Adoptive transfer of T<Subscript>reg</Subscript> depleted autologous T cells in advanced renal cell carcinoma

Purpose

CD4⁺CD25⁺ regulatory T (T_reg) cells are present in increased numbers in patients with advanced cancer and CD25⁺ T cell depletion potentiates tumour immunity in animal models. The aim of this study was to assess the feasibility and safety of adoptive transfer of CD25⁺ depleted autologous T cells in patients with advanced renal cell carcinoma and to examine resulting changes in lymphocyte subsets.

Patients and methods

Six patients with advanced renal cell carcinoma underwent leukapheresis followed by conditioning chemotherapy with cyclophosphamide and fludarabine. The autologous leukapheresis product was depleted of CD25⁺ cells using CliniMACS^® System then re-infused into the patient.

Results

Efficient CD25⁺ depletion from all leukapheresis products was achieved and 0.55–5.87 × 10⁷/kg CD3⁺ cells were re-infused. Chemotherapy related haematological toxicity was observed, but blood counts recovered in all patients allowing discharge after a mean inpatient stay of 21 days. One patient subsequently developed a rapidly progressive neurological syndrome. A transient reduction in CD25⁺ subset was noted in the peripheral blood of 5 out of 6 patients with evidence of increased T cell responses to PHA in 4 out of 6 patients. One patient showed increased specific proliferative responses to the tumour associated antigen h5T4 coinciding with the nadir of T_reg cells.

Conclusions

Given the transient nature of the reduction in CD25⁺ subset and the observed toxicity there is a need to explore further strategies to improve the safety and efficacy of this approach. Nevertheless, the results provide proof of concept in potentiation of tumour antigen T cell responses when T_reg cell levels are depleted.

     

Chemo-Cryo Combination Therapy: An Adjunctive Model for the Treatment of Prostate Cancer

Despite continuing research and the development of alternate therapeutic options, prostate cancer remains problematic. Chemotherapy has played a minor role as a treatment option due to its lack of efficacy. Whereas cryotherapy has received renewed attention as a treatment modality, it too fails to offer an absolute curative option. Previously, we reported on the utilization of a therapeutic model, which, in combination, increases cell death in a canine renal cell model. Based upon that study, we investigated a combination therapy model as an alternative for the treatment modality for prostate cancer. We hypothesized that the combination of chemotherapy and cryosurgery would result in enhanced cell death, thereby presenting a more effective treatment of prostate cancer. A human prostate cancer cell (PC-3) model was exposed to 5-fluorouracil (5-FU) for 2 and 4 days (prefreeze), freezing (-5 to -100 degrees C), or a combination of the two treatments, and each was assessed for effectiveness over a 2-week posttreatment period. Additionally, investigation into the mechanisms of cell death initiated by the respective therapies was performed through DNA cleavage analysis. For chemotherapy, cultures exposed to 5-FU (2-4 days) yielded a 15-25% loss in cell survival. For cryotherapy, cultures exposed to a temperature window of -5 to -20 degrees C yielded an initial 5-70% loss of viability but cells propagated over time. Cultures exposed to temperatures of -25 to -80 degrees C yielded a 90-99% (+/-4.5%) initial loss in viability with repopulation observed by 12 days postthaw. Cells frozen to -100 degrees C yielded 100% (+/-0.3%) loss of viability and exhibited no signs of propagation. For chemo-cryo therapy, combination treatment at milder temperatures (-5 to -25 degrees C) resulted in an enhanced loss of cell viability compared to that for either treatment alone. Combination treatment at lower temperatures (-40 to -80 degrees C) resulted in a complete loss of cell viability. DNA fragmentation analysis at 48 h posttreatment revealed that dead (detached) cells treated with 5-FU died primarily through apoptosis, whereas dead cells from freezing (-15 degrees C) alone died primarily through freeze-rupture and necrosis. Detached cell analysis from combination treatment at -15 degrees C revealed the presence of apoptotic, necrotic, and freeze-rupture cell death. Scanning electron micrographs of cells exposed to freezing contributing to cell death. These data demonstrate that the combination of 5-FU at sublethal doses and freezing temperatures improves human prostate cancer cell death efficacy. Further, we suggest that chemo-cryo therapy offers a potential alternative treatment for the control and eradication of prostate cancer.     

聚谷氨酸在医药领域的应用

聚谷氨酸是一种天然的氨基酸聚合物,具有强的水溶性、生物相容性、生物可降解性、无毒等特性,是一种新型绿色环保的生物材料。聚谷氨酸作为药物载体、疫苗佐剂、医用粘合剂和组织工程材料等应用于医药领域时,可表现出更好的生物相容性、更低的生物毒性,可提高药物的靶向性,可有效提高药效,改善药物或材料的性能,因此具有十分广阔的应用前景。本文综述了聚谷氨酸在医药领域的的应用,为下一步的研究和商业化的应用提供参考。     

六种阳离子助剂对阿维菌素的增效作用

采用浸虫法和叶片药膜法比较了6种阳离子助剂与阿维菌素混用对小菜蛾Plutella xylostella 3龄幼虫的增效作用,并通过测定助剂对阿维菌素药液物理性状的影响以及助剂对小菜蛾体内超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalases,CAT)和过氧化物酶(peroxidases,POD) 3种保护酶活性的影响等方面对其增效机制进行了初步分析,为阳离子助剂在杀虫剂领域的开发提供一定的依据。结果表明： 在400 mg/L以下,各阳离子助剂单独使用对小菜蛾3龄幼虫均无生物活性,但可明显提高阿维菌素对小菜蛾的药效。两种方法中,浸虫法的增效作用大于叶片药膜法,其中松香酸铜、1427和412103对阿维菌素的增效倍数分别为3.71、2.82和2.72。6种助剂均可明显降低阿维菌素药液的表面张力和接触角及增加药液在甘蓝叶片上的沉积量,但不同助剂间及相同助剂不同浓度间差异虽大多显著; 与增效比结果综合分析表明,助剂的润湿性能与其增效作用之间关系不大。阿维菌素药液分别加入松香酸铜、1427和412103后使试虫体内POD和SOD的活性明显提高,从侧面说明这3种助剂对虫体更具渗透性。     

Terrestrial Plant-Derived Anticancer Agents and Plant Species Used in Anticancer Research

Cancer is a major cause of death and the number of new cases, as well as the number of individuals living with cancer, is expanding continuously. Due to the enormous propensity of plants that synthesize mixtures of structurally diverse bioactive compounds, the plant kingdom is potentially a very diverse source of chemical constituents with tumor cytotoxic activity. Despite the successful utilization of few phytochemicals, such as vincristine and taxol, into mainstream cancer chemotherapy, commercial plant-derived anticancer formulations represent only one-fourth of the total repertoire of the available treatment options. Though significant progress has been made towards the characterization of isolated compounds and their structure-related activities, the complex composition of plant extracts, along with the lack of reproducibility of activity and the synergy between different, even unidentified, components of an extract, prohibits the full utilization of plants in pharmaceutical research. In this review, the results of an extensive literature survey on the anticancer properties of terrestrial plants, covering a thirty-five-year-long span (1970–2005) are presented. A total of 187 plant species, belonging to 102 genera and 61 families have been identified as an active or promising source of phytochemicals with antitumor properties, corresponding to a 41 percent increase during the last five years. Among them, only 15 species (belonging to ten genera and nine families) have been utilized in cancer chemotherapy at a clinical level, whereas the rest of the identified species are either active against cancer cell lines or exhibit chemotherapeutic properties on tumor-bearing animals under experimental conditions. Phenylpropanoids are the most widely distributed compounds (18 families), followed by terpenoids (14 families), and alkaloids (13 families). Analytical, species-specific information on bioactive constituents and target cancers is provided. The outlook of phytochemistry-based cancer therapy is discussed, particularly in the perspective of identifying immunomodulatory anticancer agents with minimal toxicity on healthy tissues.

     

Expression-System-Dependent Modulation of HIV-1 Envelope Glycoprotein Antigenicity and Immunogenicity

Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly related Env surface (gp120) antigens produced in different systems: (a) mammalian (293 FreeStyle™ cells; 293F) cells in the presence of kifunensine, which impart only high-mannose glycans; (b) insect cells (Spodoptera frugiperda, Sf9), which confer mainly paucimannosidic glycans; (c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic™), which impart high-mannose, hybrid and complex glycans without sialic acid; and (d) 293F cells, which impart high-mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9-derived and wild-type mammalian-cell-derived material that is predicted to affect ligand binding sites proximal to glycans. Modeling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4 binding and CD4-induced sites compared to those expressed in the system that do, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic-acid-containing gp120 was significantly more immunogenic than the sialylated version when administered in two different adjuvants, and induced higher titers of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic-acid-imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations that should be considered when selecting expression systems for glycosylated antigens to be used for structure-function studies and for vaccine use.     

胸腺素α原作为乙型肝炎表面抗原佐剂的研究

目的：研究胸腺素α原（ProTα）作为佐剂对重组乙型肝炎表面抗原（HBsAg）诱导小鼠产生乙型肝炎表面抗体（抗-HBs）的影响。方法：以纯系BALB/c小鼠为免疫对象,分组情况为：①HBsAg组（1μgHBsAg）;②0.1μgProTα＋1μgHBsAg组;③0.5μgProTα＋1μgHBsAg组;④1μgProTα＋1μgHBsAg组;⑤铝佐剂组（1μgVacon疫苗）;⑥1μgPro-Tα＋1μgVacon疫苗组;每组10只小鼠,分别于0、2周各肌肉注射免疫1次。采用ELISA法检测血清抗-HBs滴度（即IgG亚类IgG1和IgG2a）。结果：与单独注射HBsAg组相比,1μgProTα＋1μgHBsAg组抗-HBs总抗体滴度明显增高（P〈0.05）,抗体持续时间更长,且ProTα可以平衡Th1和Th2免疫反应;2组间IgG1/IgG2a差异显著（P〈0.05）。与铝佐剂相比,ProTα增强了小鼠对HBsAg的反应性,提高抗-HBs阳转率。结论：ProTα在增强小鼠抗-HBs产生的同时,提高细胞免疫反应,提示ProTα是一种很有潜力的HBsAg佐剂。     

编码山羊补体C3d与口蹄疫病毒VP1融合蛋白重组质粒的构建及表达

旨在构建含分子佐剂山羊补体C3d基因的O型口蹄疫病毒VP1基因真核表达质粒。克隆山羊C3d基因, 通过linker(G4S)2将3拷贝C3d基因串联; 克隆羊源O型口蹄疫病毒VP1基因, 通过linker(G4S)2与3拷贝C3d基因相连, 构建重组质粒pUC19-VP1-C3d3。将VP1-C3d3融合基因亚克隆入含有分泌表达信号肽tPA序列的pcDNA3.1(+)CMV启动子下游, 构建重组真核表达质粒pcDNA3.1-tPA-VP1-C3d3。在脂质体介导下, 将pcDNA3.1-tPA -VP1-C3d3转染HeLa细胞。间接免疫荧光分析表明, VP1- C3d3在HeLa细胞中获得了瞬时表达, Western blot分析证实转染的阳性细胞能分泌预期大小(133 kD)的融合蛋白。重组质粒pcDNA3.1-tPA-VP1-C3d3为研制以羊补体C3d为分子佐剂的口蹄疫新型疫苗奠定了基础。     

mTOR regulates TLR-induced c-fos and Th1 responses to HBV and HCV vaccines

Although IL-12 plays a critical role in priming Th1 and cytotoxic T lymphocyte(CTL) responses, Toll-like receptor(TLR) signaling only induces low amounts of IL-12 in dendritic cells and macrophages, implying the existence of stringent regulatory mechanisms. In this study, we sought to uncover the mechanisms underlying TLR-induced IL-12 expression and the Th1 response. By systemic screening, we identified a number of protein kinases involved in the regulation of TLRinduced IL-12 expression. In particular, PI3 K, ERK, and m TOR play critical roles in the TLR-induced Th1 response by regulating IL-12 and IL-10 production in innate immune cells. Moreover, we identified c-fos as a key molecule that mediates m TOR-regulated IL-12 and IL-10 expression in TLR signaling. Mechanistically, m TOR plays a crucial role in c-fos expression, thereby modulating NFκB binding to promoters of IL-12 and IL-10. By controlling the expression of a special innate gene program, m TOR can specifically regulate the TLR-induced T cell response in vivo. Furthermore, blockade of m TOR by rapamycin efficiently boosted TLR-induced antigen-specific T and B cell responses to HBV and HCV vaccines. Taken together, these results reveal a novel mechanism through which m TOR regulates TLR-induced IL-12 and IL-10 production, contributing new insights for strategies to improve vaccine efficacy.     

Quantitative aspects of the selective killing of transformed cells by methotrexate in the presence of leucovorin

Summary A quantitative study was made of the cytotoxicity of methotrexate (MTX) for nontransformed and transformed NIH 3T3 cells in the presence and absence of leucovorin. The study was preceded by an analysis of the growth rates of the cells at low and high population density combined with low and high concentrations of calf serum (CS). The reduced maximal growth rates of the transformed cells at low population densities relative to the nontransformed cells reinforced earlier evidence that heritable damage involving chromosome aberrations drives the process of transformation. When small numbers of transformed cells are cocultured with a large excess of nontransformed cells in the assay for transformed foci, the transformed cells were more readily killed by MTX than the nontransformed cells. The selectivity was increased when leucovorin (folinic acid) was present in the medium. The selective killing of the transformed cells actively multiplying in foci was most pronounced when the background of nontransformed cells had become confluent and their growth was inhibited. However, selectivity has also been demonstrated when transformed and nontransformed cells are growing at their maximum rates at low density despite the lower growth rate of the transformed cells under these conditions. The sensitivity of transformed cells in pure culture to MTX was lower during the first 3 d of subculture than in the following 6 d but decreased to zero a few d after net growth had ceased. The nontransformed cells were more susceptible to killing by MTX in Dulbecco’s modified Eagle’s medium (DMEM) than in MCDB 402, but the transformed cells were sensitive to MTX in both media. The high selectivity of MTX for transformed over nontransformed cells in MCDB 402 results from the presence of 1.0 μM leucovorin (5-formyltetrahydrofolate), a reduced form of the folic acid present in most other culture media. When leucovorin was added to DMEM with its high concentration of folic acid, the resistance to MTX of both nontransformed and transformed cells was greatly increased, but the selectivity of MTX for transformed cells was almost entirely lost. The results indicate that leucovorin protects nontransformed cells against concentrations of MTX that kill transformed cells, but the protection is dependent on the relative amounts of leucovorin to folic acid in the medium. The relative sensitivities of transformed and nontransformed cells in our system to MTX when both cell types are exhibiting their characteristic differential in growth behavior is similar to that described for tumor and normal cells in vivo. Since the unregulated growth behavior of the transformed, tumor-producing cells is efficiently and quantitatively measured in this system, it can be used to develop general principles of treatment and resolve questions of cytotoxic mechanism.