Alternative Immunomodulatory Strategies for Xenotransplantation: CD80/CD86-CTLA4 Pathway-Modified Immature Dendritic Cells Promote Xenograft Survival

Background

Xenotransplantation is a promising approach to circumventing the current organ shortage. However, T-cell-dependent anti-xenoresponses are a major challenge to successful xenografts. Given the advantages of the use of CTLA4-Ig in the survival of allografts, the purpose of the study was to investigate the therapeutic potential of CTLA4-IgG4 modified immature dendritic cells (imDCs) in the prevention of islets xenograft rejection.

Methods

CTLA4-IgG4 was constructed by the fusion of the extracellular regions of porcine CTLA4 to human the hIgG4 Fc region. The imDCs were induced and cultured from porcine peripheral blood mononuclear cells (PBMC). The CTLA4-IgG4 modified imDCs were delivered via the portal vein to the liver of diabetic mice (insulin-dependent diabetes mellitus) before islet xenografting, and mCTLA4-Ig was administered intravenously after xenotransplantation.

Results

The xenograft survival of mice receiving unmodified imDCs was approximately 30 days. However, following administration of CTLA4-IgG4 modified imDCs before grafting and mCTLA4-Ig after grafting, xenografts survived for more than 100 days. Flow cytometric analysis showed that the CD4⁺CD25⁺Foxp3⁺ Treg population was increased in spleens. The efficacy of donor CTLA4-IgG4 modified imDCs correlated partially with the amplification of Tregs.

Conclusions

These results confirm that selective inhibition of the direct and indirect pathways of T-cell activation by donor CTLA4-IgG4 modified imDCs and receptor CTLA4-Ig is a highly effective strategy to promote survival of xenografts.     

Atomic Model of Rabbit Hemorrhagic Disease Virus by Cryo-Electron Microscopy and Crystallography

Rabbit hemorrhagic disease, first described in China in 1984, causes hemorrhagic necrosis of the liver. Its etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the family Caliciviridae. The detailed molecular structure of any lagovirus capsid has yet to be determined. Here, we report a cryo-electron microscopic (cryoEM) reconstruction of wild-type RHDV at 6.5 Å resolution and the crystal structures of the shell (S) and protruding (P) domains of its major capsid protein, VP60, each at 2.0 Å resolution. From these data we built a complete atomic model of the RHDV capsid. VP60 has a conserved S domain and a specific P2 sub-domain that differs from those found in other caliciviruses. As seen in the shell portion of the RHDV cryoEM map, which was resolved to ∼5.5 Å, the N-terminal arm domain of VP60 folds back onto its cognate S domain. Sequence alignments of VP60 from six groups of RHDV isolates revealed seven regions of high variation that could be mapped onto the surface of the P2 sub-domain and suggested three putative pockets might be responsible for binding to histo-blood group antigens. A flexible loop in one of these regions was shown to interact with rabbit tissue cells and contains an important epitope for anti-RHDV antibody production. Our study provides a reliable, pseudo-atomic model of a Lagovirus and suggests a new candidate for an efficient vaccine that can be used to protect rabbits from RHDV infection.     

Role of Tomato Lipoxygenase D in Wound-Induced Jasmonate Biosynthesis and Plant Immunity to Insect Herbivores

In response to insect attack and mechanical wounding, plants activate the expression of genes involved in various defense-related processes. A fascinating feature of these inducible defenses is their occurrence both locally at the wounding site and systemically in undamaged leaves throughout the plant. Wound-inducible proteinase inhibitors (PIs) in tomato (Solanum lycopersicum) provide an attractive model to understand the signal transduction events leading from localized injury to the systemic expression of defense-related genes. Among the identified intercellular molecules in regulating systemic wound response of tomato are the peptide signal systemin and the oxylipin signal jasmonic acid (JA). The systemin/JA signaling pathway provides a unique opportunity to investigate, in a single experimental system, the mechanism by which peptide and oxylipin signals interact to coordinate plant systemic immunity. Here we describe the characterization of the tomato suppressor of prosystemin-mediated responses8 (spr8) mutant, which was isolated as a suppressor of (pro)systemin-mediated signaling. spr8 plants exhibit a series of JA-dependent immune deficiencies, including the inability to express wound-responsive genes, abnormal development of glandular trichomes, and severely compromised resistance to cotton bollworm (Helicoverpa armigera) and Botrytis cinerea. Map-based cloning studies demonstrate that the spr8 mutant phenotype results from a point mutation in the catalytic domain of TomLoxD, a chloroplast-localized lipoxygenase involved in JA biosynthesis. We present evidence that overexpression of TomLoxD leads to elevated wound-induced JA biosynthesis, increased expression of wound-responsive genes and, therefore, enhanced resistance to insect herbivory attack and necrotrophic pathogen infection. These results indicate that TomLoxD is involved in wound-induced JA biosynthesis and highlight the application potential of this gene for crop protection against insects and pathogens.     

大豆转录因子GmERF5的克隆、表达及功能分析

ERF转录因子是植物中特有的转录因子家族之一, 在植物响应生物和非生物胁迫过程中发挥重要的调控作用。通过对大豆(Glycine max)吉林32未成熟胚的表达谱分析, 利用RT-PCR技术从大豆中克隆了1个新的ERF转录因子GmERF5。GmERF5具有237个氨基酸残基, 分子量为26.09 kDa, 等电点为6.85, 其开放阅读框长714 bp。该转录因子蛋白与Gh-ERF2蛋白的同源性最高, 它们同属ERF亚家族的第IV亚类。实时荧光定量PCR分析表明, 该蛋白基因在大豆的根中表达量最高, 且受干旱、高盐、低温及乙烯、脱落酸和茉莉酸甲酯的诱导上调表达。亚细胞定位实验结果表明, GmERF5蛋白定位于细胞核中。转录激活能力分析结果显示, GmERF5可以激活报告基因的表达, 为转录激活子。综合以上结果, 认为GmERF5可能作为转录调控因子参与大豆生物和非生物胁迫的应答。     

Hela细胞HGPRT缺陷型细胞系构建

建立稳定的次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)缺陷的Hela细胞系,为细胞融合相关研究和人源化单克隆抗体制备提供有利于筛选的亲本细胞。通过诱变剂N-甲基-N′-硝基-N-亚硝基胍(MNNG)对Hela细胞进行诱变,逐步提高培养基中6-巯基鸟嘌呤(6-TG)的浓度,筛选出对6-TG稳定耐受的细胞,在次黄嘌呤-氨基喋呤-胸腺嘧啶(hypoxanthine-aminopterin-thymidine,HAT)培养基中鉴定其敏感性,最后对筛选得到的Hela-HGPRT-进行生物学鉴定。在此基础上,将Hela-HGPRT-细胞系与人淋巴细胞融合,在HAT培养基中筛选杂交细胞。筛选得到了能够长期在含20μg/mL 6-TG培养基中生长的Hela-HGPRT-细胞,并且在HAT培养基中不能存活。Hela-HGPRT-细胞与人淋巴细胞成功融合,获得能够连续传代培养的杂交瘤细胞。经MNNG诱导和6-TG筛选,得到了稳定传代的Hela-HGPRT-细胞系,该细胞系可用于细胞融合相关研究。     

豹蛙核酸酶与斑蝥酸钠对肺腺癌细胞增殖的协同抑制作用

豹蛙核酸酶(onconase,Onc)是从美洲北方豹蛙卵母细胞中提取的一种核糖核酸酶,对许多肿瘤细胞都具有杀伤作用。斑蝥素(cantharidin)是存在于芫青科昆虫斑蝥体内的一种天然防御性毒素,斑蝥酸钠(sodium cantharidate,SCA)是斑蝥素半合成衍生物。鉴于Onc与SCA对非小细胞肺癌都具有杀伤作用,采用MTT法测定Onc与SCA单独与联合作用于两株肺腺癌细胞的IC50值,运用联合作用指数(combination index,CI)和等效线分析评价两者联合作用的效果。结果表明,Onc与SCA联合作用时,CI值均小于0.7,等效线分析图显示,代表Onc与SCA联合作用的点均位于加成线下方,Onc与SCA对肺腺癌SPC-A-1、A549细胞株增殖的抑制作用具有协同效应。用流式细胞仪进行的凋亡细胞检测结果也支持上述"Onc/SCA联合使用具有协同抗癌作用"的结论。     

Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)

The presence of acetate exceeding 5 g/L is a major concern during E. coli fermentation due to its inhibitory effect on cell growth, thereby limiting high-density cell culture and recombinant protein production. Hence, engineered E. coli strains with enhanced acetate tolerance would be valuable for these bioprocesses. In this work, the acetate tolerance of E. coli was much improved by rewiring its global regulator cAMP receptor protein (CRP), which is reported to regulate 444 genes. Error-prone PCR method was employed to modify crp and the mutagenesis libraries (~3×10⁶) were subjected to M9 minimal medium supplemented with 5–10 g/L sodium acetate for selection. Mutant A2 (D138Y) was isolated and its growth rate in 15 g/L sodium acetate was found to be 0.083 h^-1, much higher than that of the control (0.016 h^-1). Real-time PCR analysis via OpenArray^® system revealed that over 400 CRP-regulated genes were differentially expressed in A2 with or without acetate stress, including those involved in the TCA cycle, phosphotransferase system, etc. Eight genes were chosen for overexpression and the overexpression of uxaB was found to lead to E. coli acetate sensitivity.     

REACTION OF ANHYDRONUCLEOSIDES WITH MAGNESIUM ALKOXIDES: REGIOSPECIFIC SYNTHESIS OF 2′-O-ALKYLPYRIMIDINE NUCLEOSIDES.

Abstract

A novel approach to 2′-O-alkylpyrimidine nucleosides involving a 3′- hydroxyl assisted intramolecular delivery of a divalent metal alkoxide leads to a regiospecific opening of the anhydropyrimidine linkage at the 2′-position. Thus, reaction of 5′-protected 2,2′-anhydrouridine with magnesium or calcium alkoxides in DMF affords exclusively the corresponding 2′-O-alkyluridines in reasonable yields.     

Interaction of cyproheptadine hydrochloride with human serum albumin using spectroscopy and molecular modeling methods

The interaction between cyproheptadine hydrochloride (CYP) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, UV–vis absorption spectroscopy, Fourier transform infrared spectroscopy (FT‐IR) and molecular modeling at a physiological pH (7.40). Fluorescence of HSA was quenched remarkably by CYP and the quenching mechanism was considered as static quenching since it formed a complex. The association constants K_a and number of binding sites n were calculated at different temperatures. According to Förster's theory of non‐radiation energy transfer, the distance r between donor (human serum albumin) and acceptor (cyproheptadine hydrochloride) was obtained. The effect of common ions on the binding constant was also investigated. The effect of CYP on the conformation of HSA was analyzed using FT‐IR, synchronous fluorescence spectroscopy and 3D fluorescence spectra. The thermodynamic parameters ΔH and ΔS were calculated to be ?14.37 kJ mol^?1 and 38.03 J mol^?1 K^?1, respectively, which suggested that hydrophobic forces played a major role in stabilizing the HSA‐CYP complex. In addition, examination of molecular modeling indicated that CYP could bind to site I of HSA and that hydrophobic interaction was the major acting force, which was in agreement with binding mode studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Combined multispectroscopic and molecular docking investigation on the interaction between strictosamide and human serum albumin

The interaction between strictosamide (STM) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, three‐dimensional fluorescence spectroscopy, ultraviolet‐visible absorption spectroscopy, circular dichroism spectroscopy and molecular modeling under physiological pH 7.4. STM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding site number n and apparent binding constant K_a were determined at different temperatures by fluorescence quenching. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated as ?3.01 kJ/mol and 77.75 J/mol per K, respectively, which suggested that the hydrophobic force played major roles in stabilizing the HSA–STM complex. The distance r between donor and acceptor was obtained to be 4.10 nm according to Förster's theory. After the addition of STM, the synchronous fluorescence and three‐dimensional fluorescence spectral results showed that the hydrophobicity of amino acid residues increased and the circular dichroism spectral results showed that the α‐helix content of HSA decreased (from 61.48% to 57.73%). These revealed that the microenvironment and conformation of HSA were changed in the binding reaction. Furthermore, the study of molecular modeling indicated that STM could bind to site I of HSA and the hydrophobic interaction was the major acting force, which was in agreement with the binding mode study. Copyright © 2013 John Wiley & Sons, Ltd.