人源化抗人肺癌单域抗体基因的构建、表达及活性分析

目的： 构建人源化的抗人肺癌单域抗体hu3D3VH基因, 在大肠杆菌中表达, 对其蛋白活性进行分析.方法： 采用CDR移植技术对mAb3D3的重链可变区进行人源化, 通过重叠PCR获得hu3D3VH的基因.构建pET22（b＋）/hu3D3VH表达载体, 并转化大肠杆菌BL21（DE3）, 在IPTG诱导下表达.表达产物通过Ni亲和层析柱纯化.采用间接ELISA和竞争抑制ELISA法进行活性分析.结果： 通过重叠PCR获得序列正确的目的基因.目的蛋白以包涵体的形式表达, 表达量占菌体总蛋白的30%以上.纯化后, 目的蛋白的纯度达95%以上.hu3D3VH具有与亲本抗体相同的抗原反应性, 并能抑制mAb3D3与L342细胞的结合.结论： 获得的人源化单域抗体hu3D3VH, 保留了与mAb3D3相同的反应性和特异性, 为进一步临床应用奠定了基础.     

医院人性化服务设施的管理与实践

医院服务设施管理分为“硬”设施管理和“软”设施管理两类。医疗服务设施管理应保证疾病诊疗、满足方便患者就医、便于医患交流、诊疗设施舒适的医学伦理要求,应遵循流程合理、提高效率和效益、无障碍设计和布置安全的原则。我院在实施人性化服务设施管理中,配备了现代化的医疗设施,设置了细致入微的就医设施,应用了自动化的智能设施,提供了方便的生活和陪护设施,完善了弱势群体的“无障碍”设施。     

心理学视角下构建医患纠纷预警机制

目的：针对国内医院和医患冲突特点,制订实用的风险预测和管理工具,改变医院的被动应对,最大限度预防冲突发生、降低冲突后果。方法综合利用文献回顾、量表检测、生化检测、行为观察、专家咨询、心理咨询与训练等方法。结果动态监测医患人际互动过程,确立医患冲突的先兆风险因素,以医院内部信息集成系统为依托,完成多向度指标的融合和预警体系的构建,并逐级、逐类分别制定针对医护人员、患者及其家属的医院管理机构的干预方案。讨论三位一体的人本化动态管理模式包含医院和患者的平等化管理,契合医学新模式;医护人员心理素质训练和心理健康保健,体现人文关怀;积极有效的微观预警与干预体系,融入医院常规活动和流程。     

医院标识导向系统现状分析及优化措施探讨

医院作为特定的公共服务空间,其规范合理的布局,有助于为就诊者提供温馨、人性化的就诊氛围。标识导向系统是提升医院整体就诊环境最便捷、最直观、最具影响力的方法和手段之一。介绍了上海市某新建国际化医院标识导向系统设计、制作、安装的经验和实践,探讨了通过优化医院标识导向系统对进一步展现医院人性化服务理念、提升医院整体品牌形象建设的意义。     

全程陪同老年病人出入院护理体会

老年门诊为了实施以人为本的全程护理,解决老年病人办理入、出院手续的种种不便,对住院的老年病人采取由护士接送入、出院,陪同办理手续,受到了病人及其家属的好评。     

治疗性单克隆抗体研究的新趋势兔源单克隆抗体

治疗性抗体正在成为新药开发的新亮点,这些都得益于它可特异的识别靶位。众所周知,目前常用的单克隆抗体多为鼠源,在临床应用方面受到多种限制。与之相比,由于兔的免疫系统自身的特点,兔源单克隆抗体(RabMAbs)具有更高的亲和力和特异性,能识别更多的抗原表位。RabMAbs已在生命科学研究领域中广泛应用,其作为生物标志物已应用到临床诊断和新药研发之中。随着谱系引导的人源化改变(mutational lineage-guided humanization)理论提出和进一步深入研究,使得RabMAbs的人源化不但相对容易还可以对抗原互补决定区与框架区进行人源化。因此,有理由相信治疗性RabMAbs的开发和应用前景更为广阔。文中就RabMAbs的特点、优势、诊断和治疗性RabMAbs的发展状况做一综述。     

人性化护理的探讨

目的探讨人性化护理的临床应用。方法了解住院病人的心理需求及人性化护理模式下指导的相应护理措施，指出目前实施人性化护理过程中存在的一些问题及其对策。结果进行人性化护理可让病人树立起战胜疾病的信心，但要克服实施过程中的一些困难，需要合理利用护理人力资源，提高护士的整体素质。结论护理人员只有急病人之所急，想病人之所想，把自己置身于病人的角度，才能为病人提供真正的人性化护理服务。     

Augmented Binary Substitution: Single-pass CDR germ-lining and stabilization of therapeutic antibodies

Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.Monoclonal antibodies are a highly established technology in drug development and the majority of currently approved therapeutic antibodies are derived from immunized rodents (1). The advent of display libraries and engineered animals that can produce “fully human” antibody v-gene sequences has had a significant positive impact on antibody drug discovery success (1), but these technologies are mostly the domain of biopharmaceutical companies. Antibodies from wild-type animals that are already extant, or can be freely developed, will therefore continue to be a rich source of therapeutic candidates. In addition, phylogenetically distant hosts such as rabbits and chickens may become a valuable source of monoclonals with clinical potential against challenging targets (2, 3).Chimerization of murine antibodies can reduce anti-IgG responses in man (4), but murine v-domains may still have provocative T-cell epitope content, necessitating “humanization” of their framework regions (5, 6). Classical humanization “grafts” murine CDRs into human v-gene sequences (7), but this typically leads to significant reduction in affinity for target, so murine residues are introduced at key positions in the frameworks (a.k.a. “back-mutations”), to restore function (8). Importantly, humanized antibodies do elicit lower immunogenicity rates in patients in comparison with chimerics (9).Alternative humanization methods have also been developed based on rational design or empirical selection (10–17), but current methods still all suffer from flaws, such as: high non-germ-line amino acid content retention (5, 6); grafting into poorly understood frameworks (13); resource-intensive, iterative methods (15, 18); requirement for homology modeling of the v-domains, which is often inaccurate (19, 20), or a cocrystal structure with the target antigen (14). Methods that allow humanization into preferred frameworks can add numerous framework mutations (18, 21), which may destabilize the v-domains (22), encode new T-cell epitopes, or introduce random amino acid mutations in CDRs (12, 13) that can drive polyspecificity and/or poor PK properties (23).Critically, testing of protein therapeutics in monkeys has been shown to be nonpredictive of immune responses in man (24) and animal immunogenicity testing has been suggested to be of little value in biosimilar development (25). Current evidence suggests that the main risk factors for antibody immunogenicity in man are human T-cell epitope content and, to a lesser extent, T-cell independent B-cell responses (6). B-cell epitopes are challenging to predict and B-cell-only responses to biotherapeutics appear to be driven by protein aggregates (26). The key attributes to reduce antibody immunogenicity risk in the clinic appear to be: low T-cell epitope content, minimized non-germ-line amino acid content and low aggregation potential (27).In recent years, several reports have strongly suggested that CDRs might be malleable in ways that could not be predicted a priori. Random mutagenesis and reselection of a classically humanized rat antibody found that individual framework back mutations and CDR residues could revert to human germ-line sequence, while maintaining or even improving the function of the antibody (28). A number of humanization studies have now also shown that a small number of positions in the CDRs could be substituted for human germ-line residues, through a rational design cycle of reversion mutations (5, 29). In addition to these observations, a number of structural analyses have illustrated the common redundancy of sequence space in antibody binding interfaces. Despite typically large buried interfaces between antibodies and protein targets, only a subset of residues in the CDRs of antibodies usually makes contact with antigen (30–32). Alanine scanning of CDR loops has also shown that only a limited number of residues directly affect antigen binding affinity (33). Indeed, it has even been shown that redundant paratope space in a single antibody may be exploited to engineer binding specificity to two separate targets (34). Additionally, CDR loop structures are known to be restricted to a limited number of canonical classes, despite amino acid variation within those classes at specific positions (35–38). These observations led us to hypothesize that, under the right experimental conditions, a large proportion of residues in grafted animal CDRs could be concurrently replaced by the residues found at the corresponding positions in a given destination human germ-line v-gene.In this study, we generated combinatorial libraries on the basis of a design principle we have named “Augmented Binary Substitution” (ABS). Each library was based on a single starting antibody: rat anti-RAGE (28), rabbit anti-A33 (2), and chicken anti-pTau (3). These libraries were built into human germ-line frameworks of high predicted stability and solubility, then interrogated via phage display and screened to identify lead clones with epitope specificity and affinity equivalent to the parental clone. ABS proved to be a facile, rapid method that retains only the functionally required CDR content of the parental animal antibody, without the need for prior crystal-structure insight. Notably, this CDR germ-lining approach generated highly stable and soluble human IgGs, from multiple key antibody discovery species, that have minimized predicted human T-cell epitope content. The reproducibility of these findings across three antibodies from three disparate species demonstrates a fundamental plasticity in antibody paratopes that can be broadly exploited in therapeutic antibody optimization.     

以人为本的护理理念在医学美容手术中的应用探讨

目的探讨将以人为本护理理念应用于医学美容手术中的临床意义。方法选取医学美容手术患者220例,随机分为有可比性的两组。其中对照组9例,给予常规护理;观察组121例,给予以人为本为理念的护理。对两组患者护理服务及技术操作满意程度进行调查比较。结果观察组患者对护理服务满意情况明显优于对照组,观察组患者对技术操作满意率为87．60％略高于对照组85．85％,两组差别不明显,无统计学意义（P〉0．05）;观察组患者对技术操作非常满意者60．33％,明显高于对照组24．24％,两组比较差异明显,有统计学意义（P〈0．05）。结论将以人为本的护理理念应用于医学美容手术,可以在更多细节上给予就医者人文关怀,使得就医者在充满人性化的环境下完成整个手术。     

Animal models of autoimmune hepatitis

Animal models of autoimmune hepatitis have been important in defining pathogenic mechanisms, and they promise to aid in the evaluation of new molecular and cellular treatments. They have evolved from models based on crude liver homogenates that produced a transient hepatitis to models that express antibodies to human antigens, manifest liver-infiltrating T cells, persist for at least 3 months and develop fibrosis. Animal models allow the study of autoimmune hepatitis from its inception, and they can detail the progression of pathological events. Key imbalances in counter-regulatory mechanisms can be isolated and manipulated. Models can be humanized by the insertion of human genetic promoters and the expression of human antigens. Genetic engineering and preconditioning have been milestones in the evolution of animal models. Vaccination or infection of murine models with viral vectors carrying human antigens are the most recent developments. Animal models promise to extend the knowledge of etiological agents and improve treatment algorithms.