Increased_expression_of_bioactive_chemokines_in_human_cerebromicrovascular_endothelial_cells_and

上海交通大学基础医学院高小玲课题组发表纳米递药系统脑内命运及其调控机制研究的新成果佚名【期刊名称】《上海交通大学学报(医学版)》【年(卷),期】2024(44)1【摘要】2023年12月29日,上海交通大学基础医学院药理学与化学生物学系高小玲教授联合上海中医药大学陈红专教授和加拿大多伦多大学郑岗教授,在国际权威杂志《自然·纳米技术》(Nature Nanotechnology)正式发表了题为Intracerebral fate of organic and inorganic nanoparticles is dependent on microglial extracellular vesicle function的研究论文。

该杂志同期配发Intracerebral fate of engineered nanoparticles新闻和评述。

该研究通过阐明不同纳米递药系统脑清除差异的原因,揭示小胶质细胞的细胞外囊泡(EVs)对于脑内纳米粒的清除至关重要;发现通过激活ERK1/2通路.【总页数】1页(P97-97)【正文语种】中文【中图分类】G64【相关文献】1.狄文课题组在纳米靶向治疗卵巢研究方面取得新进展上海交通大学医学院附属仁济医院研究人员构建新型纳米载药递送系统2.上海交通大学基础医学院高小玲课题组发表多功能仿生纳米结构靶向神经血管单元改善阿尔茨海默病认知障碍的新成果3.上海交通大学医学院附属瑞金医院蒙国宇课题组发表生物被膜形成机制研究成果4.上海交通大学医学院高小玲课题组提出“纳米刹车”新策略,阻断线粒体功能障碍级联反应并改善阿尔茨海默病认知功能障碍5.上海交通大学基础医学院方超课题组报道基于巨噬细胞的肿瘤靶向递药新技术因版权原因，仅展示原文概要，查看原文内容请购买。

Suppression of CD74Expression and Helicobacter pylori Adhesion by Auraptene Targeting Serum Starvation-Activated ERK1/2in NCI-N87Gastric Carcinoma CellsHirotaka S EKIGUCHI ,Kazuhiro I RIE ,and Akira M URAKAMI yDivision of Food Science and Biotechnology,Graduate School of Agriculture,Kyoto University,Kyoto 606-8502,JapanReceived December 9,2009;Accepted January 19,2010;Online Publication,May 7,2010[doi:10.1271/bbb.90910]Helicobacter pylori (H.pylori )is a major human pathogen and plays a central role in chronic gastritis and gastric cancer.Since the adhesion of H.pylori to the human gastric epithelium is the initial and critical step of its infection,anti-H.pylori adhesion agents may be effective for the prevention and therapy of H.pylori -associated diseases.CD74has recently been identified as a new receptor for H.pylori urease,and we have previously reported that several citrus components strongly suppressed CD74expression in NCI-N87gastric carcinoma cells.We found in this present study that auraptene (citrus coumarin)disrupted serum starvation-induced extracellular signaling-regulated kinase (ERK)1/2activation and attenuated H.pylori adhesion and IL-8production in a co-culture system.In addition,the knockdown of CD74expression led to a significant decrease of H.pylori adhesion,but unexpect-edly increased IL-8production.However,PD98059(a MEK1/2inhibitor)dramatically down-regulated this cytokine,suggesting MEK/ERK-dependent IL-8pro-duction.Our results suggest that auraptene suppressed H.pylori adhesion and resulting chemokine production by disrupting ERK1/2activation.Key words:Helicobacter pylori ;CD74;adhesion;IL-8;aurapteneHelicobacter pylori (H.pylori ),which infects approx-imately 50%of all people in the world,is one of the most widespread human pathogens and is associated with the development of chronic gastritis,and gastric and duodenal ulcers.1,2)In 1994,the organism was ranked as being class I,‘‘carcinogenic to humans,’’by the World Health Organization/International Agency for Research on Cancer (WHO/IARC).3)Although two antibiotics (amoxicillin and clarithromycin)and a proton pump inhibitor are widely used for the antimicrobial therapy of H.pylori infection,cases of resistance to clarithromycin are increasing and treatment failure has emerged,especially in Asian countries.4,5)The adhesion of H.pylori to gastric epithelial cells is recognized as one of the essential steps in the develop-ment of gastritis,which leads to injection of the definitive virulence factor,cytotoxin-associated antigenA (CagA),through a type IV secretion system.6)However,the adhesion mechanism of H.pylori to gastric epithelial cells remains to be fully understood.CD74,i.e.,the major histocompatibility complex (MHC)class II-associated invariant chain,expression has recently been detected on gastric epithelial cells and has been shown to be an adhesion molecule to urease in H.pylori .7)Urease accounts for 5–10%of bacterial whole protein and is expressed in most,if not all,H.pylori strains.8)This enzyme catalyzes the hydrolysis of urea into ammonia and carbon dioxide,while its most important role is to protect the bacteria from the acidic condition in the stomach by neutralization.9)H.pylori urease is therefore considered to be essential for bacterial colonization.Urease comprises two subunits,i.e.,urease (UreA,24kDa)and urease (UreB,68kDa).8)Beswick et al.have suggested that urease binds to CD74on gastric epithelial cells and induces nuclear factor-kappaB (NF- B)activation and thereby stimulates interleukin-8(IL-8)production.7,10)In addi-tion,cell surface-associated urease binds to MHC class II molecules,including CD74,to induce apoptosis.11)A number of adhesins on the cell surface of H.pylori ,including BabA,SabA and AlpA/B,have also been identified.BabA and SabA bind to the Lewis B blood group antigen 12)and Sialyl Lewis X,13)respectively,while the ligands to AlpA/B receptors remain un-known.14)Although being associated with H.pylori infection,these receptors do not serve as signaling molecules responsible for activating the signaling path-ways involving mitogen-activated protein kinase (MAPK)and phosphatidylinositol 3-kinase (PI3K)in host cells.Thus,natural or synthetic agents that are capable of suppressing CD74expression may be promising and effective for regulating H.pylori adhe-sion and gastritis.The coumarin derivative,auraptene (7-geranyloxy-coumarin,AUR,Fig.1A),which is present in such citrus fruit as grapefruit (Citrus paradisi )and natsudai-dai (Citrus natsudaidai ),is one of the probable chemo-preventive agents.15)Accumulative evidence from ani-mal models has indicated that the compound exerted its cancer chemopreventive ability in such organs as the tongue,16)esophagus,17)and colon.18,19)On the otheryTo whom correspondence should be addressed.Tel:+81-75-753-6282;Fax:+81-75-753-6284;E-mail:cancer@kais.kyoto-u.ac.jpAbbreviations :AUR,auraptene;CagA,cytotoxin-associated antigen A;EGFR,epidermal growth factor receptor;ERK,extracellular signaling-regulated kinase;MAPK,mitogen-activated protein kinase;MIF,macrophage migration inhibitory factor;NF- B,nuclear factor-kappaBBiosci.Biotechnol.Biochem.,74(5),1018–1024,2010hand,we have previously reported that an auraptene treatment attenuated inflammatory leukocyte activation in vivo and led to decreased inflammation,H2O2 production,and cell proliferation,while also suppressing 12-O-tetradecanoylphorbol-13-acetate-induced superox-ide in HL-60cells.20)Thesefindings suggest that auraptene would mitigate oxidative stress by suppress-ing the generation of oxygen radicals by inflammatory leukocytes.We have previously reported that auraptene notably suppressed CD74expression by an unknown mecha-nism.21)Interestingly,Takeda et al.have indicated that an oral administration of auraptene inhibited H.pylori colonization in Mongolian gerbils.22)However,this study did not clarify its mechanisms of action.We thus attempted in this present study to elucidate the molecu-lar mechanism by which auraptene suppresses CD74in NCI-N87human gastric carcinoma cells.The effects of auraptene on H.pylori adhesion and the resulting cytokine production were also investigated. Materials and MethodsChemicals.RPMI1640and fetal bovine serum(FBS)were purchased from Gibco BRL(NY,USA),and oligonucleotide primers were synthesized by Sigma Genosys(Hokkaido,Japan).Antibodies were purchased from the following sources:rabbit anti-CD74,Santa Cruz Biotechnology(Santa Cruz,CA,USA);rabbit anti-phospho-extracellular regulated kinase(ERK)1/2,Cell Signaling Technology (MA,USA);mouse anti- -tubulin,Calbiochem(San Diego,CA, USA);rabbit anti-H.pylori,anti-rabbit IgG,anti-mouse IgG and nonspecific IgG,Dako(Glostrup,Denmark).siRNA was purchased from Santa Cruz Biotechnology,and auraptene was isolated as previously described.20,23)All other chemicals were purchased from Wako Pure Chemicals(Osaka,Japan),unless specified otherwise.Gastric cell culture.NCI-N87gastric carcinoma cells were obtained from American Type Culture Collection(Rockville,MD,USA), and grown in RPMI1640supplemented with10%FBS,penicillin (100U/ml),and streptomycin(100m g/ml)at37 C in a humidified atmosphere of95%air and5%CO2.Bacterial culture.H.pylori NCTC11637was purchased from American Type Culture Collection,and grown on Columbia HP agar plates(Beckton Dickinson,CA,USA)at37 C under microaerophilic conditions.The bacteria were transferred after72h into Brucella broth containing10%FBS.After the broth had been centrifuged at4000Âg for5min,the bacteria were resuspended in serum-free RPMI1640.The bacterial density was determined by measuring the absorbance at 595nm with a microplate reader(Thermo Fisher Scientific,Waltham, MA,USA).Reverse transcription-polymerase chain reaction.The cells (1:0Â106cells/5.0ml)were seeded onto a60-mm dish and preincu-bated for24h.The medium was replaced with serum-free RPMI1640 containing the samples dissolved in DMSO.After incubating for the designated times,total RNA was extracted by using the TRIzolÒreagent(Qiagen,Hilden,Germany).cDNA was synthesized by using 1m g of total RNA and an RNA PCR AMV kit(Takara Bio,Shiga, Japan).PCR amplification was then performed with a PTC-100TM thermal cycler(MJ Research,MA,USA)under the following conditions:CD74,forward(50-TgACCAgCgCgACCTTATCT-30)and reverse(50-gAgCAggTgCATCACATggT-30)primers(0.05m M each, 384bp product size)for30cycles,with60s of denaturation at94 C, 90s of annealing at54 C,and60s of primer extension at72 C;and glyceraldehyde-3-phosphate-dehydrogenase(GAPDH),forward(50-gTgAAggTCggAgTCAACg-30)and reverse(50-ggTgAAgACgCCA-GTggACTC-30)primers(0.05m M each,300bp product size)for23 cycles,with30s of denaturation at95 C,60s of annealing at58 C, and60s of primer extension at72 C.Amplified cDNA was subjected to electrophoresis on3%agarose gel and stained with SYBRÒGold (Molecular Probes,CA,USA).Image analysis was performed with NIH image,and GAPDH was used as the internal control.Western blotting.After the cells(3:4Â105cells/1.7ml)had been seeded onto a35-mm dish and preincubated for24h,the medium was replaced with serum-free RPMI1640containing the samples dissolved in DMSO.After incubating for the designated times,the cells were lysed with a lysis buffer(1%protease and phosphatase inhibitor cocktail(Sigma-Aldrich,MO,USA),10m M Tris(pH7.4),1%sodium dodecyl sulfate(SDS),and1m M sodium orthovanadate)and the lysate was sonicated.The denatured proteins(30m g)were separated by using SDS-polyacrylamide gel electrophoresis on10%polyacrylamide gel and transferred onto an ImmobilonÔ-P transfer membrane(Millipore, MA,USA).After blocking for1h at room temperature in Block Ace (Dainippon Pharmaceutical,Osaka,Japan),the membrane was incubated with each primary antibody at a dilution of1:1000,and then a second incubation was performed with each HRP conjugated secondary antibody at a dilution of1:1000(anti-mouse)and1:2000 (anti-rabbit).The blots were developed with the ECL western blotting detection reagent(Amersham Biosciences,Buckinghamshire,UK), and each band intensity was analyzed by NIH Image. -Tubulin was used as the internal control.H.pylori adhesion assay.The adhesion assay was performed as described previously.24,25)Briefly,the cells(5:0Â105cells/1.0ml) were grown on a24-well microplate and preincubated for24h.The medium on the plate was then replaced with serum-free RPMI1640 containing the samples dissolved in DMSO for48h.The cells were exposed to H.pylori in serum-free RPMI1640(5:0Â107cells/1.0ml) after the medium had been changed.After6h,the medium and unbound H.pylori were washed out three times with phosphate-buffered saline(PBS).The cells were thenfixed with8%formaldehyde and exposed to a quenching buffer(0.6%H2O2in the washing buffer) for20min at room temperature.The plate was incubated with the anti-H.pylori antibody or nonspecific IgG(1m g/ml each)for1h after washing.After further washing,the horseradish-peroxidase(HRP)-conjugated secondary antibody was added to the plate which was incubated for1h at room temparature.A color reaction was initiated for15min by adding a substrate solution(o-phenylethylenediamine (OPD)in a citrate buffer(1mg/ml))and terminated by a stop solution (1N sulfuric acid in PBS).Visible absorption at492nm was recorded with a microplate reader.ELISA.The cells(5:0Â105cells/1.0ml)were seeded onto a24-well microplate.After24h of incubation,the medium on the plate was replaced with serum-free RPMI1640containing the samples dissolved in DMSO.The medium was replaced after48h of incubation,and the cells were stimulated with or without H.pylori(5:0Â107cells/1.0ml) for another24h.The medium was then collected to measure the H.pylori-induced IL-8and macrophage migration inhibitory factor (MIF)levels.Each supernatant was centrifuged at1000Âg at4 C for 5min,and stored atÀ80 C until being used.The MIF(R&D Systems) and IL-8(Beckton Dickinson)levels in each culture supernatant were analyzed by kits according to the manufacturer’s instructions.Knockdown of CD74.siRNA was transfected with LipofectamineÔ2000(Invitrogen,CA,USA)according to the manufacturer’s instruc-tions.Briefly,24h before transfection,the cells(2:0Â105cells/ 1.0ml)were seeded onto a24-well microplate in RPMI1640with10% FBS.The cells(about70%confluent)were transfected with siRNA (the non-specific control and CD74;45pmol/0.6ml)by using Lip-ofectamineÔ2000dissolved in Opti-MEM IÒfor7h.The cells were then incubated for48h after the medium had been replaced by1ml of RPMI1640containing10%FBS.After being washed twice with PBS, the cells were treated with serum-free RPMI1640containing H.pylori (2:0Â107cells/1.0ml).H.pylori adhesion and the quantification of IL-8were respectively evaluated after6h and24h as already described.Cell viability.Cell viability was measured by using3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT).26)After being incubated,the cells were washed twice with PBS,then110m l of serum-free RPMI1640containing10m l of an MTT solution(5mg/ml)Auraptene Suppresses CD74and H.pylori Adhesion1019was added to each culture,and the culture was further incubated at 37 C for 2h.Next,200m l of DMSO was added,the culture was sonicated for 5min,and then 100m l of HCl/2-propanol (3.4m l/ml)was added to each well.The visible absorbance was measured at 570nm and 630nm with a microplate reader.The cell viability in each experiment was >70%.Statistical analysis.Each experiment was performed at least three times and the data are shown as the mean Æstandard deviation (SD),where appropriate.A one-way analysis of variance (ANOVA)was performed,followed by Tukey’s multiple-comparison test to determine whether the differences among the groups were significant.The differences are considered significant at p <0:05or 0.01.ResultsSuppressive effects of auraptene on ERK1/2activation As shown in Fig.1B,serum starvation for 1h resulted in marked dephosphorylation of ERK1/2,after which it was time-dependently restored during 3–12h.It is notable that auraptene significantly attenuated ERK1/2activation after 6h (p38MAPK and JNK 1/2,data not shown).Auraptene inhibited H.pylori adhesion and resulting IL-8productionWe wanted to find if auraptene could suppress H.pylori adhesion and the resulting production of pro-inflammatory cytokines in a co-culture system.The level of H.pylori adhesion to NCI-N87cells was slightly but significantly decreased by auraptene at a concentration of 50m M (Fig.2A).We then assessed whether the auraptene treatment could affect the production of IL-8and MIF in NCI-N87cells exposed to H.pylori .Exposure to H.pylori for 24h increased the level of IL-8production by 2.1-fold.Interestingly,pretreating with auraptene for 48h significantly inhibited the production in concentration-dependent fashion,the level being re-versed to the control value (Fig.2B).The MIF level was not affected with any statistical significance (Fig.2C).Knockdown of CD74inhibited H.pylori adhesion To ascertain the involvement of CD74in H.pylori adhesion,we examined how CD74down-regulation when using siRNA affected this adhesion and cytokineproduction (Fig.3A).As shown in Fig.3B,H.pylori adhesion was significantly suppressed by 20%in CD74-silenced cells as compared with the siRNA-transfected control cells.In contrast,IL-8production was unexpect-edly increased in CD74-silenced cells (Fig.4).It has recently been suggested that MIF up-regulated both CD74expression and that of the epidermal growth factor receptor (EGFR)which in turn phosphorylated ERK1/2.27,28)Furthermore,Kim et al.have recently reported that NCI-N87cells exhibited the highest expression level of EGFR when compared with several gastric carcinoma cell lines.29)MEK inhibitor PD98059(MEK1/2inhibitor)reduced the IL-8production in both CD74-silenced and control cells which had been stimulated with H.pylori ,while the EGFR tyrosine kinase inhibitor,AG1478,reduced it only in the former cells (Fig.4).CD74deficiency did not affect either the spontaneous or H.pylori -induced MIF release level (data not shown).DiscussionCD74was detected on gastric epithelial cells and shown to be a new receptor of H.pylori .7,10)CD74is a nonpolymorphic type II integral membrane protein that functions on the signaling pathways for malignant B-cell proliferation and survival.30,31)Interestingly,it has also been reported as a high-affinity binding protein for the multifunctional pro-inflammatory cytokine,MIF,32–36)which binds to the extracellular domain of CD74,and its interaction with CD44is required to activate such proliferative and pro-inflammatory signaling molecules as ERK1/2and prostaglandin E 2.37)In addition,once released,MIF mediates the expression and production of numerous pro-inflammatory cytokines/proteins includ-ing IL-1,IL-2,tumor necrosis factor- (TNF- ),IL-6,IL-8,interferon- (IFN- ),inducible nitric oxide syn-thase (iNOS)and cyctooxygenase-2(COX-2).These subsequently act to recruit and activate macrophages and T cells,resulting in inflammatory and immune diseases.33,38,39)Taken together,targeting CD74sup-pression may not only disrupt H.pylori adhesion,but also mitigate the resulting pro-inflammatory events.InA O OO pi-ERK 1/244B(kDa)42ERK 1/24442α-Tubulin600AUR (µM )02002002020(h)13612Fig.1.Structure of Auraptene and Effects of Auraptene on Serum Starvation-Induced ERK1/2Phosphorylation in NCI-N87Human GastricCarcinoma Cells.Cells treated with auraptene (AUR,20m M )or DMSO were lysed in a lysis buffer for the designated times and analyzed by western blotting as described in the Materials and Methods section.The experiments were independently conducted at least three times.1020H.S EKIGUCHI et al.this context,we have previously reported that citrus compounds including auraptene exhibited significant inhibitory activity toward CD74expression at the protein level.21)Takeda et al.have recently shown that auraptene exhibited anti-H.pylori colonization activity in Mongolian gerbils.22)In this present study,auraptene inhibited H.pylori adhesion to NCI-N87cells by only 27%(Fig.2A).As already mentioned,the adhesion of H.pylori to gastric epithelial cells does not only depend on CD74,but also on many other adhesins and their receptors.However,this coumarin was able to abolish H.pylori adhesion-dependent IL-8production (Fig.2B),suggesting that this effect,at least in part,could be associated with CD74suppression.On the other hand,RNAi for CD74significantly reduced the rate of H.pylori adhesion by 22%(Fig.3B).Our present findings suggest that the mechanism by which auraptene mitigated gastritis was associated,at least in part,with CD74repression.IL-8plays a crucial role in H.pylori -associated gastritis by recruiting,activating and infiltrating neu-trophils to the site of infection.In addition to its chemotactic potential,it is notable that the level of IL-8in gastric mucosa is associated with the histological severity in patients with H.pylori -induced gastritis.40)Although CD74is one of the mediators of H.pylori for promoting IL-8production,other mediators have also been suggested in many studies.For example,several12R e l a t i v e H . p y l o r iAI L -8 (p g / m l)100200300400B12345CM I F (n g / m l )AUR (µM )420500.8H. pyloriFig.2.Effects of Auraptene on H.pylori Adhesion,and on H.pylori -Induced IL-8and MIF Production in NCI-N87Gastric Carcinoma Cells.Cells were treated with auraptene (AUR,0–50m M )for 48h and then co-cultured with H.pylori for 6h (A)or 24h (B and C).H.pylori adhesion was determined by using an anti-Helicobacter pylori antibody and HRP-conjugated antibody as determined by ELISA (A),and the levels of IL-8production (B)and MIF secretion (C)were quantified by ELISA as described in the Materials and Methods section.The experiments were independently conducted at least three times,and the data are presented as the mean ÆSD .Bars not sharing a letter differ,p <0:05(A)and p <0:01(B and C),by Tukey’s multiple-comparisontest.0.511.5CD74GAPDHABControlCD74H. pyloriR e l a i v e H . p y l o r i l e v e l(siRNA)384300ControlCD7448(h)(siRNA)(bp)Fig.3.Effects of CD74Knockdown on H.pylori Adhesion in NCI-N87Gastric Carcinoma Cells.Cells were treated with the control or CD74siRNA for 7h by using Lipofectamine Ô2000and then the medium were replaced with RPMI1640including 10%FBS for 48h,before exposure to H.pylori for 6h.Total RNA was subjected to RT-PCR (A),and H.pylori adhesion was determined by using an anti-Helicobacter pylori antibody and HRP-conjugated antibody as determined by ELISA (B)and described in the Materials and Methods section.The experiments were independently conducted at least three times and the data are presented as the mean ÆSD (B).Bars not sharing a letter differ,p <0:05,by Tukey’s multiple-comparison test.Auraptene Suppresses CD74and H.pylori Adhesion 1021reports have proposed that H.pylori inoculated CagA into gastric epithelial cells to induce the phosphorylation of MEK,Src and SHP-2,thereby promoting IL-8production.41–43)On the other hand,Tabassam et al.have proposed that although the activation of EGFR-PI3K signaling,at least in part,positively regulated H.pylori -mediated IL-8production,CagA was involved in EGFR-dependent IL-8production.44)EGFR is over-expressed in various cancer cells and involved in pro-inflammatory responses and procarcinogenic events including cell proliferation,migration,aggregation,and invasion.45,46)The expression of this receptor is well known for many cancer tissues including gastric cancer.47–50)In the present study,CD74down-regulation by siRNA did not suppress,but rather increased IL-8production when co-cultured with H.pylori (Fig.4).Several studies so far have suggested that H.pylori or urease binding to CD74increased IL-8production,7,10,51)while H.pylori infection induced the expression and activation of EGFR in gastric epithelial cells.44)Fur-thermore,MIF appears to induce EGFR phosphorylation through multiple receptors,e.g.,CD74,CXCR2,and CXCR4.27)We have therefore hypothesized that MIF would be associated not only with serum starvation-induced CD74expression but also that of EGFR.27,28)Both PD98059(the MEK1/2inhibitor)and AG1478(the EGFR tyrosine kinase inhibitor)significantly inhibited IL-8production by H.pylori in CD74-silenced cells as compared with the control in this study.Previous work has suggested that ERK1/2played a central role on the signaling pathway linking the activation of EGFR and increased IL-8expression.28)Kim et al.have shown that treating AGS cells being transfected with mutant genes for Ras,c -Jun and I -B with MAPK inhibitors led to the suppression of these H.pylori -induced tran-scription factors and the resulting expression of IL-8.52,53)The MAPK cascade may therefore be responsible for inducing chemokine expression through NF- B and activator protein-1(AP-1)in H.pylori -infected gastric epithelial cells.54)Interestingly,Kawabata et al.have demonstrated that auraptene strongly dephosphorylated constitutively activated ERK1/2in HT-29human colonadenocarcinoma cells;55)this is consistent with our present findings (Fig.1B).Dual-specificity phosphatases (DUSP)are a heterogeneous group of protein phospha-tases that can dephosphorylate both phosphotyrosine and phosphoserine/phosphothreonine residues within the one substrate.DUSP can be divided into six subgroups on the basis of sequence similarity that include sling-shots such as phosphatase and tensin homologues deleted on chromosome 10(PTEN)and MAPK phos-phatases (MKP).56)For example,Zhu et al.have suggested that MKP-6up-regulation may act as a negative feedback loop,inhibiting excessive activation of ERK1/2in cagA -transfected B1lymphocytes,since MKP-6can dephosphorylate ERK1/2.57)Thus,the molecular target of auraptene may be MKP whichhas100200300400500600I L -8 (p g / m l )H. pyloriControlCD74ControlCD74(siRNA)Fig.4.Effects of CD74Knockdown and Specific Inhibitors on H.pylori -Increased IL-8Production in NCI-N87Gastric Carcinoma Cells.Cells were treated with the control or CD74siRNA for 7h,and then the medium were replaced with RPMI1640including 10%FBS.After 48h,the cells were exposed to H.pylori for 24h with DMSO,PD98059(PD:MEK inhibitor,50m M )or AG1478(AG:EGFR tyrosine kinase inhibitor,10m M ).The concentration of IL-8in the supernatant was determined by ELISA as described in the Materials and Methods section.The experiments were independently conducted at least three times,and the data are presented as the mean ÆSD .Bars not sharing a letter differ,p <0:05,by Tukey’s multiple-comparisontest.Fig.5.Proposed Molecular Mechanisms by Which Auraptene Suppresses H.pylori Adhesion and IL-8Production in NCI-N87Cells.The adhesion of H.pylori to NCI-N87cells is partially mediated by cell surface CD74.This attachment may activate the MEK/ERK pathway for increased IL-8production.Auraptene,on the other hand,inactivates ERK1/2which leads to the down-regulation of both CD74and IL-8.In contrast,CD74knockdown by siRNA did not,but rather potentiated IL-8production.This could be related to the possibility that MIF,a CD74ligand,may bind to such a surrogate receptor as EGFR.27)When CD74is abolished by siRNA,MIF binding to EGFR has previously been shown to activate ERK/IL-8.27,28)1022H.S EKIGUCHI et al.been reported to dephosphorylate ERK1/2.In any case, our results suggest that EGFR was partly involved in MIF-induced ERK1/2activation and IL-8production (Fig.5),although other factors such as CagA may also activate ERK1/2as already mentioned.43)As illustrated in Fig.5,while CD74mediated H.pylori-induced IL-8 production via the MEK/ERK pathway,MIF-activated EGFR may play a similar role in IL-8production when CD74is down-regulated.In conclusion,our results show that citrus auraptene notably suppressed CD74expression as well as H. pylori adhesion and IL-8production by regulating ERK1/2.To the best of our knowledge,this is thefirst report describing a food phytochemical that has the potential for regulating H.pylori adhesion by targeting a specific molecule.AcknowledgmentsThis study was supported in part by grant-aid from the Council for Advancement of Fruit Tree 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Journal of Clinical Laboratory Analysis00:1–6(2014) Determination of Anticyclic Citrullinated Peptide Based on Biotin–Streptavidin-Amplified Time-ResolvedFluoroimmunoassayHuiming Sheng,1Zhi-Gang Hu,2,3∗Jie Liu,2Fenghong Yuan,2Mei Li,2Yaohong Zou,2and Yu Chen31Shanghai Tongren Hospital Affiliated to Shanghai Jiaotong University,School of Medicine,Shanghai,China2Affiliated Wuxi People’s Hospital of Nanjing Medical University,Wuxi,China 3Department of Laboratory Medicine,The First Affiliated Hospital,College of Medicine,ZhejiangUniversity,Hangzhou,ChinaBackground:A rapid and sensitive time-resolvedfluoroimmunoassay(TRFIA) based on the biotin–streptavidin amplifi-cation system was developed for the deter-mination of anticyclic citrullinated peptide (anti-CCP).Methods:Europium-labeled streptavidin derivatives combined with eu-ropium and anhydride of diethylene triamine pentaacetic acid were used to label strep-tavidin,biotin was coupled with rabbit anti-human IgG to form a biotin–anti-human IgG bridge between streptavidin–europium and the anti-CCP antibody in the immunoas-say.The anti-CCP assay was carried out by measuring thefluorescence of Eu3+–streptavidin at615nm.Results:The pre-sented method produced a wide linear range from0.58to9,463U/ml,while itwas only591.4–18.48U/ml when using anELISA kit,and featured a detection limit upto0.5U/ml for anti-CCP.The values deter-mined by the biotin–streptavidin–TRFIA andELISA correlated well(R2=0.8927).The method was applied to determine anti-CCPin serum samples with satisfied recoveriesof96.45–104.63%.Conclusion:The assayresults obtained by the present methodshowed that biotin–streptavidin-amplifiedTRFIA improve the traditional ELISA kit foranti-CCP detection.Therefore,it offers abetter alternative immunoassay in rheuma-toid arthritis b.Anal.00:1–6,2014.C 2014Wiley Periodi-cals,Inc.Key words:cyclic citrullinated peptide;rheumatoid arthritis;time-resolvedfluoroim-munoassayINTRODUCTIONRheumatoid arthritis(RA)is an inflammatory autoim-mune disease,with a prevalence of0.3–1%worldwide, which leads to progressive joint erosion and substantial disability if not treated early.A reliable and specific test for a marker present early in the disease would be use-ful for identifying RA patients prior to the occurrence of joint damage(1).Antibodies to citrullinated proteins(ACPA)are specific serological markers for RA(2).ACPA constitute a grow-ing family of autoantibodies,and the major breakthrough came with the realization of the crucial role of peptide epitopes,which were citrullinated posttranslationally,and the subsequent development of the anticyclic citrullinated peptide antibodies(anti-CCP)test(3).The anti-CCP an-tibodies can be detected in up to80%of patients with RA,which is highly specific for the disease,and may be of value for both the diagnosis and prognosis of RA, particularly implicated in more erosive disease course(4). Grant sponsor:Wuxi’s Hospital Administration Center Key Subject;Grant number:YGZ1101;Grant sponsor:Directive Planning Project of Wuxi Science and Technology Bureau,China;Grant number:CSE31N1302.∗Correspondence to:Zhigang Hu,Affiliated Wuxi People’s Hospital of Nanjing Medical University,214023,Wuxi,China.E-mail:jswx-hzg@Received24November2013;Accepted25June2014DOI10.1002/jcla.21796Published online in Wiley Online Library().C 2014Wiley Periodicals,Inc.2Sheng et al.Time-resolvedfluoroimmunoassay(TRFIA),a nonra-dioimmunoassay,offers better sensitivity than other tradi-tional FIA methods due to the uniquefluorescence prop-erties of lanthanide chelates and the time-resolved mea-surement mode,which allows the specificfluorescence to be measured after the backgroundfluorescence has al-ready declined(5).Bioanalytical assays based on TRFIA have made a substantial impact on routine and research areas(6).Up to now,the use of Eu3+-labeled streptavidin(Eu3+–streptavidin)and time-resolvedfluorometry has been well suited as an indirect label for its relatively high affinity to biotin and low nonspecific binding properties.As a tetrameric structure with four binding sites for biotin,it can be used for amplifying signal(7).Moreover,a univer-sal detection reagent,europium chelate,as afluorescent marker has attracted much attention for high-throughput screening of large populations of biologically active sam-ples(8,9).In this study,the europium chelate of Eu3+–streptavidin was prepared and used as a signal generation reagent after its binding to the biotin moieties of the surface immuno-complex.A novel,rapid,biotin–streptavidin-amplified TRFIA for the determination of anti-CCP was developed and is described here.This method was applied to deter-mine anti-CCP in human serum samples with satisfactory results.MATERIALS AND METHODSChemicals and InstrumentationAuto DELFIA1235,Eu3+-labeled regent,and Eu3+-labeled enhancement solution were purchased from Wallac Oy(Turku,Finland).The96-well polystyrene microtiter plates and recombinant CCP antigen were ob-tained from AESKU(Dresden,Germany).Monoclonal rabbit anti-human IgM was purchased from Sigma(St. Louis,MO).N2-[p-isocyanate-benzyl]diethylenetriamine tetraacetic acid(DTTA)was purchased from Sigma Aldrich(St.Louis,MO).Bovine serum albumin(BSA) was purchased from Institute of Biological Products Min-istry of Health(Shanghai,China).Sephadex G25was pur-chased from Pharmacia(Piscataway,NJ).Concentrated wash buffer and enhancement solution were provided by Institute of Nuclear Medicine(Jiangsu,China).Other reagents used were of analytical reagent grade.ELISA Kit and Reference Standard of the Anti-CCP PreparationCommercial ELISA kits of the anti-CCP IgG were ob-tained from AESKU,Dr.Fenning(Kirchzarten,Ger-many),and ORGENTEC(Mainz,Germany),respec-tively.Reference standard of the anti-CCP was obtained from AESKU.Preparation of Solid AntigenEach well of polystyrene microtiter plates was coated with100μl of recombinant CCP antigen diluted with coating buffer(pH9.6,50mmol/l Na2CO3-NaHCO3) by incubating at4°C overnight.The coating buffer was discarded.After three times wash,some binding sites not occupied by the coating buffer were then blocked by250μl 3g/l BSA(Institute of Biological Products Department of Health,Shanghai,China)in phosphate buffer solu-tion(PBS;pH7.4)and the plates were incubated at4°C overnight.After the blocking buffer was discarded,the plates were vacuum-dried and stored at−20°C until anal-ysis.Biotin Labeling of Anti-Human IgGOne milligram of N-hydroxysuccinimidobitin (Promega,Madison,Wisconsin)was added to0.5mg of a1mg/ml solution of rabbit anti-human IgG(St. Louis,MO)in pH7.2,0.05M PBS.The reaction mixture was incubated at37°C for1h and then dialyzed overnight at4°C with PBS.The biotin-labeled antibody was stored at−20°C until use.Eu3+-Labeled Streptavidin PreparationEu3+labeling of streptavidin(Sigma)was performed us-ing a Eu3+-labeling Kit(EG&G-Wallac,Turku,Finland) strictly according to the manufacture’s instructions.One milligram of streptavidin was loaded on a PD10column and eluted using carbonate buffer(pH8.5,50mmol/l Na2CO3-NaHCO3)containing0.155mol/l NaCl.Then streptavidin was mixed with0.2mg Eu3+-DTPAA(di-ethylenetriamine pentaacetic acid;Sigma Aldrich)fol-lowed by vigorous stirring at30°C for20h.The resulting mixture was then fractionated on a Sepharose CL-6B(1×40cm;Pharmacia)with pH7.8,80mmol/l Tris-HCl buffer.The absorbance values of the elution were measured at280nm to obtain protein concentra-tions.After purification,equal amount of AR glycerin was added before subpackaging and stored at−20°C until use.Anti-CCP Antibody AssayThe procedures for BAS-TRFIA of serum anti-CCP antibody were performed using an indirect ELISA tech-nique.Hundred microliters of serum diluted with assay buffer was pipetted into each coated microtiter well,and incubated for30min at25°C.The plates were washed fourb.Anal.Determination of Anticyclic Citrullinated Peptide3times with wash buffer,then100μl of rabbit anti-humanIgG was added to each well,and incubated with shakingfor30min at25°C.The plates were washed again fourtimes,then pipetted with100μl buffer,diluted with Eu3+-labeled streptavidin to each well,and incubated with shak-ing for15min.After washing six times with wash buffer,100μL of enhancement solution was pipetted into eachwell.The plates were shaken for5min.Eu3+fluorescenceintensity(cps)was measured with auto DELFIA1235.The concentrations of anti-CCP antibody in respectivesamples were determined based on the calibration curvesusing Multicalc software program.Evaluation on the Kits(1)Precision testing.Three pools of mixed serum speci-mens with high,intermediate,and low concentrationof anti-CCP antibody were subpackaged and stored at −20°C.Mixed with the serum specimens and evaluat-ing their concentrations with samples,the coefficientof variations(CVs)was calculated.(2)Linear range testing.Serum from patient with thehighest anti-CCP antibody concentration was dilutedin a twofold serial dilution manner,and the anti-CCPantibody concentrations of diluted specimens weredetected using the established BAS-TRFIA kit and acommercial ELISA one,respectively.The linear curveof dilution concentration was obtained with dilutionmultiple as the horizontal abscissa andfluorescentintensity as the vertical ordinate.(3)Sensitivity testing.The sensitivity of the assay wasback-calculated by the obtained meanfluorescentcounts(n=20)of the zero standard plus2SD inthe calibration curve.(4)Clinical applications.The specificity of anti-CCP an-tibody detection was evaluated by testing serum from52healthy blood donors,32systemic lupus erythe-matosus(SLE),27Sjogren’s syndrome(SS),10sclero-derma,20mixed connective tissue disease(MCTD),23multiple sclerosis(MS)patients.(5)Correlation test.The anti-CCP antibody concentra-tions of32serum specimens from patients with RAwere detected using the established BAS-TRFIA kitand a commercial ELISA kit,respectively,and thenthe values obtained were analyzed.(6)Stability testing.New BAS-TRFIA kit and threeELISA kits were put into the37°C water box for7days and then compared with the one stored in rou-tine conditions.(7)Coefficient of recovery.Three sera with high antibodyconcentrations were diluted with sample buffer andassayed in the new TRFIAkit.Fig.1.Serial dilutions of a specimen with the highest anti-CCP IgG concentration were tested with TRFIA and ELISA,respectively.Double Y-axis curve was determined according to dilution strength(X-axis)as well as BAS-TRFIAfluorescence intensity and ELISA absorbance(the two Y-axes).Statistical AnalysesData were analyzed using the SPSS13.0software.Re-gression analysis and analysis of variance were used to calculate correlation and test linearity,respectively.In-terassay and intra-assay variation was calculated using the CV.The statistical significance level was set at0.05. RESULTSLinearityA strong positive specimen from9,081to0.55U/ml was diluted and the serial dilutions with the established TRFIA as well as ELISA were detected.The curve of de-tectable range of the two methods is shown in Figure1,in which we observe that for the established TRFIA kit there was a good linear range within9,463–0.58U/ml,whereas it was within591.4–18.48U/ml when using an ELISA kit,indicating that the method of TRFIA we established has a wider detectable range than the commercial ELISA. Calibration Curve for Determination of Anti-CCP IgGAs shown in Figure2,the time-resolvedfluorescence intensities are directly proportional to the concentrations of anti-CCP IgG.The concentrations of anti-CCP IgG were2,365.8,591.44,147.86,36.96,9.24,0.58U/ml.The line equation for the calibration curve of anti-CCP IgG was Y=3.79071+0.79505X+0.01094X2,where X is the concentration of anti-CCP IgG(GPL U/ml)and Y is fluorescence intensity.The sensitivity of the assay,defined as the mean signal of the zero standard plus2SD,was 0.5U/ml.b.Anal.4Sheng et al.Y : A x i s T i t l eX:The concentration for anti-CCP IgG (U/ml)Fig.2.Standard curves for anti-CCP IgG by TRFIA at different concentrations (2,365.8,591.44,147.86,36.96,9.24,0.58U/ml).TABLE 1.Intrabatch and Interbatch Precision AnalysisBAS-TRFIA (U/ml)ELISA (U/ml)Mean ±SDCV (%)Mean ±SD CV (%)HighIntrabatch (n =20)241.5±7.63 3.16242.8±14.86 6.12Interbatch (n =10)240.9±10.33 4.29243.1±17.647.26IntermediateIntrabatch (n =20)72.7±2.78 3.8272.6±5.948.18Interbatch (n =10)72.3±3.38 4.6873.1±7.269.93LowIntrabatch (n =20)14.15±0.60 4.2414.32±1.389.61Interbatch (n =10)14.23±0.714.9714.37±1.5410.74Assay PrecisionThe precision of the assay was also analyzed by mea-suring three pools of mixed serum specimens with high,intermediate,and low concentration of anti-CCP IgG 20times in one series (intra-assay)and in duplicate in eight different series,and data were compared with those ob-tained by the ELISA kit.As shown in Table 1,the intra-assay CVs detected by TRFIA at mean anti-CCP IgG concentrations of 241.5,72.7,14.15U/ml were 3.16%,3.82%,and 4.24%,respectively,and the interassay CVs at mean concentrations of 240.9,72.7,14.23U/ml were 4.29%,4.68%,and 4.97%,respectively.In comparison,the intra-assay CVs detected by ELISA at mean concen-trations of 242.8,72.6,14.32U/ml were 6.12%,8.18%,and 9.61%,respectively,and the interassay CVs at mean concentrations of 243.1,73.1,14.37U/ml were 7.268%,9.93%,and 10.74%,respectively.Obviously,the TRFIA kit we established for anti-CCP IgG detection was better than the commercial ELISA kit in intra-and interassay precision with either rank of concentration.TABLE 2.Specificity of Anti-CCP Antibody in Different Types of PatientsCase and Negative control 15Positive ࣙSpecificity groupn U/ml U/ml(%)Healthy blood donors 5251198.1SLE 3231196.9SS2726196.3Scleroderma 10100100MCTD 2019195.0MS2322195.7Clinical ApplicationEach well of polystyrene microtiter plates was coated with recombinant CCP antigen.The specificity of anti-CCP antibody detection was evaluated by testing serum from 52healthy blood donors,32SLE,27SS,10sclero-derma,20MCTD,and 23MS patients.Cross-reactivities are shown in Table 2.Correlation With ELISAConcentration results of 32serum samples determined by TRFIA were compared with those obtained by an ELISA kit.The scatter plots showed that the two methods correlated well (Fig.3)and the correlation coefficient of the results obtained from the two assays was 0.8927.Stability of the TRFIA KitsThe assay kits were stored at 4°C and water bathed at 37°C for 7days.Then,the difference of fluores-cence intensity or absorbance—that is decline rate ofb.Anal.Determination of Anticyclic Citrullinated Peptide5Fig.3.Correlation between the BAS-TRFIA and ELISA. TABLE3.Stability of the BAS-TRFIA and ELISAGroups4°Cpreserve7days37°Cwater bath7daysThe declinedrate ofcombination(%)ELISA1(abs) 2.56 1.6336.3 ELISA2(abs) 2.39 1.6929.3 ELISA3(abs) 2.71 2.2317.7 BAS-TRFIA(cps)2,451,2362,153,41512.1 combination—was measured both by TRFIA and ELISA.We observed that it exhibited no visible change except for a decline by12.1%for TRFIA(17.7%,29.3%, 36.3%for the commercial ELISA)in the rate of combina-tion when compared with the one stored in routine con-ditions,suggesting that the established assay kit had good stability and could be stored at4°C for1year(Table3). Moreover,the accuracy decreased as time was extended by ELISA,but after enhancement solution was added the detection offluorescent results was not affected up to the next day.Thus,it can be seen that TRFIA is more stable.Coefficient of RecoveryThe recoveries of thefirst sample were100.98%, 102.25%,and96.45%,respectively.The recoveries of the second sample were100.46%,98.18%,and101.59%, respectively.The recoveries of the third sample were 104.63%,97.18%,and98.17%,respectively.The average recovery rate calculated by the average of nine individ-ual values by the formula of observed concentrations×100/expected concentration in Table4)was99.98%.TABLE4.Coefficient of Recovery:Three Sera With High Anti-body Concentrations Were Diluted With Sample Buffer and As-sayed in the New BAS-TRFIA KitObserved ExpectedSample Dilution(U/ml)(U/ml)O/E(%) 11:100266.41:200134.5133.2100.981:40068.166.6102.251:80032.133.396.40 21:100175.61:20088.287.8100.461:40043.143.998.181:80022.321.95101.59 31:10088.51:20046.344.25104.631:40021.522.12597.181:80010.8611.062598.17DISCUSSIONTo facilitate early diagnosis,a serological marker of RA that is detectable very early in the disease is needed.As mentioned above,several recent studies have shown that anti-CCP can be detected very early in the course of RA and may be helpful in early diagnosis.Herein,we developed a new sandwich TRFIA with biotin–streptavidin system as a quantitative and rapid immunological assay for measuring anti-CCP in clini-cal serum samples,since the new method combined the advantages of both high sensitivity of traditional TR-FIA and high affinity of biotin to streptavidin tetrameric bond.Moreover,the detectable signal can be amplified because of the tetrameric structure with four binding sites of streptavidin for biotin.The experimental results indi-cated that the new TRFIA is a promising immunofluoro-metric method with highly sensitive and specific,mean-while,the assay provided wider dynamic working ranges and better reproducibility.In Figure1,TRFIA demon-strated a good linear range within9,463–0.58U/ml,by contrast,within591.4–18.48U/ml of an ELISA kit.The expanded detection range will improve earlier detection and higher sensitivity in RA diagnosis(10,11).Further-more,the serologic-positive patients will be treated timely and effectively,subsequently,preventing irreversible joint damage(12).By comparison,the new assay has satisfied correlation with general commercial ELISA kit with the R2value 0.8927(Fig.3),moreover,its stability performance is bet-ter than the commercial ones,which will guarantee the more stable yield(Table3).In sum,the new TRFIA with biotin–streptavidin system as a quantitative and rapid im-munological assay for measuring anti-CCP is a promising alternative assay for RA diagnosis and monitor.b.Anal.6Sheng et al.CONFLICT OF INTERESTAll authors declare that they have no competing inter-ests.REFERENCES1.Raptopoulou A,Sidiropoulos P,Katsouraki M,Boumpas DT.Anti-citrulline 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24.755 23.917 23.654 23.565 23.333 23.194 22.929 22.864 22.864 22.864 22.490 22.345 22.333 21.757 21.543 21.543 20.833 20.761 20.614 19.966 19.795 19.547 19.352 18.571 18.571 18.038 17.983 17.983 17.949 17.689 17.689 17.689 17.436 17.313 17.215 16.417 16.238 16.179 16.008 16.008 15.766 15.575 15.518 15.389 15.389 15.389 15.333 15.333 15.280 15.280 15.265 15.253 15.251 15.202
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