Construction,expression and binding specificity of bispecific CD3×VEGFR-2 and CD3×NCAM antibodies in the single chain and diabody format

Bispecific antibodies are recombinant proteins with novel immunological properties and therapeutic potential. Recombinant protein quality and activity of several bispecific antibodies comprising different variable domain combinations with respect to the parental monospecific single chain fragments (scFv) were evaluated after expression in bacteria or mammalian cells. The parental scFv proteins humanized anti-NCAM scFv, murine anti-VEGFR-2 scFv, murine and humanized anti-CD3 scFv, respectively, could successfully be expressed in E. coli, whereas the murine anti-NCAM scFv version could not be reliably detected. Bispecific CD3 × VEGFR-2 and CD3 × NCAM anti-bodies were expressed in the bispecific single chain and the single chain diabody format. However, the diabody derived from the murine anti-NCAM scFv could not efficiently be expressed in E. coli or in mammalian cells. Significant binding of the CD3 × NCAM single chain diabody comprising the humanized version of anti-CD3 and humanized version of anti-NCAM was efficient to both antigens. Nevertheless, binding of the bispecific single chain version to the NCAM antigen was inefficient in comparison to CD3 binding. In conclusion, the data could indicate that the result of scFv expression in bacteria may be predictive for the chances of success for functional expression of more complex bispecific derivatives.

金雀异黄素对巨噬细胞RAW 264.7增殖、形态和细胞周期的影响

目的研究金雀异黄素对巨噬细胞RAW264.7增殖、细胞形态及细胞周期的影响,以探讨其对免疫系统的作用。方法以鼠源巨噬细胞系RAW264.7为细胞模型,将金雀异黄素用二甲基亚砜(DMSO)溶解,再用培养液稀释成所需终浓度为12.5、25、50、100μmol·L-1的4组及溶剂对照组(DMSO体积含量比低于0.1%)。通过WST-1细胞增殖实验、倒置相差显微镜及流式细胞仪分别检测金雀异黄素对巨噬细胞的增殖、细胞形态及细胞周期的影响。结果金雀异黄素对巨噬细胞的影响呈时间和剂量依赖关系。50～100μmol·L-1浓度的金雀异黄素作用巨噬细胞24h和48h后均能抑制细胞增殖,细胞增殖率分别为77%～81%、58%～73%(P<0.01);并能影响细胞形态改变,表现为细胞体积增大,并形成较长伪足。流式细胞结果显示,该浓度金雀异黄素可将巨噬细胞阻滞在S-G2/M期。结论金雀异黄素能抑制巨噬细胞细胞增殖,影响巨噬细胞形态,可能与干扰细胞周期有关。

脾酪氨酸激酶抑制剂对脂多糖诱导巨噬细胞炎症反应的影响及机制

目的研究脾酪氨酸激酶（Syk）在LtX3诱导的Toll样受体4（TLR4）信号转导通路的作用。方法用100ng／mL脂多糖和不同浓度Syk抑制剂分别处理RAW264．7巨噬细胞后，采用Alarmar blue方法检测细胞增殖；采用Griess试剂检测细胞培养液中NO的含量；Westernblot法检测诱导型一氧化氮合成酶（iNOS）的蛋白表达；ELISA检测巨噬细胞中TNF-α和IL-6含量，RAW—Blue巨噬细胞检测核因子KB（NF—KB）和激活子蛋白-1（AP-1）的活化。结果Syk抑制剂作用于RAW264．7巨噬细胞24h，（0．5～14．0）μmol／L的Syk抑制剂对细胞增殖无明显影响，但当剂量超过1．5μmol／L时，能显著抑制LPS刺激的RAW264．7巨噬细胞NO的产生和iNOS的蛋白表达，抑制TNF-α和IL-6的产生（P〈0．01）以及NF—KB和AP-1的激活。结论Syk抑制剂降低LPS诱导的巨噬细胞分泌NO和iNOS的蛋白表达及TNF-α和IL-6的释放，通过下调Syk下游TLR4受体信号传导通路的蛋白，进而显著降低转录因子NF-KB和AP-1的激活有关。

金雀异黄素对脂多糖诱导RAW264.7细胞分泌功能的影响

目的:研究金雀异黄素对脂多糖(LPS)诱导巨噬细胞RAW264.7分泌功能的影响,探讨金雀异黄素抗炎作用及其机制,为临床应用提供理论基础。方法:用脂多糖诱导巨噬细胞株Raw264.7建立体外细胞炎症模型,金雀异黄素与巨噬细胞株Raw264.7温孵1h后,加入100ng·mL-1脂多糖以诱导巨噬细胞的活化,采用Griess试剂检测细胞培养液中NO的含量,通过双抗体夹心酶联分析法和细胞转染技术观察不同浓度金雀异黄素对脂多糖刺激巨噬细胞分泌细胞因子TNF-α、IL-6和IL-10含量及对核转录因子NF-κB和AP-1表达的影响。结果:金雀异黄素(4～111μmolL-1)能明显抑制脂多糖刺激巨噬细胞生成一氧化氮产生(P<0.05);显著抑制脂多糖诱导巨噬细胞分泌细胞因子TNF-α、IL-6和IL-10(P<0.01),并能抑制核转录因子NF-κB和AP-1的表达。结论:本实验证明了金雀异黄素有抗炎作用,其能显著降低脂多糖诱导巨噬细胞分泌NO及细胞因子TNF-α、IL-6和IL-10的释放,通过抑制这些炎症因子的释放来达到抗炎作用,金雀异黄素抑制核转录因子NF-κB和AP-1的表达来实现的。

Helicobacter pylori vac A genotype is a predominant determinant of immune response to Helicobacter pylori CagA

To evaluate the frequency of Helicobacter pylori (H. pylori) CagA antibodies in H. pylori infected subjects and to identify potential histopathological and bacterial factors related to H. pylori CagA-immune response. METHODSSystematic data to H. pylori isolates, blood samples, gastric biopsies for histological and molecular analyses were available from 99 prospectively recruited subjects. Serological profile (anti-H. pylori, anti-CagA) was correlated with H. pylori isolates (cagA, EPIYA, vacA s/m genotype), histology (Sydney classification) and mucosal interleukin-8 (IL-8) mRNA and protein expression. Selected H. pylori strains were assessed for H. pylori CagA protein expression and IL-8 induction in co-cultivation model with AGS cells. RESULTSThirty point three percent of microbiologically confirmed H. pylori infected patients were seropositive for CagA. Majority of H. pylori isolates were cagA gene positive (93.9%) with following vacA polymorphisms: 42.4% vacA s1m1, 23.2% s1m2 and 34.3% s2m2. Anti-CagA-IgG seropositivity was strongly associated with atrophic gastritis, increased mucosal inflammation according to the Sydney score, IL-8 and cagA mRNA expression. VacA s and m polymorphisms were the major determinants for positive (vacA s1m1) or negative (vacA s2m2) anti-CagA serological immune response, which also correlated with the in vitro inflammatory potential in AGS cells. In vitro co-cultivation of representative H. pylori strains with AGS cells confirmed functional CagA translocation, which showed only partial correlation with CagA seropositivity in patients, supporting vacA as major co-determinant of the immune response. CONCLUSIONSerological immune response to H. pylori cagA+ strain in H. pylori infected patients is strongly associated with vacA polymorphism, suggesting the crucial role of bacterial factors in immune and clinical phenotype of the infection.

Nobachelins,new siderophores from Nocardiopsis baichengensis protecting Caenorhabditis elegans from Pseudomonas aeruginosa infection

The biosynthetic potential of actinobacteria to produce novel natural products is still regarded as immense.In this paper,we correlated a cryptic biosynthetic gene cluster to chemical molecules by genome mining and chemical analyses,leading to the discovery of a new group of catecholate-hydroxamate siderophores,nobachelins,from Nocardiopsis baichengensis DSM 44845.Nobachelin biosynthesis genes are conserved in several bacteria from the family Nocardiopsidaceae.Structurally,nobachelins feature fatty-acylated hydroxy-ornithine and a rare chlorinated catecholate group.Intriguingly,nobachelins rescued Caenorhabditis elegans from Pseudomonas aeruginosa-mediated killing.

Integrating Culture-based Antibiotic Resistance Profiles with Whole-genome Sequencing Data for 11,087 Clinical Isolates

Emerging antibiotic resistance is a major global health threat. The analysis of nucleic acid sequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistance determinants to inform molecular diagnostics and drug development. We collected genetic data (11,087 newly-sequenced whole genomes) and culture-based resistance profiles (10,991 out of the 11,087 isolates comprehensively tested against 22 antibiotics in total) of clinical isolates including 18 main species spanning a time period of 30 years. Species and drug specific resistance patterns were observed including increased resistance rates for Acinetobacter baumannii to carbapenems and for Escherichia coli to fluoroquinolones. Species-level pan-genomes were constructed to reflect the genetic repertoire of the respective species,including conserved essential genes and known resis-tance factors. Integrating phenotypes and genotypes through species-level pan-genomes allowed to infer gene–drug resistance associations using statistical testing. The isolate collection and the analysis results have been integrated into GEAR-base,a resource available for academic research use free of charge at https://gear-base.com.

Fungal diversity notes 929-1035:taxonomic and phylogenetic contributions on genera and species of fungi

This article is the ninth in the series of Fungal Diversity Notes,where 107 taxa distributed in three phyla,nine classes,31 orders and 57 families are described and illustrated.Taxa described in the present study include 12 new genera,74 new species,three new combinations,two reference specimens,a re-circumscription of the epitype,and 15 records of sexualasexual morph connections,new hosts and new geographical distributions.Twelve new genera comprise Brunneofusispora,Brunneomurispora,Liua,Lonicericola,Neoeutypella,Paratrimmatostroma,Parazalerion,Proliferophorum,Pseudoastrosphaeriellopsis,Septomelanconiella,Velebitea and Vicosamyces.Seventy-four new species are Agaricus memnonius,A.langensis,Aleurodiscus patagonicus,Amanita flavoalba,A.subtropicana,Amphisphaeria mangrovei,Baorangia major,Bartalinia kunmingensis,Brunneofusispora sinensis,Brunneomurispora lonicerae,Capronia camelliaeyunnanensis,Clavulina thindii,Coniochaeta simbalensis,Conlarium thailandense,Coprinus trigonosporus,Liua muriformis,Cyphellophora filicis,Cytospora ulmicola,Dacrymyces invisibilis,Dictyocheirospora metroxylonis,Distoseptispora thysanolaenae,Emericellopsis koreana,Galiicola baoshanensis,Hygrocybe lucida,Hypoxylon teeravasati,Hyweljonesia indica,Keissleriella caraganae,Lactarius olivaceopallidus,Lactifluus midnapurensis,Lembosia brigadeirensis,Leptosphaeria urticae,Lonicericola hyaloseptispora,Lophiotrema mucilaginosis,Marasmiellus bicoloripes,Marasmius indojasminodorus,Micropeltis phetchaburiensis,Mucor orantomantidis,Murilentithecium lonicerae,Neobambusicola brunnea,Neoeutypella baoshanensis,Neoroussoella heveae,Neosetophoma lonicerae,Ophiobolus malleolus,Parabambusicola thysanolaenae,Paratrimmatostroma kunmingensis,Parazalerion indica,Penicillium dokdoense,Peroneutypa mangrovei,Phaeosphaeria cycadis,Phanerochaete australosanguinea,Plectosphaerella kunmingensis,Plenodomus artemisiae,P.lijiangensis,Proliferophorum thailandicum,Pseudoastrosphaeriellopsis kaveriana,Pseudohelicomyces menglunicus,Pseudoplagiostoma mangiferae,Robillarda mangiferae,Roussoella elaeicola,Russula choptae,R.uttarakhandia,Septomelanconiella thailandica,Spencermartinsia acericola,Sphaerellopsis isthmospora,Thozetella lithocarpi,Trechispora echinospora,Tremellochaete atlantica,Trichoderma koreanum,T.pinicola,T.rugulosum,Velebitea chrysotexta,Vicosamyces venturisporus,Wojnowiciella kunmingensis and Zopfiella indica.Three new combinations are Baorangia rufomaculata,Lanmaoa pallidorosea and Wojnowiciella rosicola.The reference specimens of Canalisporium kenyense and Tamsiniella labiosa are designated.The epitype of Sarcopeziza sicula is re-circumscribed based on cyto-and histochemical analyses.The sexual-asexual morph connection of Plenodomus sinensis is reported from ferns and Cirsium for the first time.In addition,the new host records and country records are Amanita altipes,A.melleialba,Amarenomyces dactylidis,Chaetosphaeria panamensis,Coniella vitis,Coprinopsis kubickae,Dothiorella sarmentorum,Leptobacillium leptobactrum var.calidus,Muyocopron lithocarpi,Neoroussoella solani,Periconia cortaderiae,Phragmocamarosporium hederae,Sphaerellopsis paraphysata and Sphaeropsis eucalypticola.