半乳糖结合位点封闭式免疫毒素的制备及其特性

本文报道B链半乳糖结合位点封闭的蓖麻毒素—胃癌单克隆抗体(McAb)结合物的制备及其细胞毒特征.首先将引入碘乙酰基团的毒素与引入巯基的抗体反应,得到的结合物通过琼脂糖亲和层析柱除去仍保留半乳糖结合能力的部分.ELISA结果表明,结合物稀释至2 nmol/L水平,仍保留抗体结合活性.结合物对靶细胞在体外有较强的选择性杀伤作用,在0.1 pmol/L水平,对人胃癌细胞KATOⅢ的杀伤率为46％,优于McAb—蓖麻毒素A链结合物.结果表明,免疫毒素中B链的存在有助于增强A链的细胞杀伤作用。     

Variants of lymphoid lines produced with ricin A-chain monoclonal antibody conjugates

Conjugates of ricin A-chain with monoclonal anti-light chain antibodies specifically killed cells hearing kappa or lambda immunoglobulin (Ig) light chains. Exposure of cells from B-lymphoblastoid cell lines (B-LCL) to conjugate for less than 30 h had only a slight effect on cell growth, but on 48 h exposure a marked killing effect was achieved. After recovery of growth, cells were re-exposed to conjugate for 9-14 days. Treatment of cells from the EB4 line (sIgG lambda) in this way yielded 4 variants which showed a marked reduction in levels of surface Ig lambda and secreted Ig lambda with slight, or no, reduction in MHC class II expression and similar growth rates to the parent line. Variant lines retained their phenotype over long periods of culture.     

Structures of Eukaryotic Ribosomal Stalk Proteins and Its Complex with Trichosanthin,and Their Implications in Recruiting Ribosome-Inactivating Proteins to the Ribosomes

Ribosome-inactivating proteins (RIP) are RNA N-glycosidases that inactivate ribosomes by specifically depurinating a conserved adenine residue at the α-sarcin/ricin loop of 28S rRNA. Recent studies have pointed to the involvement of the C-terminal domain of the eukaryotic stalk proteins in facilitating the toxic action of RIPs. This review highlights how structural studies of eukaryotic stalk proteins provide insights into the recruitment of RIPs to the ribosomes. Since the C-terminal domain of eukaryotic stalk proteins is involved in specific recognition of elongation factors and some eukaryote-specific RIPs (e.g., trichosanthin and ricin), we postulate that these RIPs may have evolved to hijack the translation-factor-recruiting function of ribosomal stalk in reaching their target site of rRNA.     

Diverse Profiles of Ricin-Cell Interactions in the Lung Following Intranasal Exposure to Ricin

Ricin, a plant-derived exotoxin, inhibits protein synthesis by ribosomal inactivation. Due to its wide availability and ease of preparation, ricin is considered a biothreat, foremost by respiratory exposure. We examined the in vivo interactions between ricin and cells of the lungs in mice intranasally exposed to the toxin and revealed multi-phasic cell-type-dependent binding profiles. While macrophages (MΦs) and dendritic cells (DCs) displayed biphasic binding to ricin, monophasic binding patterns were observed for other cell types; epithelial cells displayed early binding, while B cells and endothelial cells bound toxin late after intoxication. Neutrophils, which were massively recruited to the intoxicated lung, were refractive to toxin binding. Although epithelial cells bound ricin as early as MΦs and DCs, their rates of elimination differed considerably; a reduction in epithelial cell counts occurred late after intoxication and was restricted to alveolar type II cells only. The differential binding and cell-elimination patterns observed may stem from dissimilar accessibility of the toxin to different cells in the lung and may also reflect unequal interactions of the toxin with different cell-surface receptors. The multifaceted interactions observed in this study between ricin and the various cells of the target organ should be considered in the future development of efficient post-exposure countermeasures against ricin intoxication.     

Anti‐cancer activity of anti‐p185HER‐2 ricin A chain immunotoxin on gastric cancer cells

Background and Aim: Overexpression of the human epidermal growth factor receptor 2 (HER‐2) protein has been detected in gastric cancer and has been associated with an unfavorable prognosis. We investigated the anti‐cancer effects of anti‐p185^HER‐2 ricin A chain (RTA) immunotoxin, alone or in combination with 5‐flurouracil on SGC7901‐HER‐2+ cells. Methods: SGC7901‐HER‐2+ cells were obtained by transfecting SGC7901 cells with HER‐2‐pcDNA3.1. Anti‐p185^HER‐2‐RTA was prepared by chemical conjugation of anti‐HER‐2 monoclonal antibody (mAb) and RTA. The SGC7901‐HER‐2+ cells were incubated with RTA, anti‐p185^HER‐2‐RTA, and/or 5‐flurouracil. The effects of drugs on cells were evaluated by MTT assay and Annexin V‐fluorescein isothiocyanate and propidium iodide double staining flow cytometry. The expression of caspase‐3, caspase‐9, cyclooxygenase‐2, and nuclear factor‐κB/p65 were assayed by western blot. SGC7901‐HER‐2+ cells were transplanted into BALB/c nude mice to produce solid tumors in an attempt to study the immunotoxin activity in vivo. Results: In vitro, anti‐p185^HER‐2‐RTA inhibited cell growth and induced apoptosis in SGC7901‐HER‐2+ cells. Anti‐p185^HER‐2‐RTA enhanced caspase‐3 and caspase‐9 activity, while downregulating the expression of cyclooxygenase‐2 and nuclear factor‐κB/p65. Its combination with 5‐flurouracil further inhibited the growth of SGC7901‐HER‐2+ cells. In vivo, our data showed that anti‐p185^HER‐2‐RTA significantly inhibited the growth of SGC7901‐HER‐2+ cells‐transplanted tumors. Conclusions: Anti‐p185^HER‐2‐RTA inhibits the growth of SGC7901‐HER‐2+ cells. The effect may be related to the activation of caspase‐3 and caspase‐9 and inhibition of cyclooxygenase‐2 and nuclear factor‐κB/p65. Anti‐p185^HER‐2‐RTA plus 5‐FU enhance anti‐cancer activity, suggesting useful clues for further study for the treatment of HER‐2 positive gastric cancers.     

Anti-tumor effects of toxins targeted to the prostate specific membrane antigen

BACKGROUND: There is presently no effective therapy for relapsing, metastatic, androgen-independent prostate cancer. Immunotherapy with monoclonal antibody-vehicled toxins (Immunotoxins, ITs) may be a promising novel treatment option for the management of prostate cancer in these cases. METHODS: Three anti-prostate specific membrane antigen (anti-PSMA) monoclonals (J591, PEQ226.5, and PM2P079.1) were cross-linked to ricin A-chain (RTA; native or recombinant), and their cytotoxic effects were investigated in monolayer and three-dimensional (3-D) cell cultures of prostate carcinoma cells (LNCaP). RESULTS: The various Immunotoxins showed effects in the nanomolar range (IC(50s) of 1.6-99 ng/ml) against PSMA+ cells (IC(50) being the concentration inhibiting 50% cell proliferation or protein synthesis). PSMA(-) cell lines were 62- to 277-fold less sensitive to anti-PSMA ITs, evidencing an appreciable therapeutic window. Treatment with J591-smpt-nRTA (0.35-31.7ng/ml) resulted in complete eradication of 3-D tumor micromasses or in 1.46- to 0.35-log reduction of target cells number, depending on the dose. CONCLUSION: Anti-PSMA ITs appear to be promising for use in the eradication of small prostate tumor cell aggregates present in tissues and in the bone marrow.     

Passive antibody administration (immediate immunity) as a specific defense against biological weapons

The potential threat of biological warfare with a specific agent is proportional to the susceptibility of the population to that agent. Preventing disease after exposure to a biological agent is partially a function of the immunity of the exposed individual. The only available countermeasure that can provide immediate immunity against a biological agent is passive antibody. Unlike vaccines, which require time to induce protective immunity and depend on the host's ability to mount an immune response, passive antibody can theoretically confer protection regardless of the immune status of the host. Passive antibody therapy has substantial advantages over antimicrobial agents and other measures for postexposure prophylaxis, including low toxicity and high specific activity. Specific antibodies are active against the major agents of bioterrorism, including anthrax, smallpox, botulinum toxin, tularemia, and plague. This article proposes a biological defense initiative based on developing, producing, and stockpiling specific antibody reagents that can be used to protect the population against biological warfare threats.     

蓖麻毒素及其SPDP修饰物对小鼠肝脏GSH含量的影响

目的：观察麻毒素（ＲＴ）修饰前后对小鼠肝脏毒性的影响。方法：ＲＴ用３－（２－吡啶二巯基）丙酸Ｎ－羟基琥珀酰亚胺酯中（ＳＰＤＰ）－－一种异型双功能交联剂,进行化学修饰,自下而上的ＳＰＤＰ衍生物（ＰＤＰ－Ｒ）,测定两者在不同剂量和时间对小鼠肝脏还原型谷胱甘肽（ＧＳＨ）含量的影响,以比较两者的毒性差别,结果：随着中毒剂量的增加和时间的延长,两者与可使小鼠肝脏ＧＳＨ含量下降,但ＲＴ组下降程度较ＰＤＰ－Ｒ     

Interstitial chemotherapy with ricin-loaded thermosensitive hydrogel in pancreatic cancer xenograft

BACKGROUND:Pancreatic cancer is one of the most aggressive malignancies,and has a poor prognosis. Despite efforts made in multiple fields,there has been little success in improving the disease-free survival rate of patients.This study was undertaken to investigate the effectiveness and feasibility of using intra-tumoral injection of ricin-loaded thermosensitive hydrogel for treatment of pancreatic cancer xenografts,attempting to develop a new treatment for human pancreatic cancer. METHODS:BALB/c-(nu/nu)nude...     

Interstitial chemotherapy using thermosensitive gel‐coated ricin in nude mice bearing a human hepatoma

Aim: To investigate the anti‐tumor effects and mechanisms of interstitial chemotherapy using intra‐tumor injection of thermosensitive gel‐coated ricin in nude mice bearing a human hepatoma. Methods: In a subcutaneous mouse model of hepatoma, saline, blank gel, ricin, or thermosensitive gel‐coated ricin (TGR) was injected directly into tumors. Fourteen days later, eight mice in each group were sacrificed. The tumors were removed and weighed for calculating tumor growth inhibition rate. Serum alpha‐fetoprotein levels, as well as hepatic and renal functions, were measured. Tumor tissue was analyzed under an optical microscope. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling was used to detect the apoptotic index. Moreover, caspase‐3 activity and protein expression in tumor tissue were examined. The survival time of the tumor bearing mice was determined. Results: Following interstitial chemotherapy by intra‐tumor injection of TGR in nude mice, serum alpha‐fetoprotein levels were significantly reduced with no significant impact on hepatic or renal functions. The rate of tumor growth inhibition was 58.5% following a single, local injection. Histological analysis revealed abundant necrosis. The apoptotic index was 45.96 ± 7.41%. Caspase‐3 activity was increased, and caspase‐3 protein was significantly activated in tumor cells. Compared to the saline group, the survival time of mice in the TGR group was significantly extended. At the observation terminal time, day 120, two mice were still alive and fully recovered. Conclusion: Interstitial chemotherapy by intra‐tumor injection of TGR was highly efficient and safe for the treatment of nude mice bearing a human hepatoma. Interstitial chemotherapy exhibits inhibitory effects by inducing apoptosis and directly killing tumor cells.