表面等离子共振技术在中医药研究中应用进展

利用表面等离子共振现象制备的生物传感技术具有无标记、灵敏度高、通量大、特异性强，样品耗损少等优点，在生命科学等众多学科中得到了广泛的关注和应用。近年来该技术也被运用在不少中医药研究的重要领域。本文简要介绍了表面等离子共振原理，总结并评述了在中医药新靶标发现、优效小分子寻找等方向的研究进展和前景。     

Design,display and immunogenicity of HIV1 gp120 fragment immunogens on virus-like particles

The broadly neutralizing antibody against HIV-1, b12, binds to the CD4 binding site (CD4bs) on the outer domain (OD) of the gp120 subunit of HIV-1 Env. We have previously reported the design of an E. coli expressed fragment of HIV-1 gp120, b122a, containing about 70% of the b12 epitope with the idea of focusing the immune response to this structure. Since the b122a structure was found to be only partially folded, as assessed by circular dichroism and protease resistance, we attempted to stabilize it by the introduction of additional disulfide bonds. One such mutant, b122a1-b showed increased stability and bound b12 with 30-fold greater affinity as compared to b122a. Various b122a and OD fragment proteins were displayed on the surface of Qβ virus-like particles. Sera raised against these particles in six-month long rabbit immunization studies could neutralize Tier1 viruses across different subtypes with the best results observed with b122a1-b displayed particles. Significantly higher amounts of antibodies directed towards the CD4bs were also elicited by particles displaying b122a1-b. This study highlights the ability of fragment immunogens to focus the antibody response to the conserved CD4bs of HIV-1.     

Target-mediated clearance and bio-distribution of a monoclonal antibody against the Kunitz–type protease inhibitor 2 domain of Tissue Factor Pathway Inhibitor

Introduction

A humanised monoclonal antibody, concizumab, that binds with high affinity to the Kunitz-type protease inhibitor (KPI) 2 domain of human tissue factor pathway inhibitor (TFPI) is in clinical development. It promotes coagulation by neutralising the inhibitory function of TFPI and may provide a subcutaneous prophylaxis option for patients with haemophilia. We aimed to study biodistribution and pharmacokinetics (PK) of concizumab.

Materials and Methods

Blockage of cellular TFPI by concizumab was measured by tissue factor/Factor VIIa-mediated Factor X activation on human EA.hy926 cells. Biodistribution of concizumab was analysed in rabbits by immunohistology, and the PK was measured in rabbits and rats.

Results and Conclusions

Concizumab bound to cell surface TFPI on EA.hy926 cells and neutralised TFPI inhibition of Factor X activation. The antibody cross-reacted with rabbit TFPI, but not with rat TFPI, allowing for comparative PK studies. PK data in rats described a log-linear profile typical for a non-binding antibody, whereas PK data in rabbits revealed a non-linear, dose-dependent profile, consistent with a target-mediated clearance mechanism. Immunohistology in rabbits during target-saturation showed localisation of the antibody on the endothelium of the microvasculature in several organs. We observed a marked co-localisation with endogenous rabbit TFPI, but a negligible sub-endothelial build-up. Concizumab binds and neutralises the inhibitory effect of cell surface-bound TFPI. The PK profile observed in rabbits is consistent with a TFPI-mediated drug disposition. Double immunofluorescence shows co-localisation of the antibody with TFPI on the endothelium of the microvasculature and points to this TFPI as a putative target involved in the clearance mechanism.     

A proteomic approach to investigate AuNPs effects in Balb/3T3 cells

Although gold nanoparticles (AuNPs) are currently used in several industrial products and biomedical applications, information about their biological effects is very limited. Thus, it is becoming crucial to assess their safety and adequately investigate the complexity of cell–nanoparticles interactions. In this work, the Balb/3T3 mouse fibroblast cell line was selected as an in vitro model to study the effects of AuNPs. Alteration of cellular processes and biochemical pathways caused by AuNPs exposure was investigated by analysing the differentially expressed proteome. Of interest was the difference observed in the protein pattern expression of cells exposed to AuNPs. It was found that 88 and 83 proteins were de-regulated after exposure to 5 and 15 nm AuNPs, respectively. Analysis of the proteome revealed that AuNPs triggers several pathways related to cellular growth and proliferation, cell morphology, cell cycle regulation, cellular function and maintenance, oxidative stress, and inflammatory response. Moreover, SPR analysis showed an increase of ECM proteins biosynthesis in cells exposed to AuNPs. We observed by TEM analysis that NPs are internalized and confined mainly in autophagosomes. Endoplasmic reticulum stressed and modification at mitochondrial level occurred. This study aims to improve existing knowledge necessary for a correct assessment of the balance between AuNPs potential adverse and beneficial effects and might have important implications for biomedical applications (e.g. nanomedicine).     

DNA Quadruplex–Based Inhibitor With Flexible Fragments at the 3′ Terminal Shows Enhanced Anti–HIV-1 Fusion Activity

Chemically optimizing the molecular structure of aptamers may enhance properties such as biological activity or metabolic stability. DNA quadruplex–based HIV-1 fusion inhibitors were found to interact with HIV-1 surface glycoprotein in aptamer mode. In this work, a series of quadruplex-based HIV-1 fusion inhibitors with flexible oligodeoxynucleotide fragments at the 3′ terminal was discovered. The flexible extension did not greatly influence quadruplex formation at the 5′-end. Increasing the length of the flexible fragment may increase antifusion activity. Compared with a traditional inhibitor, d(^5′TGGGAG^3′)₄, these novel inhibitors showed enhanced interaction with HIV-1 glycoproteins gp120 and gp41, which increased inhibition of 6-helical bundle formation during the course of virus fusion. These inhibitors also showed improved stability, compared with natural oligodeoxynucleotide. This work may inform the design of anti–HIV-1 DNA helix-based inhibitors with new structures or mechanisms.     

Immunogenicity and safety of the new intradermal influenza vaccine in adults and elderly: A randomized phase 1/2 clinical trial

BackgroundRecent clinical evidence indicates that an intradermal (ID) delivery of vaccines confers superior immunogenicity as compared to a standard intramusclular or subcutaneous (SC) delivery.MethodsIn this exploratory study, 600 healthy adults were randomized to 6 study groups with subgroups of young adults (20–64 years old) and older adults (65 years and older). The subjects were either injected by a novel ID injection system with a single dose of 6, 9, or 15 μg HA or two doses (21 days apart) of 15 μg HA per strain or injected by an SC injection method with a single or two doses (21 days apart) of 15 μg HA per strain. Immunogenicity was assessed using hemagglutination inhibition (HAI) titer and microneutralization titer on Days 0, 10, 21, and 42. Solicited and unsolicited adverse events were recorded for 7 and 21 days post-vaccination, respectively.ResultsIn both young adults and older adults groups, the geometric titer (GMT) ratios of HAI in the ID 15 μg HA group were higher than those in the SC 15 μg HA group on both Day 10 and Day 21, while those in the ID 6 and ID 9 μg HA groups were comparable with those in the SC 15 μg HA group. The kinetics of GMTs of HAI suggested that the ID vaccine has the potential to induce the prompt immune response, which is rather hampered in older adults as seen in the SC vaccine groups. The injection-site AEs were generally mild and transient, and did not occur in a dose or dosage-dependent manner.ConclusionsThe results of this study clearly suggest that the immunologic profile of the ID vaccine is better than that of the SC vaccine, while the safety profile of the ID vaccine is similar to that of the SC vaccine. In this exploratory study with almost 100 subjects per each group, single or two-dose administration of the ID vaccine containing 15 μg HA was suggested to be an appropriate regimen in order to prevent influenza and to reduce the associated disease burden.Trial registrationJAPIC Clinical Trials Information (JapicCTI-132096).     

Oversulfated chondroitin sulfate interaction with heparin-binding proteins: New insights into adverse reactions from contaminated heparins

An oversulfated chondroitin sulfate (OSCS) was identified as a contaminant to pharmaceutical heparin and severe anaphylactoid reactions were ascribed to this contaminant. An examination of the biochemistry underlying both the anticoagulant activity and the toxic effects of oversulfated chondroitin sulfate was undertaken. This study demonstrates that the anticoagulant activity of this oversulfated chondroitin sulfate is primarily dependent on heparin cofactor II mediated inhibition of thrombin. Heparin and oversulfated chondroitin sulfate binding to coagulation, kinin-kallikrein and complement proteins were studied by surface plasmon resonance. While oversulfated chondroitin sulfate binds tightly to antithrombin III, unlike heparin, OSCS does not induce antithrombin III to undergo the conformational change required for its inactivation of thrombin and factor Xa. In contrast to heparin, oversulfated chondroitin sulfate tightly binds factor XIIa suggesting a biochemical mechanism for the factor XIIa-based enhancement of vasoactive bradykinin production.     

脑瘫开槽式与椎板开窗式SPR术式优缺点及术后护理的比较

脑性瘫痪是出生前到出生后一个月内发育时期非进行性脑损伤所致的综合征，主要表现为中枢性运动障碍及姿势异常。由于不同的病因、类型、受累部位和程度，临床上表现差异较大，主要症状有：肌张力异常增高与痉挛；不随意运动和共济失调；智力低下及情绪障碍；其他可伴有视、听力功能障碍，吞咽困难，流涎，牙齿发育不良，斜视，部分伴有癫痫，少数还表现为肌张力低下等。本文通过两种手术方法优缺点及术后的护理比较，得出开窗式SPR术式不仅减轻了患儿术后痛苦，同时给术后患儿的精神安慰和功能煅炼带来积极影响。     

马尾近端SPR的临床应用解剖学

目的：为限制性椎板切除的ＳＰＲ提供解剖学依据。方法：在２０例成人防腐灌注红色乳胶的脊柱标本上，对近端马尾神经进行巨—微解剖学观察。结果：马尾近端，Ｌ１前后根之间有最下一对齿状韧带，前根位于相应后根的前内侧。各节段后根在马尾近端呈冠状位的内外平排，从内向外依次为Ｓ２～Ｌ１的后根；各节段后根进脊髓处，Ｌ５和Ｓ１后根之间有明显分界线，相邻节段后根之间，均有恒定小静脉分隔。以终丝起点为定点，各节段后根与终丝之间的距离和成角从Ｓ２到Ｌ１逐渐增加。结论：在限制性椎板切除的ＳＰＲ术中，前、后根的鉴别主要依靠脊髓的外侧索和Ｌ１前后根之间的最下一对齿状韧带；后根节段的鉴别主要依靠后根连脊髓处，Ｌ５和Ｓ１之间有明显分界、相邻节段后根间有恒定小静脉来定性鉴别。也可根据终丝起点水平，各节段后根与终丝之间的距离和成角来鉴别后根节段     

Summary of knowledge gaps related to quality and efficacy of current influenza vaccines

Influenza viruses are a public health threat, as they are pathogenic, highly transmissible and prone to genetic changes. For decades vaccination strategies have been based on trivalent inactivated vaccines, which are regulated by specific guidelines. The progress in scientific knowledge and the lessons learned from the A(H1N1)2009 pandemic have highlighted further the need to improve current guidelines, including the immunogenicity criteria set by the CHMP in 1997, and to promote the discussion on the shortcomings encountered, e.g. the evaluation of vaccine efficacy in the paediatric and elderly populations, the measurement of the naivety of a population, the impact of prior immunity on subsequent vaccinations, and the technical issues with the serological assays for detection of immunity and immunogenicity.