Enhanced lymph node retention of subcutaneously injected IgG1-PEG2000-liposomes through pentameric IgM antibody-mediated vesicular aggregation

An efficient strategy for enhancing the lymph node deposition of rapidly drained liposomes from the interstitial injection site is described. Subcutaneously injected small-sized immuno-poly(ethyleneglycol)-liposomes (immuno-PEG-liposomes), containing 10 mol% mPEG₃₅₀-phospholipid and 1 mol% PEG₂₀₀₀-phospholipid in their bilayer and where IgG1 is coupled to the distal end of PEG₂₀₀₀, not only drain rapidly from the interstitial spaces into the initial lymphatic system, but also accumulate efficiently among the lymph nodes draining the region when compared with non-PEG-bearing immunoliposomes where IgG is directly coupled to the phospholipid. Liposome deposition among the draining lymph nodes, however, was further enhanced dramatically following an adjacent subcutaneous injection of a pentameric IgM against the surface attached IgG molecules (IgM:IgG, 10:1) without compromising vesicle drainage from the interstitium. This is suggested to arise either as a result of formation of large immuno-aggregates within the lymphatic vessels with subsequent transport to and trapping among the regional lymph nodes and/or following IgM binding to Fc receptors of the lymph node sinus macrophages forming a platform for subsequent trapping of drained IgG-coupled liposomes. This lymph node targeting approach may be amenable for the design and surface engineering of any rapidly drained nanoparticulate system bearing peptides and proteins that can be aggregated with a desired monoclonal pentameric IgM.     

Developmental patterning deciphered in avian chimeras

I started my scientific carer by investigating the development of the digestive tract in the laboratory of a well-known embryologist, Etienne Wolff, then professor at the Collège de France. My animal model was the chick embryo. The investigations that I pursued on liver development together with serendipity, led me to devise a cell-marking technique based on the construction of chimeric embryos between two closely related species of birds, the Japanese quail ( Coturnix coturnix japonica ) and the chick ( Gallus gallus ).
The possibility to follow the migration and fate of the cells throughout development from early embryonic stages up to hatching and even after birth, was a breakthrough in developmental biology of higher vertebrates.
This article describes some of scientific achievements based on the use of this technique in my laboratory during the last 38 years.     

腹腔镜下行子宫切除术及淋巴清扫术治疗老年子宫颈癌的疗效

目的:探讨腹腔镜与开腹下行子宫切除术及淋巴清扫术治疗老年子宫颈癌患者的临床疗效。方法:选取2014年1月至2016年1月我院收治的60例老年子宫颈癌患者,随机分为两组,每组30例,A组患者接受开放性子宫切除术及淋巴清扫术,B组患者在腹腔镜下行子宫切除术及淋巴清扫术,比较两组患者的手术情况、术后恢复情况以及术中、术后并发症的发生情况和随访期间的生活质量。结果:B组患者手术中淋巴结的清扫数目明显比A组多(P0.05),术中出血量和术后使用镇痛泵的次数明显少于A组(P0.05),抗生素的使用时间、术后排气时间、膀胱功能恢复时间、引流管滞留时间、住院时间较A组患者明显缩短(P0.05),术中大出血以及术后尿潴留、淋巴囊肿的发生率显著低于A组(P0.05);术后3个月和6个月的I-QOL以及FACT-G评分显著高于A组(P0.05)。结论:腹腔镜下行子宫切除术加盆腔淋巴清扫术治疗老年子宫颈癌患者的临床疗效显著,有利于患者术后恢复,并有效提高患者术后生活质量。     

农杆菌介导的大豆高频遗传转化

大豆(Glycine max(L.)Merrill)遗传转化目前常用的两种方法为农杆菌介导的子叶节转化系统和基因枪介导的体细胞胚转化,但这两种转化系统都存在转化频率低、难于重复及依赖于特定的基因型等问题.为了提高农杆菌介导的大豆子叶节的转化频率,采用了一种基于bar基因作为筛选标记基因的固体-液体筛选系统,与农杆菌共培养3d的大豆子叶节在MS添加2 mg/L 6-BA和5 mg/L的glufosinate的筛选培养基培养2周后,再转到含有0.01 mg/L TDZ和2mg/L glufosinate的液体培养基中筛选,并每周更换一次培养液.得到的再生芽首先经GUS分析为阳性后再转入生根培养基得到完整转化植株,然后通过Southern杂交分析证实外源基因整合到大豆基因组,转化植物含有1～2个基因拷贝数.该转化系统具有转化频率高、转化周期短以及不依赖于大豆基因型等优点,对影响该转化系统的一些因子进行了讨论.     

A proteomic view of the facultatively chemolithoautotrophic lifestyle of Ralstonia eutropha H16

Ralstonia eutropha H16 is an H₂‐oxidizing, facultative chemolithoautotroph. Using 2‐DE in conjunction with peptide mass spectrometry we have cataloged the soluble proteins of this bacterium during growth on different substrates: (i) H₂ and CO₂, (ii) succinate and (iii) glycerol. The first and second conditions represent purely lithoautotrophic and purely organoheterotrophic nutrition, respectively. The third growth regime permits formation of the H₂‐oxidizing and CO₂‐fixing systems concomitant to utilization of an organic substrate, thus enabling mixotrophic growth. The latter type of nutrition is probably the relevant one with respect to the situation faced by the organism in its natural habitats, i.e. soil and mud. Aside from the hydrogenase and Calvin‐cycle enzymes, the protein inventories of the H₂‐CO₂‐ and succinate‐grown cells did not reveal major qualitative differences. The protein complement of the glycerol‐grown cells resembled that of the lithoautotrophic cells. Phosphoenolpyruvate (PEP) carboxykinase was present under all three growth conditions, whereas PEP carboxylase was not detectable, supporting earlier findings that PEP carboxykinase is alone responsible for the anaplerotic production of oxaloacetate from PEP. The elevated levels of oxidative stress proteins in the glycerol‐grown cells point to a significant challenge by ROS under these conditions. The results reported here are in agreement with earlier physiological and enzymological studies indicating that R. eutropha H16 has a heterotrophic core metabolism onto which the functions of lithoautotrophy have been grafted.     

Brevican distinctively assembles extracellular components at the large diameter nodes of Ranvier in the CNS

Brevican is known to be an abundant extracellular matrix component in the adult brain and a structural constituent of perineuronal nets. We herein show that brevican, tenascin-R (TN-R) and phosphacan are present at the nodes of Ranvier on myelinated axons with a particularly large diameter in the central nervous system. A brevican deficiency resulted in a reorganization of the nodal matrices, which was characterized by the shift of TN-R, and concomitantly phosphacan, from an axonal diameter-dependent association with nodes to an axonal diameter independent association. Supported by the co-immunoprecipitation results, these observations indicate that the presence of TN-R and phosphacan at nodes is normally brevican-dependent, while in the absence of brevican these molecules can also be recruited by versican V2. The versican V2 and Bral1 distribution was not affected, thus indicating a brevican-independent role of these two molecules for establishing hyaluronan-binding matrices at the nodes. Our results revealed that brevican plays a crucial role in determining the specialization of the hyaluronan-binding nodal matrix assemblies in large diameter nodes.     

Incipient forebrain boundaries traced by differential gene expression and fate mapping in the chick neural plate

We correlated available fate maps for the avian neural plate at stages HH4 and HH8 with the progress of local molecular specification, aiming to determine when the molecular specification maps of the primary longitudinal and transversal domains of the anterior forebrain agree with the fate mapped data. To this end, we examined selected gene expression patterns as they normally evolved in whole mounts and sections between HH4 and HH8 (or HH10/11 in some cases), performed novel fate-mapping experiments within the anterior forebrain at HH4 and examined the results at HH8, and correlated grafts with expression of selected gene markers. The data provided new details to the HH4 fate map, and disclosed some genes (e.g., Six3 and Ganf) whose expression domains initially are very extensive and subsequently retract rostralwards. Apart from anteroposterior dynamics, some genes soon became downregulated at the prospective forebrain floor plate, or allowed to identify an early roof plate domain (dorsoventral pattern). Peculiarities of the telencephalon (initial specification and differentiation of pallium versus subpallium) are contemplated. The basic anterior forebrain subdivisions seem to acquire correlated specification and fate mapping patterns around stage HH8.     

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g. size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.