SURAMIN ACTIVIY IN HUMAN HEAD AND NECK SQUAMOUS CELL CARCINOMA——Ⅱ.EFFECT OF SURAMIN ON HUMAN HEAD AND NECK CARCINOMA XENOGRAFTS AND RELATED TOXICITY AND PHARMACOKINETIC STUDIES IN NUDE MICE

In part Ⅰ of the study,we have demonstrated that suraminhas a marked antiproliferative effect on human head and neck squamous cellcarcinoma cell lines,In the present study,both toxicity and pharmacokineticstudies in nude mice were done.The treatment of nude mice bearing head andneck cancers with suramin showed that suramin had a minimal antitumor effect.The possible mechanism of suramin lacking antitumor effect is discussed.     

珊瑚人工骨复合游离骨膜瓣的肌内植入试验

珊瑚人工骨复合游离骨膜植入兔腓肠肌内，所有试样均有新生骨骼和血管组织生成，３周时新骨量占试样的１３．３±７．０，６周时为２２．３±７．０％，新骨于植入５天后开始发生，并持续６周以上。而单纯人工骨或骨膜肌内植入均无新骨产生。全部植入物未发现炎症和排斥反应。     

微囊化人嗜铬细胞对大鼠异种移植的免疫隔离作用

目的探讨微囊化人嗜铬细胞(ME-HCC)移植于大鼠眼前房或足胝部的免疫隔离作用。方法雌性SD大鼠48只,3月龄,体重180-250g。随机分为3组,HCC组、空微囊组、ME-HCC组,每组16只。HCC组、空微囊组、ME-HCC组分别将HCC、空微囊、ME-HCC移植于大鼠的眼前房(n=8)或足胝部(n=8)。移植术后7d大鼠断尾取血,测定白细胞介素-2(IL-2)、IgG和IgM浓度。移植术后28d 处死大鼠,光镜下观察右侧眼球或左侧足组织形态学变化。结果与HCC组比较,空微囊组和ME- HCC组大鼠血清IL-2、IgG、IgM浓度均降低(P<0.01);ME-HCC、空微囊组血清IL-2、IgG、IgM浓度比较差异无统计学意义(P>0.05)。HCC组大鼠眼前房和足胝部可见大量淋巴细胞和中性粒细胞浸润。空微囊组、ME-HCC组大鼠眼前房和足胝部仅见少量淋巴细胞和中性粒细胞。结论微囊化人HCC 对大鼠异种移植的免疫排斥具有隔离作用。     

Intranasal vaccination with influenza HA/GO-PEI nanoparticles provides immune protection against homo- and heterologous strains

Intranasal (i.n.) immunization is a promising vaccination route for infectious respiratory diseases such as influenza. Recombinant protein vaccines can overcome the safety concerns and long production phase of virus-based influenza vaccines. However, soluble protein vaccines are poorly immunogenic if administered by an i.n. route. Here, we report that polyethyleneimine-functionalized graphene oxide nanoparticles (GP nanoparticles) showed high antigen-loading capacities and superior immunoenhancing properties. Via a facile electrostatic adsorption approach, influenza hemagglutinin (HA) was incorporated into GP nanoparticles and maintained structural integrity and antigenicity. The resulting GP nanoparticles enhanced antigen internalization and promoted inflammatory cytokine production and JAWS II dendritic cell maturation. Compared with soluble HA, GP nanoparticle formulations induced significantly enhanced and cross-reactive immune responses at both systemic sites and mucosal surfaces in mice after i.n. immunization. In the absence of any additional adjuvant, the GP nanoparticle significantly boosted antigen-specific humoral and cellular immune responses, comparable to the acknowledged potent mucosal immunomodulator CpG. The robust immune responses conferred immune protection against challenges by homologous and heterologous viruses. Additionally, the solid self-adjuvant effect of GP nanoparticles may mask the role of CpG when coincorporated. In the absence of currently approved mucosal adjuvants, GP nanoparticles can be developed into potent i.n. influenza vaccines, providing broad protection. With versatility and flexibility, the GP nanoplatform can be easily adapted for constructing mucosal vaccines for different respiratory pathogens.

Influenza remains one of the leading infectious diseases causing morbidity and mortality worldwide. Vaccination is the most cost-effective approach to preventing influenza virus infection. However, current virus-based seasonal influenza vaccines induce strain-specific immunity and are less effective against mismatched strains that may cause influenza epidemics (1). Furthermore, there is no vaccine countermeasure available for new pandemic strains. Intranasal (i.n.) immunization is a promising vaccination route for infectious respiratory diseases, such as influenza. This vaccination route can induce both systemic and mucosal immune responses. Secretory immunoglobulin A (sIgA) and immunoglobulin G (IgG) may prevent influenza infection at the portal of virus entry. Influenza mucosal immunity has been reported to confer cross-protection against heterologous and heterosubtypic viruses (2). Moreover, needle-free i.n. influenza vaccines possess superior logistical advantages over traditional injectable vaccines, such as easy administration with high acceptance for recipients and avoidance of biohazardous sharps waste.However, the development of i.n. influenza vaccines has progressed slowly. The cold-adapted live-attenuated influenza virus (LAIV) vaccine is the only available human i.n. influenza vaccine up to date. Studies have shown that the LAIV vaccine could provide heterologous immunity (3). Nevertheless, safety concerns of LAIV are raised, especially in high-risk populations, such as infants under 2 y old and the elderly over 50. LAIV could undergo genetic reassortments and revert into a virulent form, thus posing a risk. Besides, the suboptimal protective efficacy of LAIV vaccines was reported in children in the 2009 H1N1 pandemic. A new generation of virus-independent, safe, and efficient influenza i.n. vaccines that induce broader cross-protection with high efficacy is urgently needed.Intranasal vaccination with recombinant protein/peptide-based vaccines is an attractive strategy with high safety (4). Purified protein/peptide antigens eliminate safety concerns in individuals with egg allergies, possess minimal side effects, and enable quick and cost-effective production. However, soluble protein vaccines are poorly immunogenic by i.n. immunization due to the harsh and tolerogenic mucosal environment (5). The selection of appropriate formulations and adjuvants is crucial for successful i.n. vaccines. Nanoparticle vaccine platforms have been applied for i.n. vaccine development in recent years (6, 7). Nanoparticles serve as antigen and adjuvant carriers and immunostimulants themselves to enhance immune responses. The immunoenhancing effects of various nanoparticles have been reported (8–13). However, most nanoparticle vaccines suffer from low antigen-loading capacity, complicated and lengthy preparation procedures, and structural complexity because of covalent conjugation.Two-dimensional (2D) graphene oxide (GO) nanoparticles have great potential as a novel vaccine platform due to their extraordinary attributes. These features include the high aspect ratio and ultra-large surface area for high-density antigen association, wealthy chemical groups for flexible surface modification and noncovalent antigen loading via electrostatic adsorption, hydrogen bond, and hydrophobic and π–π stacking interactions. Besides, GO nanoparticles themselves are biocompatible and nonimmunogenic (14, 15). Various GO vaccine formulations were demonstrated to induce improved immune responses by activating immune cells or triggering innate signaling (16–18). However, most prior studies were limited to conventional routes with tumor antigens for cancer immunotherapies. Studies on GO-based influenza i.n. vaccines are lacking.We developed a polyethyleneimine (PEI)-functionalized GO (GO-PEI, GP) influenza vaccine nanoplatform, prepared influenza GP nanoparticles by incorporating recombinant influenza hemagglutinin (HA) and investigated their immunoenhancing effects (Fig. 1). Our work revealed that influenza GP nanoparticles enhanced antigen internalization and promoted the production of inflammatory cytokines and the maturation of JAWS II dendritic cells (DCs) during in vitro experiments. i.n. vaccination with influenza GP nanoparticles induced robust humoral and cellular immune responses, conferring broader protection against challenges by homologous and heterologous influenza viruses in mice.Open in a separate windowFig. 1.Schematic illustration of the preparation and performance of influenza GP nanoparticles. (A) Preparation of GP and GP-HA/adjuvant nanoparticles. (B) Immunoenhancing effects of GP nanoparticle vaccines. GP nanoparticle vaccines showed enhanced cellular uptake in DCs and promoted inflammatory cytokine secretion and DC maturation. i.n. vaccination with influenza GP nanoparticles induced significantly enhanced and broad immune protection against homo- and heterologous influenza virus challenges. CTL, cytotoxic T lymphocyte; SR, survival rate.     

Terpene synthases are widely distributed in bacteria

Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes.Some 50,000 terpenoid metabolites, including monoterpenes, sesquiterpenes, and diterpenes representing nearly 400 distinct structural families, have been isolated from both terrestrial and marine plants, liverworts, and fungi. In contrast, only a relatively minor fraction of these widely occurring metabolites has been identified in prokaryotes. The first study of bacterial terpenes grew out of an investigation of the characteristic odor of freshly plowed soil reported in 1891 by Berthelot and André (1). Berthelot and André noted that a volatile substance apparently responsible for the typical earthy odor of soil could be extracted from soil by steam distillation. Their attempts to assign a structure to the isolated odor constituent failed;, however, when the neutral alcohol resisted oxidative degradation or other conventional chemical modification. The first modern studies of volatile bacterial terpenes were carried out some 75 years later by Gerber and Lechevalier (2) and Gerber (3–7), who speculated that the characteristic odor of cultures of Actinomycetales microorganisms, which are widely distributed in soil, might be caused by volatile terpenes. In addition to determining the structure of Berthelot’s geosmin, shown to be a C₁₂ degraded sesquiterpene alcohol (and giving it its name, which means earth odor) (2, 3), Gerber (4) also isolated and determined the structures of the methylated monoterpene 2-methylisoborneol as well as several other cyclic sesquiterpenes produced by streptomycetes (5–7). In subsequent years, numerous volatile terpenes have been detected in streptomycetes (8–16). The three most commonly detected streptomycetes terpenoids, geosmin, and 2-methylisoborneol and the tricyclic α,β-unsaturated ketone albaflavenone (Fig. 1) are well-known as volatile odoriferous microbial metabolites. The two terpene alcohols are, in fact, the most frequently found secondary metabolites in actinomycetes (8, 11, 17), filamentous Cyanobacteria (18–20), and Myxobacteria (21), and they are also produced by a small number of fungi (22–24). The production of 2-methylisoborneol is associated with a characteristic scent, whereas albaflavenone, which was first isolated from cultures of a highly odoriferous Streptomyces albidoflavus species, is best described as earthy and camphor-like (25).Open in a separate windowFig. 1.The structures of the major known terpenes produced by bacteria.Cyclic monoterpene, sesquiterpene, and diterpene hydrocarbons and alcohols are formed by variations of a universal cyclization mechanism that is initiated by enzyme-catalyzed ionization of the universal acyclic precursors geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) to form the corresponding allylic cations. These parental branched, linear isoprenoid precursors are themselves synthesized by mechanistically related electrophilic condensations of the 5-carbon building blocks dimethylallyl diphosphate and isopentenyl diphosphate. The several thousand known or suspected terpene synthases from plants and fungi have a strongly conserved level of overall amino acid sequence similarity, thus making possible the application of local alignment methods, such as the widely used BLAST algorithm, for the discovery of genes encoding presumptive terpene synthases from plant and fungal sources. Despite the relatively high level of overall sequence conservation, however, assignment of the actual biosynthetic cyclization product of each fungal or plant terpene synthase has remained beyond the reach of available bioinformatic methods. The discovery and biochemical characterization of bacterial terpene synthases represent an even greater challenge, because unlike the plant and fungal enzymes, bacterial terpene synthases not only exhibit no significant overall amino acid sequence similarity to those from plants and fungi but typically display relatively low levels of mutual sequence similarity. To address this challenge, we recently described the successful application of an alternative genome mining strategy for the discovery of previously unidentified bacterial terpene synthases based on the use of hidden Markov models (HMMs) and protein families database (Pfam) searching methods (26). These initial efforts identified a large number of previously unrecognized bacterial terpene synthase candidates, including the discovery of the previously unidentified synthase for the methylated monoterpene 2-methylisoborneol, and led to the heterologous expression of the relevant genes that produce 2-methylisoborneol and 2-methylenebornane from 2-methylgeranyl diphosphate (27). We subsequently refined and expanded the set of HMM parameters using as a reference set exclusively the group of newly predicted bacterial terpene synthases in distinction to the original HMM model (PF03936), which had been based on plant terpene synthases. Using these newly refined parameters, we then succeeded in identifying a previously unrecognized ortholog of 2-methylisoborneol synthase in the cyanobacterium Pseudanabaena limnetica str. Castaic Lake (28). Application of this second generation HMM model allowed, in total, the discovery of 140 predicted terpene synthases of bacterial origin.We now report the development of a third generation HMM model trained by the previously identified 140 bacterial terpene synthases that has expanded the number of predicted bacterial terpene synthases to 262 from within the most complete set of predicted proteins incorporated in the most recent collection of public databases and in-house draft genome sequences of streptomycete microorganisms. Among the newly identified gene sequences, a subset selected by phylogenetic analysis has been expressed in a specially engineered heterologous Streptomyces host, and the resultant terpenes have been identified and structurally characterized.     

异种角膜移植抗排斥的研究进展

异种角膜移植术由于角膜供体来源广、取材易,可以解决多种角膜疾病,如角膜严重瘢痕变性、圆锥变形等,但术后强烈的免疫排斥反应严重阻碍此项技术的发展,现就其免疫排斥反应的机制及相应预防措施做一综述如下。     

心得安对糖尿病大鼠节段胰腺移植作用的初步观察

运用显微外科技术给29只糖尿病大鼠行节段胰腺移植术,20只有(?)7天以上,有效地控制了糖尿病,成功率为69%。实验对胰腺移植大鼠作了小剂量心得安治疗(每日1mg/kg(?)重)的观察,结果显示,移植胰存活时间心得安治疗组比对照组平均延长1.8天(P<0.05),说明心得安有延长大鼠移植胰存活的作用.     

异种肾移植超急性排斥反应时血液流变学的变化

采用猪-犬肾移植建立超急性排斥反应模型，以研究此时的血液流变学变化。分别在移植前、移植肾超急性排斥发生时和排斥肾切除后10～15分钟检测外周血的血液流变学参数变化。结果异种肾移植发生超急性排斥时，血小板聚集显著增高，移植肾切除后血中的血小板数和纤维蛋白原明显下降，血小板聚集也恢复原有水平。提示血小板、纤维蛋白原参与了异种移植超急性排斥反应的发生过程，而血小板聚集可能是诊断异种移植超急性排斥反应一个有价值的指标。     

玻璃体内注射他克莫司对异种移植的视网膜色素上皮细胞存活的影响

目的 观察少量、多次玻璃体内注射免疫抑制剂他克莫司（FK506）对移植到兔眼视网膜下间隙内的人视网膜色素上皮（retinal pigment epithelium, RPE）细胞存活情况的影响。 方法 采用绿色荧光蛋白（green fluorescent protein, GFP）逆转录病毒感 染人RPE细胞。将50 μl（4×10³个/μl）表达GFP的人RPE细胞悬液注射到18只白兔及1 0只灰兔双眼视网膜下间隙，手术后所有兔左眼玻璃体内注射5 μl FK506（5 μg/ μl），每周1次，连续5周，然后间周一次至20周;右眼不注射作为对照。眼球壁铺片倒置荧光 显微镜观察移植细胞的存活状态。 结果 白兔于移植后1、2、3、4 、6、10、11、14、18、20、23、24、25、33、54周，灰兔于移植后4、5、6、7、14、18、20、26周双眼视网膜下均可见表达的GFP细胞，但移植术后1~14周内玻璃体内注射了FK506的左眼视网膜下RPE-GFP细胞形态、细胞膜的完整程度均比未注射的右眼好。18周后，7只白兔和3只灰兔双眼视网膜下移植细胞的状态差异不明显。眼球切片苏木素-伊红（hematoxylin-eosin, HE）染色观察结果显示，移植术后1~6周，6只白兔和3只灰兔右眼脉络膜小血管周围可见灶性或弥散的淋巴细胞，应用了FK506的左眼淋巴细胞的浸润明显减少。 结论 移植术后早期玻璃体内使用小剂量免疫抑制剂可减轻局部炎症反应，有利于维持视网膜下间隙移植的异种细胞的存活。 （中华眼底病杂志，2003，19：333-404）     

大鼠胚胎小脑组织移植的研究 Ⅰ.大鼠胚胎小脑组织移植的形态学观察

取孕17～20天的Wistar大鼠胚胎小脑组织植入同种大鼠小脑内。移植后10天,移植块与宿主脑组织融为一体,两者之间无隔膜,也无疤痕组织;移植物明显变大,充满预制脑腔,其外颗粒层细胞分裂相多,普肯野细胞结构特征出现,分子层、普肯野细胞层及内颗粒层依次排列,小脑皮质形成。移植后20天,普肯野细胞明显排成一层,尼氏体出现。移植后30天,外颗粒层消失,小脑皮质成熟。实验证明,同种胚胎脑组织移植,移植块可成活并按其固有模式生长成熟。