BACKGROUND & AIMS: BACKGROUND:The purpose of this study was to determine the distribution of radioiodinated estramustine (RI-EMP) and a radioiodinated antibody against estramustine binding protein (RI-EMBP-AB) in mice. METHODS:RI-EMP and RI-EMBP-AB were injected in male mice intravenously, and the activities of tissue samples were measured 1-31 hr from the injection. Pure iodine-125 (RI) was used as a control. RESULTS:RI-EMP accumulated in the prostate, which contained 2.6% of injected activity (ID) per gram tissue at 7 hr. The liver had an activity of 21.4% ID/g at 1 hr, which decreased as RI-EMP was secreted in bile. The lung contained 2.3% ID/g at 7 hr, and it retained the activity longer than the prostate. RI-EMBP-AB accumulated in the prostate: The activity was 2.9% ID/g at 7 hr. The gallbladder contained 6.5% ID/g at 7 hr. CONCLUSIONS:Due to its cytotoxic and radiosensitizing properties, RI-EMP can possibly be used for treating prostate cancer and other tumors.

背景与目标：

BACKGROUND & AIMS: :We have studied by electron microscopy a fascinating series of antidansyl monoclonal antibodies developed by Dangl et al. (Cytometry 2, 395-401, 1982) which have the same variable domain but different constant domains. Three of the four subclasses of mouse IgG were represented, IgG1, IgG2a and IgG2b. Previously, Oi et al. (Nature 307, 136-140, 1984) had examined the flexibilities of these antibodies by time-resolved fluorescence depolarization and found that IgG1 was least flexible, IgG2a was intermediate and IgG2b was the most flexible. In this communication we examine the conformations of circular complexes formed between these antibodies and a bivalent hapten, bis-dansyl cadaverine. The circular complexes were predominantly composed of two antibodies linked into a ring by two bivalent haptens, and are referred to as dimers, since only the antibody molecules are seen with the electron microscope. A few trimers and an occasional tetramer were also present in these preparations. For the least flexible IgG1, almost all (greater than 99%) of the circular dimers were "open-hinge" complexes with a hinge angle between the Fab arms of 100-120 degrees. For the intermediate IgG2a, most of the dimers were "open-hinge" complexes, but a larger percentage, 4 to 5%, had closed hinges with a hinge angle approaching 0 degrees. For the most flexible IgG2b, over 40% of the dimers were "closed-hinge" complexes. A model is proposed to explain these differences based upon orientation of the hapten in the combining site and differences in hinge structure.

背景与目标： : 我们通过电子显微镜研究了由Dangl等人开发的一系列令人着迷的抗dansyl单克隆抗体 (Cytometry 2，395-401，1982)，它们具有相同的可变结构域但具有不同的恒定结构域。代表了小鼠IgG的四个亚类中的三个，IgG1，IgG2a和IgG2b。以前，Oi等人 (Nature 307，136-140，1984) 通过时间分辨荧光去极化检查了这些抗体的柔韧性，发现IgG1是最不柔韧的，IgG2a是中间的，IgG2b是最柔韧的。在本交流中，我们检查了这些抗体与二价半抗原bis-dansyl尸胺之间形成的圆形复合物的构象。圆形复合物主要由两种通过两个二价半抗原连接成一个环的抗体组成，并且被称为二聚体，因为在电子显微镜下只能看到抗体分子。这些制剂中还存在一些三聚体和偶尔的四聚体。对于柔性最小的IgG1，几乎所有 (大于99%) 的圆形二聚体是 “开铰链” 复合物，其Fab臂之间的铰链角度为100-120度。对于中间体IgG2a，大多数二聚体是 “开铰链” 复合物，但较大的百分比 (4至5%) 具有铰链角度接近0度的闭合铰链。对于最灵活的IgG2b，超过40% 的二聚体是 “闭合铰链” 复合物。提出了一个模型来解释这些差异，该模型基于结合部位中半抗原的方向和铰链结构的差异。

BACKGROUND & AIMS: :Data are presented indicating that the growth of 5 out of 5 murine lymphoid tumors can be inhibited in a synergistic fashion in vitro by combined treatment with the iron chelator deferoxamine (DFO) and an immunoglobulin G (IgG) monoclonal anti-transferrin receptor antibody (ATRA). A two-way dose/response analysis shows that the ATRA becomes more efficient as an inhibitor with increasing doses of DFO. Flow cytometric studies further support the view that IgG ATRAS impair transferrin receptor (TR) function by causing TR down-modulation and degradation, even when the presence of DFO acts to promote increased cell surface TR expression. It is also shown that an IgG ATRA is nearly as effective as an IgM ATRA in inhibiting tumor cell growth when used in combination with DFO. Finally, studies with the iron chelator picolinic acid show that it produces only additive, or very slightly supra-additive, effects when used in combination with the ATRA. Therefore, these studies not only continue to suggest that combination chelator/ATRA therapy warrants further investigation as a tool in the therapy of hematopoietic malignancies, but also make the following new points: (1) the clinically familiar iron chelator deferoxamine, but not all iron chelators, produces synergistic inhibition of tumor growth in vitro with ATRAS; and (2) IgG ATRAS, which may be clinically more attractive reagents than IgA or IgM ATRAS because of better access to extra vascular tissue spaces, have unexpectedly been found to function as powerful growth inhibitors when used in combination with DFO.

背景与目标： : 提供的数据表明，通过与铁螯合剂去铁胺 (DFO) 和免疫球蛋白G (IgG) 单克隆抗转铁蛋白受体联合治疗，可以在体外以协同方式抑制5种鼠淋巴样肿瘤中的5种的生长抗体 (ATRA)。双向剂量/反应分析表明，随着DFO剂量的增加，ATRA作为抑制剂变得更有效。流式细胞术研究进一步支持以下观点: 即使DFO的存在促进细胞表面TR表达增加，IgG ATRAS也会通过引起TR下调和降解而损害运铁蛋白受体 (TR) 的功能。还显示，当与DFO组合使用时，IgG ATRA在抑制肿瘤细胞生长方面几乎与IgM ATRA一样有效。最后，对铁螯合剂吡啶甲酸的研究表明，与ATRA结合使用时，它仅产生添加剂或非常轻微的超添加剂效应。因此，这些研究不仅继续表明，螯合剂/ATRA联合治疗值得进一步研究作为治疗造血系统恶性肿瘤的工具，而且还提出了以下新的观点 :( 1) 临床上熟悉的铁螯合剂去铁胺，但不是所有的铁螯合剂，与ATRAS在体外产生肿瘤生长的协同抑制; 和 (2) IgG ATRAS在临床上可能比IgA或IgM ATRAS更具吸引力，因为更好地进入额外的血管组织空间，当与DFO组合使用时，已意外地发现IgG ATRAS具有强大的生长抑制剂的功能。

BACKGROUND & AIMS: :Oncolytic viruses exploit the cancer cell phenotype to complete their lytic life cycle, releasing progeny virus to infect nearby cells and repeat the process. We modified the oncolytic group B adenovirus EnAdenotucirev (EnAd) to express a bispecific single-chain antibody, secreted from infected tumour cells into the microenvironment. This bispecific T-cell engager (BiTE) binds to EpCAM on target cells and cross-links them to CD3 on T cells, leading to clustering and activation of both CD4 and CD8 T cells. BiTE transcription can be controlled by the virus major late promoter, limiting expression to cancer cells that are permissive for virus replication. This approach can potentiate the cytotoxicity of EnAd, and we demonstrate using primary pleural effusions and peritoneal malignant ascites that infection of cancer cells with the BiTE-expressing EnAd leads to activation of endogenous T cells to kill endogenous tumour cells despite the immunosuppressive environment. In this way, we have armed EnAd to combine both direct oncolysis and T cell-mediated killing, yielding a potent therapeutic that should be readily transferred into the clinic.

背景与目标： : 溶瘤病毒利用癌细胞表型来完成其裂解生命周期，释放后代病毒以感染附近的细胞并重复该过程。我们修改了溶瘤组B腺病毒EnAdenotucirev (EnAd) 表达双特异性单链抗体，该抗体从感染的肿瘤细胞分泌到微环境中。这种双特异性T细胞接合器 (BiTE) 与靶细胞上的EpCAM结合，并将它们与T细胞上的CD3交联，从而导致CD4和CD8 T细胞的聚集和激活。BiTE转录可以由病毒主要的晚期启动子控制，从而限制了对允许病毒复制的癌细胞的表达。这种方法可以增强EnAd的细胞毒性，并且我们证明使用原发性胸腔积液和腹膜恶性腹水，尽管存在免疫抑制环境，但用BiTE表达的EnAd感染癌细胞会导致内源性T细胞激活以杀死内源性肿瘤细胞。通过这种方式，我们已经武装EnAd将直接溶解和T细胞介导的杀伤结合起来，产生了一种有效的治疗方法，可以很容易地转移到临床中。

BACKGROUND & AIMS: :Successful translation of findings derived from preclinical studies into effective therapies is critical in biomedical research. Lack of robustness and reproducibility of the preclinical data, due to insufficient number of repeats, inadequate cell-based and mouse models contribute to the poor success rate. Antibodies are widely used in preclinical research, notably to determine the expression of potential therapeutic targets in tissues of interest, including tumors, but also to identify disease and/or treatment response biomarkers. We sought to determine whether the current antibody characterization standards in preclinical research are sufficient to ensure reliability of the data found in peer-reviewed publications. To address this issue, we used detection of the protein c-FLIP, a major factor of resistance to apoptosis, as a proof of concept. Accurate detection of endogenous c-FLIP levels in the preclinical settings is imperative since it is considered as a potential theranostic biomarker. Several sources of c-FLIP antibodies validated by their manufacturer and recommended for western blotting were therefore rigorously tested. We found a wide divergence in immune recognition properties. While these antibodies have been used in many publications, our results show that several of them failed to detect endogenous c-FLIP protein by Western blotting. Our results suggest that antibody validation standards are inadequate, and that systematic use of genetic knockdowns and/or knockouts to establish proof of specificity is critical, even for antibodies previously used in the scientific literature. Because antibodies are fundamental tools in both preclinical and clinical research, ensuring their specificity is crucial.

背景与目标： : 成功地将临床前研究的发现转化为有效的疗法在生物医学研究中至关重要。由于重复次数不足，基于细胞的和小鼠模型不足，缺乏临床前数据的稳健性和可重复性导致成功率低。抗体广泛用于临床前研究，特别是用于确定感兴趣组织 (包括肿瘤) 中潜在治疗靶标的表达，还用于鉴定疾病和/或治疗反应生物标志物。我们试图确定临床前研究中当前的抗体表征标准是否足以确保同行评审出版物中发现的数据的可靠性。为了解决这个问题，我们使用了检测蛋白c-FLIP (抗凋亡的主要因素) 作为概念的证明。临床前环境中内源性c-FLIP水平的准确检测势在必行，因为它被认为是潜在的肿瘤生物标志物。因此，对其制造商验证并推荐用于蛋白质印迹的c-FLIP抗体的几种来源进行了严格测试。我们发现免疫识别特性存在很大差异。尽管这些抗体已在许多出版物中使用，但我们的结果表明，其中一些未能通过Western印迹检测到内源性c-FLIP蛋白。我们的结果表明，抗体验证标准是不充分的，并且系统地使用基因敲除和/或敲除来建立特异性证明是至关重要的，即使对于以前在科学文献中使用的抗体也是如此。由于抗体是临床前和临床研究的基本工具，因此确保其特异性至关重要。

BACKGROUND & AIMS: :Although many multiple myeloma (MM) patients initially respond to cytotoxic therapy, most eventually relapse. Novel therapeutic strategies employing a combination of chemotherapy with targeted biologics may significantly enhance the response of tumor cells to treatment. We tested a fully human anti-IGF-IR antibody (A12) against MM, and showed specific inhibition of IGF-I or serum-induced IGF-IR signaling in MM cells in vitro. The A12 as a single agent was demonstrated to exert modest to significant inhibition of tumor growth in vivo in various subcutaneous xenograft MM models. The A12 was also evaluated in a disseminated xenograft MM.1S NOD/SCID model as monotherapy or in combination with other drugs (bortezomib, melphalan) currently in clinical use. The tumor burden, as determined by luciferase bioimaging, was sharply decreased, and overall survival significantly prolonged when the therapies were combined. Immunohistochemical analysis demonstrated that the A12 treated tumors had significantly decreased vascularization compared to control tumors. Furthermore, most MM lines constitutively secreted significant quantities of VEGF, and this was enhanced following IGF-I treatment. Inhibition of IGF-IR by the A12 in vitro suppressed both constitutive and IGF-I-induced secretion of VEGF, indicating that a putative anti-angiogenic mechanism associated with the A12 treatment may contribute to its anti-tumor effect.

背景与目标： : 尽管许多多发性骨髓瘤 (MM) 患者最初对细胞毒性治疗有反应，但大多数最终复发。采用化疗与靶向生物制剂相结合的新型治疗策略可能会显着增强肿瘤细胞对治疗的反应。我们测试了针对MM的全人抗igf-ir抗体 (A12)，并在体外显示了对MM细胞中igf-i或血清诱导的igf-ir信号的特异性抑制。在各种皮下异种移植MM模型中，A12作为单一药物被证明对体内肿瘤生长具有适度至显着的抑制作用。还在播散性异种移植MM.1S NOD/SCID模型中评估了A12，作为单一疗法或与目前临床使用的其他药物 (硼替佐米，美法仑) 联合使用。通过荧光素酶生物成像确定的肿瘤负荷急剧降低，并且当联合治疗时，总生存期显着延长。免疫组织化学分析表明，与对照肿瘤相比，A12治疗的肿瘤血管形成明显减少。此外，大多数MM系组成型分泌了大量的VEGF，并且在igf-i治疗后这种情况得到了增强。体外A12抑制igf-ir抑制了组成型和igf-i诱导的VEGF分泌，表明与A12治疗相关的推定抗血管生成机制可能有助于其抗肿瘤作用。

BACKGROUND & AIMS: :A major traditional of antibacterial drugs is antibiotic which promotes more rapid release of the toxins from bacteria cells in human body, which causes severe infection. The thermostable direct hemolysin (TDH) has been proposed as a major virulence factor of Vibrio parahaemolyticus (Vp). This study covers the preparation of polymer microparticle-antibody conjugate for the development of a drug targeting approach for antibacterial drug delivery. The chemical binding of antibodies (ab) to latex bead of 0.2 mum diameter was performed by using a water-soluble carbodiimide technique. Confocal microscopy revealed that the bacteria were strongly absorbed by the latex beads with bound anti-Vp polyclonal antibody (pAb). Treatment with a latex bead bound both anti-Vp pAb and anti-TDH monoclonal antibody (mAb) significantly inhibited bacterial adherence to the Caco-2 cells (p < 0.01), and reduced TDH-induced cytotoxicity in histology. These preliminary results suggest that it may be possible to effectively protect against Vp infection by using this microparticle-antibody conjugate delivery system.

背景与目标： 抗菌药物的主要传统是抗生素，它促进人体细菌细胞中毒素的更快释放，从而引起严重的感染。已提出热稳定的直接溶血素 (TDH) 作为副溶血性弧菌 (Vp) 的主要毒力因子。这项研究涵盖了聚合物微粒-抗体偶联物的制备，用于开发用于抗菌药物递送的药物靶向方法。通过使用水溶性碳二亚胺技术进行抗体 (ab) 与直径为0.2的乳胶珠的化学结合。共聚焦显微镜显示，细菌被结合了抗Vp多克隆抗体 (pAb) 的乳胶珠强烈吸收。用结合抗Vp pAb和抗TDH单克隆抗体 (mAb) 的乳胶珠处理可显着抑制细菌对Caco-2细胞的粘附 (p <0.01)，并降低组织学中TDH诱导的细胞毒性。这些初步结果表明，通过使用这种微粒-抗体缀合物递送系统，有可能有效地预防Vp感染。

BACKGROUND & AIMS: :Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 x 10(6) cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotype-matched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-Mr MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.

背景与目标： : 在第1、4、7、10和14天，将5 mg/kg甲氨蝶呤 (MTX) 与人高分子量黑色素瘤相关抗原 (hmw-maa)，单克隆抗体 (mAb) 225.28 (IgG2a) 连接的抗体静脉注射到裸鼠中，在皮下接种2 × 10(6) 培养的人M21黑色素瘤细胞后24小时开始，在治疗开始后第14天90% 抑制平均肿瘤体积，并在第50天65% 抑制平均肿瘤体积。注射等摩尔量的游离MTX和MTX与正常小鼠IgG或同种型匹配的骨髓瘤蛋白连接不会显着抑制肿瘤生长。与mAb 225.28连接的MTX不抑制人黑素瘤细胞系M21的亚系的异种移植，而没有可检测到的hmw-maa表达。在克隆形成测定中，MTX-225.28缀合物在抑制M21黑色素瘤细胞生长方面的效力是游离MTX的三倍，但Caki-1不抑制不表达高Mr MAA的肾癌细胞的生长。相反，与mAb DAL K29连接的MTX与Caki-1的肾癌细胞反应，抑制了它们的生长，但不影响黑色素瘤细胞的生长。与未处理小鼠的M21细胞相比，从用MTX-225.28缀合物处理的小鼠的残留肿瘤中分离的M21黑色素瘤细胞在与mAb 225.28的反应性和对MTX的敏感性方面没有差异。

BACKGROUND & AIMS: :Internalization by cells of radiolabeled protein ligands bound to the cell surface is frequently analyzed by extraction of the cells with low pH buffers. This treatment supposedly strips the ligands from the cell surface, and remaining molecules are considered to be internalized. However, we show herein that: (1) low molecular weight catabolic products that are trapped within lysosomes (residualizing radiolabels) are efficiently extracted by low pH buffers, under the same conditions used to remove cell surface-bound material, and (2) low pH treatment lyses the majority of the cells, as shown with both a nonadherent and an adherent cell line, with the release of most of a (51)Cr label. Still, low pH extraction was effective at demonstrating Ab internalization, as has been demonstrated many times. These effects of low pH treatment may be attributed to the fixative properties of these buffers. Regardless of the mechanism, these data must be taken into consideration in interpreting the results of such experiments.

背景与目标： : 通过细胞内化结合到细胞表面的放射性标记蛋白配体经常通过用低pH缓冲液提取细胞来分析。据推测，这种处理将配体从细胞表面剥离，剩余的分子被认为是内在化的。然而，我们在本文中显示 :( 1) 在用于去除细胞表面结合材料的相同条件下，通过低pH缓冲液有效地提取了溶酶体中捕获的低分子量分解代谢产物 (残留放射性标记)，以及 (2) 低pH处理裂解大多数细胞，如非粘附细胞系和粘附细胞系所示，大部分 (51)Cr标记的释放。尽管如此，低pH萃取仍可有效证明Ab内化，正如多次证明的那样。低pH处理的这些影响可能归因于这些缓冲液的固定性。无论机制如何，在解释此类实验结果时必须考虑这些数据。

BACKGROUND & AIMS: :In a small number of patients treated with botulinum toxin (BT) antibody (Ab) formation occurs. BT Ab can be detected by the mouse protection assay (MPA) or by the mouse diaphragm assay (MDA). Both methods, however, have major drawbacks. We tested a method for detecting BT Ab which measures the BT-induced reduction in the electromyographic amplitude of the mean maximal voluntary activation (M-EMG) of the sternocleidomastoid muscle. The M-EMG reduction was compared in 17 patients with cervical dystonia and secondary BT therapy failure to the M-EMG reduction previously measured in controls. Values more than 2 SD below the mean of controls were considered abnormal. Six patients showed BT Ab on the MPA and MDA; all of these had abnormal M-EMG reductions. Eleven patients showed no BT Ab on MPA and MDA testing; in ten of these the M-EMG reduction was normal, and in one it was pathological, but MDA testing later changed to positive under continued BT therapy. The sternocleidomastoid test is easy to perform and produces quantitative results. Since its sensitivity and specificity are at least as good as those of the MDA and the MPA, it can replace them.

背景与目标： : 在少数接受肉毒杆菌毒素 (BT) 治疗的患者中，抗体 (Ab) 形成。BT Ab可以通过小鼠保护试验 (MPA) 或小鼠隔膜试验 (MDA) 检测。然而，这两种方法都有主要缺点。我们测试了一种检测BT Ab的方法，该方法测量了BT引起的胸锁乳突肌平均最大自愿激活 (m-emg) 的肌电图幅度的降低。将17例宫颈肌张力障碍和继发性BT治疗失败的患者的m-emg降低与先前在对照组中测量的m-emg降低进行了比较。低于对照组平均值超过2 SD的值被认为是异常的。六名患者在MPA和MDA上显示BT Ab; 所有这些都有异常的M-EMG降低。11例患者在MPA和MDA测试中没有显示BT Ab; 其中10例m-emg降低是正常的，其中1例是病理性的，但随后在持续的BT治疗下MDA测试变为阳性。胸锁乳突肌试验易于进行并产生定量结果。由于其敏感性和特异性至少与MDA和MPA一样好，因此可以替代它们。

BACKGROUND & AIMS: OBJECTIVE:Cytotoxic T lymphocytes (CTL) initially recognize target cells using the T-cell receptor (TCR), then strongly adhere to these cells by accessory molecules, and finally induce apoptosis by Fas ligand (FasL)/Fas or lyse by the granzyme/perforin system. We describe the development of gelatin beads carrying anti-tumor monoclonal antibody (mAb) and anti-Fas mAb mimicking the TCR and FasL, respectively. We hypothesized that these antibody-coated beads can be therapeutically utilized for the elimination of tumor cells. MATERIALS AND METHODS:We evaluated the cytotoxic activity of gelatin beads bearing CH11 (anti-Fas mAb) after incubation with several human leukemia cell lines. Cytotoxic activities were measured using colorimetric DNA fragmentation assay and lactate dehydrogenase (LDH) release assay. RESULTS:We demonstrated that the cytotoxic effects of anti-Fas mAb were markedly enhanced by fixation on gelatin beads. Microscopic examination showed that the beads attached to the target cells and induced their apoptosis. These effects were enhanced further by adding tumor-specific mAb. These in vitro properties of the beads were well reconstituted in the peritoneal cavity of mice. CONCLUSION:Although antibody-coated gelatin beads lack several important properties of natural CTL, such as differentiation, proliferation, and the functions of adhesion molecules, they mimic well the targeting and cytotoxic functions of natural CTL. Our findings suggest that antibody-carrying gelatin beads may be the first step toward the development of artificial CTL and can be applied, for example, to artificial dendritic and stroma cells for the development of novel biotherapeutic approaches.

背景与目标：

BACKGROUND & AIMS: :Protein isoprenylation is a lipid posttranslational modification required for the function of many proteins that share a carboxyl-terminal CAAX motif. The X residue determines which isoprenoid will be added to the cysteine. When X is a methionine or serine, the farnesyl-transferase transfers a farnesyl, and when X is a leucine or isoleucine, the geranygeranyl-transferase I, a geranylgeranyl group. But despite its CKVL motif, RhoB was reported to be both geranylgeranylated and farnesylated. Thus, the determinants of RhoB prenylation appear more complex than initially thought. To determine the role of RhoB CAAX motif, we designed RhoB mutants with modified CAAX sequence expressed in baculovirus-infected insect cells. We demonstrated that RhoB was prenylated as a function of the three terminal amino acids, i.e., RhoB bearing the CAIM motif of lamin B or CLLL motif of Rap1A was farnesylated or geranylgeranylated, respectively. Next, we produced a specific polyclonal antibody against farnesyl cysteine methyl ester allowing prenylation analysis avoiding the metabolic labeling restrictions. We confirmed that the unique modification of the RhoB CAAX box was sufficient to direct the RhoB distinct prenylation in mammalian cells and, inversely, that a RhoA-CKVL chimera could be alternatively prenylated. Moreover, the immunoprecipitation of endogenous RhoB from cells with the anti-farnesyl cysteine antibody suggested that wild-type RhoB is farnesylated in vivo. Taken together, our results demonstrated that the three last carboxyl amino acids are the main determinants for RhoB prenylation and described an anti-farnesyl cysteine antibody as a useful tool for understanding the cellular control of protein farnesylation.

背景与目标： : 蛋白质异戊二烯化是一种脂质翻译后修饰，是许多具有羧基末端CAAX基序的蛋白质的功能所需的。X残基决定了哪些类异戊二烯将被添加到半胱氨酸中。当X是蛋氨酸或丝氨酸时，法尼基转移酶转移法尼基，而当X是亮氨酸或异亮氨酸时，香叶香叶基转移酶I，香叶香叶基。但是，尽管有CKVL基序，但据报道RhoB既被香叶基化又被法尼基化。因此，RhoB异戊烯化的决定因素似乎比最初认为的更复杂。为了确定RhoB CAAX基序的作用，我们设计了具有在杆状病毒感染的昆虫细胞中表达的修饰CAAX序列的RhoB突变体。我们证明了RhoB作为三个末端氨基酸的函数被异戊二烯化，即带有lamin B的CAIM基序或Rap1A的CLLL基序的RhoB分别被法呢基化或香叶基化。接下来，我们产生了针对法呢基半胱氨酸甲酯的特异性多克隆抗体，从而可以进行异戊烯化分析，从而避免了代谢标记的限制。我们证实，RhoB CAAX盒的独特修饰足以在哺乳动物细胞中引导RhoB独特的异戊烯化，并且相反，RhoA-CKVL嵌合体可以替代异戊烯化。此外，用抗法尼基半胱氨酸抗体从细胞中免疫沉淀内源性RhoB表明，野生型RhoB在体内被法尼基化。合在一起，我们的结果表明，最后三个羧基氨基酸是RhoB异戊烯化的主要决定因素，并描述了抗法尼基半胱氨酸抗体作为了解蛋白质法尼基化的细胞控制的有用工具。

BACKGROUND & AIMS: :The immune response to different dosage schedules of oral live Salmonella typhi Ty21a vaccines was studied by enumeration of specific antibody-secreting cells (ASC) in the peripheral blood believed to have been stimulated by the vaccine antigen on mucosal surfaces and to be on their way back to those sites for local antibody secretion. Four groups of subjects were vaccinated with either three (3 x S), two (2 x S) or one (1 x S) dose of a suspension-formulated vaccine, or with three doses of vaccine in enteric-coated capsules (3 x E). The ASC-responses were highest in group 3 x S, followed by 3 x E, 2 x S and 1 x S, in this order. These differences parallel differences in protection from disease as observed in field trails with these regimens. This assay might therefore be useful for presumptive assessment of the protective ability of new vaccines or vaccine regimens. It certainly can be used to measure the immunogenicity of an oral vaccine.

背景与目标： : 通过计数外周血中的特异性抗体分泌细胞 (ASC)，研究了对不同剂量方案的口服伤寒沙门氏菌Ty21a活疫苗的免疫反应，这些细胞被认为是由粘膜表面的疫苗抗原刺激的，并且正在返回到那些局部抗体分泌的部位。四组受试者接种了三种 (3 x S)，两种 (2 x S) 或一种 (1 x S) 剂量的悬浮液配制疫苗，或在肠溶胶囊中接种了三种疫苗 (3 x E)。ASC反应在3 x S组中最高，依次为3 x E，2 x S和1 x S。这些差异与这些方案在野外试验中观察到的疾病保护差异平行。因此，此测定法可能有助于推定评估新疫苗或疫苗方案的保护能力。当然，它可以用于测量口服疫苗的免疫原性。

BACKGROUND & AIMS: The murine IgG3 monoclonal antibody NCC-ST-421 (ST-421), raised against human gastric cancer, shows strong reactivity with the Le(a)/Le(a) (al-fucosylated extended type 1 chain) antigen expressed on gastrointestinal (GI) cancer cells. ST-421 is capable of mediating both antibody-dependent cellular cytotoxicity (ADCC) by human peripheral blood lymphocytes (PBL), and complement dependent cytotoxicity (CDC). We investigated combination immunotherapy with OK-432, a streptococcal preparation, and ST-421 in vitro and in vivo. ADCC against Colo 205 (a human colon cancer cell line) was enhanced 2 to 3 fold after preincubation of PBL with OK-432 in vitro. These effect's were strongest when PBL were preincubated with OK-432 at a concentration of 0.5 ng/ml for 24 hours. In vivo, a human colon cancer xenograft model exhibited significant growth suppression after combined treatment with ST-421 and OK-432. Such combination immunotherapy may therefore be clinically useful in GI cancer.

背景与目标： 抗人胃癌的鼠IgG3单克隆抗体NCC-ST-421 (ST-421) 与在胃肠道 (GI) 癌细胞上表达的Le(a)/Le(a) (al-岩藻糖基化的延伸1型链) 抗原显示出强反应性。ST-421能够介导人外周血淋巴细胞 (PBL) 的抗体依赖性细胞毒性 (ADCC) 和补体依赖性细胞毒性 (CDC)。我们研究了与OK-432 (一种链球菌制剂) 的联合免疫疗法，并在体外和体内ST-421。在体外用OK-432预孵育PBL后，针对Colo 205 (人结肠癌细胞系) 的ADCC增强2至3倍。当PBL与0.5 ng/ml浓度的OK-432预孵育24小时时，这些作用最强。在体内，人结肠癌异种移植模型在用ST-421和OK-432联合治疗后表现出显著的生长抑制。因此，这种联合免疫疗法可能在胃肠道癌症中具有临床意义。

BACKGROUND & AIMS: :The current method for in vitro immunization (IVI) uses several antigens including toxins, food allergens, pathogenic bacteria, and self-antigen-derived peptides that induce an antigen-specific immune response in peripheral blood mononuclear cells (PBMCs). This protocol, however, requires donor blood collection and preparation of PBMCs before every IVI. In the present study, we aimed to design a more efficient system utilizing B cells immortalized with Epstein-Barr virus (EBV-B) as host cells for IVI to make antigen-specific antibodies. Results showed that previously antigen-sensitized, EBV-B cells exposed to the antigen along with IL-6, CpG oligonucleotides, and CD40 ligand signal produced antigen-specific antibodies. These results provide evidence for a novel and easy method to expand memory-type B cells and produce antigen-specific antibodies.

背景与目标： : 当前的体外预防接种 (IVI) 方法使用多种抗原，包括毒素，食物过敏原，致病菌和自身抗原衍生的肽，它们在外周血单核细胞 (pbmc) 中诱导抗原特异性免疫反应。但是，该方案要求在每次IVI之前进行供体血液采集和pbmc准备。在本研究中，我们旨在设计一种更有效的系统，该系统利用爱泼斯坦-巴尔病毒 (ebv-b) 永生化的b细胞作为IVI的宿主细胞来制造抗原特异性抗体。结果显示先前抗原致敏的ebv-b细胞与IL-6、CpG寡核苷酸和CD40配体信号一起暴露于抗原产生抗原特异性抗体。这些结果为扩展记忆型b细胞并产生抗原特异性抗体的新方法提供了证据。