⁸Y-(IB4M)-B3 and 0.84, 1.23, 1.56, and 1.31 for ¹¹¹In/⁸⁸Y (DOTA)-B3, respectively, ratios 1 were observed between ¹¹¹In-(IB4M)-B3 and ⁸⁸Y-(DOTA)-B3. In summary, while neither IB4M nor DOTA was equally stable for ¹¹¹In and ⁸⁸Y, the fate of ⁸⁸Y- (DOTA)-B3 could be closely traced by that of ¹¹¹ In-(IB4M)-B3.     

Differential effects of anxiogenic central and peripheral benzodiazepine receptor ligands in tests of learning and memory

Previous research has demonstrated that low doses of anxiogenic central benzodiazepine receptor (CBR) ligands, the beta-carbolines, improve performance in various learning and memory tests in animals if administered prior to training. The present experiments compared the effect of a beta-carboline (FG 7142) with that of a pharmacologically distinct anxiogenic compound, a peripheral benzodiazepine receptor (PBR) ligand, 4-chlorodiazepam (Ro5-4864), in two tests of learning and memory in rats. As expected, FG 7142 significantly improved performance in a passive avoidance test. Ro5-4864 was without effect. In a shuttlebox escape test, Ro5-4864 significantly impaired performance while FG 7142 had no effect. The effect of Ro5-4864 was antagonized by the specific peripheral benzodiazepine receptor antagonist, PK 11195. These results indicate that the differential impact of CBR and PBR anxiogenic ligands on performance in aversively-motivated learning tests may be a reflection of their distinct pharmacologies.     

亲环素配体研究进展

综述近年来亲环素配体在构效关系研究、合成及应用方面的进展,展望了今后的研究动向。亲环素是一类具有肽基脯氨酸顺反异构酶活性的免疫亲和素,广泛分布于各种组织细胞内。它参与脯氨酸肽键的顺反异构化、线粒体膜通透性转变、细胞内及细胞间信号传导等多种生理活动。亲环素配体是与亲环素有一定结合能力的化合物。目前研究的亲环素配体主要是具有免疫抑制或细胞保护作用的抑制剂。     

兔纹状体多巴胺受体的测定及应用

采用~3H-Spiperone作放射性配体,测定了兔纹状体细胞膜多巴胺受体的特异性结合特性,证明特异性结合可达饱和状态,并有两种不同的结合位点。作者用该受体蛋白建立了测定血清中氟哌啶醇的放射受体分析法,最小可测浓度为1.8±0.5ng/ml,批内和批间变异系数分别为6.5%和10.54%。放射受体法测定的结果与高压液相测定的结果密切相关,因前者测定的是血清中游离的氟哌啶醇,后者测定血清中氟哌啶醇的总浓度,故前者测定值低于后者。     

Multi-Target-Directed Ligands and other Therapeutic Strategies in the Search of a Real Solution for Alzheimer's Disease

The lack of an adequate therapy for Alzheimer''s Disease (AD) contributes greatly to the continuous growing amount of papers and reviews, reflecting the important efforts made by scientists in this field. It is well known that AD is the most common cause of dementia, and up-to-date there is no prevention therapy and no cure for the disease, which contrasts with the enormous efforts put on the task. On the other hand many aspects of AD are currently debated or even unknown. This review offers a view of the current state of knowledge about AD which includes more relevant findings and processes that take part in the disease; it also shows more relevant past, present and future research on therapeutic drugs taking into account the new paradigm “Multi-Target-Directed Ligands” (MTDLs). In our opinion, this paradigm will lead from now on the research toward the discovery of better therapeutic solutions, not only in the case of AD but also in other complex diseases. This review highlights the strategies followed by now, and focuses other emerging targets that should be taken into account for the future development of new MTDLs. Thus, the path followed in this review goes from the pathology and the processes involved in AD to the strategies to consider in on-going and future researches.     

XLP1 inhibitory effect by 2B4 does not affect DNAM‐1 and NKG2D activating pathways in NK cells

X‐linked lymphoproliferative disease 1 (XLP1) is a rare congenital immunodeficiency caused by SH2D1A (Xq25) mutations resulting in lack or dysfunction of SLAM‐associated protein adaptor molecule. In XLP1 patients, upon ligand (CD48) engagement, 2B4 delivers inhibitory signals that impair the cytolytic activity of NK (and T) cells. This causes the selective inability to control EBV infections and the occurrence of B‐cell lymphomas. Here, we show that in the absence of SLAM‐associated protein, co‐engagement of 2B4 with different activating receptors, either by antibodies or specific ligands on target cells, inhibits different ITAM‐dependent signaling pathways including activating killer Ig‐like receptors. In XLP1 NK cells, 2B4 affected both the cytolytic and IFN‐γ production capabilities, functions that were restored upon disruption of the 2B4/CD48 interactions. Notably, we provide evidence that 2B4 dysfunction does not affect the activity of DNAM‐1 and NKG2D triggering receptors. Thus, while CD48⁺ B‐EBV and lymphoma B cells devoid of NKG2D and DNAM‐1 ligands were resistant to lysis, the preferential usage of these receptors allowed XLP1 NK cells to kill lymphomas that expressed sufficient amounts of the specific ligands. The study sheds new light on the XLP1 immunological defect and on the cross‐talk of inhibitory 2B4 with triggering NK (and T) receptors.     

大环内酯类免疫抑制剂的研究进展

FK506结合蛋白12(FK506 binding protein12,FKBP12)是FK506结合蛋白的重要成员,目前对其参与免疫抑制剂抗器官移植后排斥反应的研究较多。他克莫司(FK506,tacrolimus)、雷帕霉素(rapamycin)和子囊霉素(ascomycin)是FKBP12的三个天然存在的大环内酯类配体,除了免疫抑制外还具有其他生理功能。这三个配体的衍生物也对FKBP12有一定的亲和力,且有些已经作为药物被应用于临床上。本文针对FKBP12的配体及其治疗作用进行综述。     

结直肠癌Dll-1/Notch信号传导通路的研究

目的 研究Dll-1/Notchl信号传导通路与结直肠癌病理学特征的关系,明确此通路对结直肠癌细胞增殖及凋亡的影响.方法 应用固定化蛋白质印迹法检测63例结直肠癌组织及其邻近正常肠黏膜中Dll-1及Notch1蛋白表达;用Notch1通路中γ-分泌酶抑制剂DAPT作用于结肠癌细胞系SW480,MTT法检测细胞增殖状态,流式细胞仪观察其对细胞凋亡的影响,固定化蛋白质印迹法检测Notch1胞内活性段及其靶基因产物Hes-1和Bcl-2蛋白的表达.分别采用独立样本t检验、配对样本t检验及单因素方差分析.结果 结直肠癌组织中Notch1和Dll-1蛋白表达水平分别高于正常肠黏膜的1.75及2.21倍(t=2.554,P=0.012及t=3.565,P=0.005);二者表达与肿瘤分化程度(t =2.463,P=0.017及t=2.390,P=0.019)、分期(t=2.675,P=0.007及f=2.310,P=0.021)及淋巴结转移(t =2.229,P=0.021及t=2.210,P=0.023)有关.用γ-分泌酶抑制剂DAPT阻断Notch1通路可抑制SW480细胞的增殖,诱导其凋亡;同时NICD和Bcl-2的表达水平随作用时间延长而降低.结论 Dll-1及Notch1的高表达与结直肠癌病理学特征密切相关,阻断Notch1通路可抑制Bcl-2表达,同时可抑制结肠癌细胞的增殖,并诱导其凋亡.