2008 Screening microalgae for biodiesel production

Microbiota Modulate Behavioral and Physiological Abnormalities Associatedwith Neurodevelopmental DisordersElaine Y.Hsiao,1,2,*Sara W.McBride,1Sophia Hsien,1Gil Sharon,1Embriette R.Hyde,3Tyler McCue,3Julian A.Codelli,2 Janet Chow,1Sarah E.Reisman,2Joseph F.Petrosino,3Paul H.Patterson,1,4,*and Sarkis K.Mazmanian1,4,*1Division of Biology and Biological Engineering,California Institute of Technology,Pasadena,CA91125,USA2Division of Chemistry and Chemical Engineering,California Institute of Technology,Pasadena,CA91125,USA3Alkek Center for Metagenomics and Microbiome Research,Baylor College of Medicine,Houston,TX77030,USA4These authors contributed equally to this work*Correspondence:ehsiao@(E.Y.H.),php@(P.H.P.),sarkis@(S.K.M.)/10.1016/j.cell.2013.11.024SUMMARYNeurodevelopmental disorders,including autism spectrum disorder(ASD),are defined by core behav-ioral impairments;however,subsets of individuals display a spectrum of gastrointestinal(GI)abnormal-ities.We demonstrate GI barrier defects and micro-biota alterations in the maternal immune activation (MIA)mouse model that is known to display features of ASD.Oral treatment of MIA offspring with the hu-man commensal Bacteroides fragilis corrects gut permeability,alters microbial composition,and ame-liorates defects in communicative,stereotypic,anxi-ety-like and sensorimotor behaviors.MIA offspring display an altered serum metabolomic profile,and B.fragilis modulates levels of several metabolites. Treating naive mice with a metabolite that is increased by MIA and restored by B.fragilis causes certain behavioral abnormalities,suggesting that gut bacterial effects on the host metabolome impact behavior.Taken together,thesefindings support a gut-microbiome-brain connection in a mouse model of ASD and identify a potential probiotic therapy for GI and particular behavioral symptoms in human neurodevelopmental disorders.INTRODUCTIONNeurodevelopmental disorders are characterized by impaired brain development and behavioral,cognitive,and/or physical abnormalities.Several share behavioral abnormalities in socia-bility,communication,and/or compulsive activity.Most recog-nized in this regard is autism spectrum disorder(ASD),a serious neurodevelopmental condition that is diagnosed based on the presence and severity of stereotypic behavior and deficits in lan-guage and social interaction.The reported incidence of ASD has rapidly increased to1in88births in the United States as of2008(Autism and Developmental Disabilities Monitoring Network Sur-veillance Year2008Principal Investigators and CDC,2012),rep-resenting a significant medical and social problem.However, therapies for treating core symptoms of autism are limited. Much research on ASD has focused on genetic,behavioral, and neurological aspects of disease,though the contributions of environmental risk factors(Hallmayer et al.,2011),immune dysregulation(Onore et al.,2012),and additional peripheral dis-ruptions(Kohane et al.,2012)in the pathogenesis of ASD have gained significant attention.Among several comorbidities in ASD,gastrointestinal(GI) distress is of particular interest,given its reported prevalence (Buie et al.,2010;Coury et al.,2012)and correlation with symp-tom severity(Adams et al.,2011).While the standardized diag-nosis of GI symptoms in ASD is yet to be clearly defined,clinical as well as epidemiological studies have reported abnormalities such as altered GI motility and increased intestinal permeability (Boukthir et al.,2010;D’Eufemia et al.,1996;de Magistris et al.,2010).Moreover,a recent multicenter study of over 14,000ASD individuals reveals a higher prevalence of inflamma-tory bowel disease(IBD)and other GI disorders in ASD patients compared to controls(Kohane et al.,2012).GI abnormalities are also reported in other neurological diseases,including Rett syn-drome(Motil et al.,2012),cerebral palsy(Campanozzi et al., 2007),and major depression(Graff et al.,2009).The causes of these GI problems remain unclear,but one possibility is that they may be linked to gut bacteria.Indeed,dysbiosis of the microbiota is implicated in the patho-genesis of several human disorders,including IBD,obesity,and cardiovascular disease(Blumberg and Powrie,2012),and several studies report altered composition of the intestinal mi-crobiota in ASD(Adams et al.,2011;Finegold et al.,2010;Fine-gold et al.,2012;Kang et al.,2013;Parracho et al.,2005;Williams et al.,2011;Williams et al.,2012).Commensal bacteria affect a variety of complex behaviors,including social,emotional,and anxiety-like behaviors,and contribute to brain development and function in mice(Collins et al.,2012;Cryan and Dinan, 2012)and humans(Tillisch et al.,2013).Long-range interactions between the gut microbiota and brain underlie the ability of microbe-based therapies to treat symptoms of multiplesclerosis Cell155,1451–1463,December19,2013ª2013Elsevier Inc.1451and depression in mice (Bravo et al.,2011;Ochoa-Repa´raz et al.,2010),and the reported efficacy of probiotics in treating emotional symptoms of chronic fatigue syndrome and psycho-logical distress in humans (Messaoudi et al.,2011;Rao et al.,2009).Based on the emerging appreciation of a gut-microbiome-brain connection,we asked whether modeling some of the behavioral features of ASD in a mouse model also causes GI abnormalities.Several mouse models of genetic and/or environ-mental risk factors are used to study ASD.We utilize the maternal immune activation (MIA)model,which is based on large epide-miological studies linking maternal infection to increased autismrisk in the offspring (Atlado´ttir et al.,2010;Gorrindo et al.,2012).A number of studies link increased ASD risk to familial autoim-mune disease (Atlado´ttir et al.,2009;Comi et al.,1999)and elevated levels of inflammatory factors in the maternal blood,placenta,and amniotic fluid (Abdallah et al.,2013;Brown et al.,2013;Croen et al.,2008).Modeling MIA in mice by injecting preg-nant dams with the viral mimic poly(I:C)yields offspring that exhibit the core communicative,social,and stereotyped impair-ments relevant to ASD,as well as a common autism neuro-pathology—a localized deficiency in cerebellar Purkinje cells (Malkova et al.,2012;Shi et al.,2009).Furthermore,pregnant monkeys exposed to poly(I:C)yield offspring with symptoms of ASD (Bauman et al.,2013).Although several environmental and genetic risk factors for ASD have been investigated in pre-clinical models,GI abnormalities have not been reported.We show herein that offspring of MIA mice,which display behavioral abnormalities,have defects in intestinal integrity and alterations in the composition of the commensal microbiota that are analo-gous to features reported in human ASD.To explore the potential contribution of GI complications to these symptoms,weF I T C i n t e n s i t y /m l s e r u m (f o l d c h a n g e )Adult (8-10 weeks)F I T C i n t e n s i t y /m l s e r u mAdolescent (3 weeks)S O C S N O S I L 1T N F I L 1I L m R N A /A C T B (f o l d c h a n g e )*S PF G F -b a s iI F N -I L -1I L -I L -I L -I L -12p 40/p 7I L -1I P -1K M C P -M I M I P -1T N F -V E G 300350400450p g /m g p r o t e i nT J P T J P O C L C L D N C L D N C L D N C L D N C L D N C L D N 1C L D N 1m R N A /A C T B (f o l d c h a n g e )S PABCDFigure 1.MIA Offspring Exhibit GI Barrier Defects and Abnormal Expression of Tight Junction Components and Cytokines(A)Intestinal permeability assay,measuring FITC intensity in serum after oral gavage of FITC-dextran.Dextran sodium sulfate (DSS):n =6,S (saline+vehicle):adult n =16;adolescent n =4,P (poly(I:C)+vehicle):adult n =17;adolescent n =4.Data are normalized to saline controls.(B)Colon expression of tight junction components relative to b -actin.Data for each gene are normalized to saline controls.n =8/group.(C)Colon expression of cytokines and inflamma-tory markers relative to b -actin.Data for each gene are normalized to saline controls.n =6–21/group.(D)Colon protein levels of cytokines and chemo-kines relative to total protein content.n =10/group.For each experiment,data were collected simul-taneously for poly(I:C)+B.fragilis treatment group (See Figure 3).See also Figure S1.examine whether treatment with the gut bacterium Bacteroides fragilis ,demon-strated to correct GI pathology in mouse models of colitis (Mazmanian et al.,2008)and to protect against neuroinflam-mation in mouse models of multiple sclerosis (Ochoa-Repa´raz et al.,2010),impacts ASD-related GI and/or behavioral abnormalities in MIA offspring.Our study reflects a mechanistic investigation of how alterations in the commensal microbiota impact behavioral abnormalities in a mouse model of neurodeve-lopmental disease.Our findings suggest that targeting the microbiome may represent an approach for treating subsets of individuals with behavioral disorders,such as ASD,and comor-bid GI dysfunction.RESULTSOffspring of Immune-Activated Mothers Exhibit GI Symptoms of Human ASDSubsets of ASD children are reported to display GI abnormal-ities,including increased intestinal permeability or ‘‘leaky gut’’(D’Eufemia et al.,1996;de Magistris et al.,2010;Ibrahim et al.,2009).We find that adult MIA offspring,which exhibit a number of behavioral and neuropathological symptoms of ASD (Malkova et al.,2012),also have a significant deficit in intestinal barrier integrity,as reflected by increased translocation of FITC-dextran across the intestinal epithelium,into the circulation (Figure 1A,left).This MIA-associated increase in intestinal permeability is similar to that of mice treated with dextran sodium sulfate (DSS),which induces experimental colitis (Figure 1A,left).Defi-cits in intestinal integrity are detectable in 3-week-old MIA offspring (Figure 1A,right),indicating that the abnormality is established during early life.Consistent with the leaky gut phenotype,colons from adult MIA offspring contain decreased gene expression of TJP1,TJP2,OCLN ,and CLDN8and increased expression of CLDN15(Figure 1B).Deficient expres-sion of TJP1is also observed in small intestines of adult MIA1452Cell 155,1451–1463,December 19,2013ª2013Elsevier Inc.offspring(Figure S1A available online),demonstrating a wide-spread defect in intestinal barrier integrity.Gut permeability is commonly associated with an altered immune response(Turner,2009).Accordingly,colons from adult MIA offspring display increased levels of interleukin-6(IL-6) mRNA and protein(Figures1C and1D)and decreased levels of the cytokines/chemokines IL-12p40/p70and MIP-1a(Fig-ure1D).Small intestines from MIA offspring also exhibit altered cytokine/chemokine profiles(Figure S1C).Changes in intestinal cytokines are not accompanied by overt GI pathology,as as-sessed by histological examination of gross epithelial morphology from hematoxylin-and eosin-stained sections (data not shown).Overall,wefind that adult offspring of im-mune-activated mothers exhibit increased gut permeability and abnormal intestinal cytokine profiles.MIA Offspring Display Dysbiosis of the Gut Microbiota Abnormalities related to the microbiota have been identified in ASD individuals,including disrupted community composition (Adams et al.,2011;Finegold et al.,2010;Finegold et al., 2012;Parracho et al.,2005;Williams et al.,2011;Williams et al.,2012),although it is important to note that a well-defined ASD-associated microbial signature is lacking thus far.To eval-uate whether MIA induces microbiota alterations,we surveyed the fecal bacterial population by16S rRNA gene sequencing of samples isolated from adult MIA or control offspring.Alpha di-versity,i.e.,species richness and evenness,did not differ signif-icantly between control and MIA offspring,as measured by several indices(Figures S2A and S2B).In contrast,unweighted UniFrac analysis,which measures the degree of phylogenetic similarity between microbial communities,reveals a strong ef-fect of MIA on the gut microbiota of adult offspring(Figure2). MIA samples cluster distinctly from controls by principal coordi-nate analysis(PCoA)and differ significantly in composition(Ta-ble S3,with ANOSIM R=0.2829,p=0.0030),indicating robust differences in the membership of gut bacteria between MIA offspring and controls(Figure2A).The effect of MIA on altering the gut microbiota is further evident when sequences from the classes Clostridia and Bacteroidia,which account for approxi-mately90.1%of total reads,are exclusively examined by PCoA(R=0.2331,p=0.0070;Figure2B),but not when Clostri-dia and Bacteroidia sequences are specifically excluded from PCoA of all other bacterial classes(R=0.1051,p=0.0700;Fig-ure2C).This indicates that changes in the diversity of Clostridia and Bacteroidia operational taxonomic units(OTUs)are the pri-mary drivers of gut microbiota differences between MIA offspring and controls.Sixty-seven of the1,474OTUs detected across any of the samples discriminate between treatment groups,including those assigned to the bacterial families Lachnospiraceae, Ruminococcaceae,Erysipelotrichaceae,Alcaligenaceae,Por-phyromonadaceae,Prevotellaceae,Rikenellaceae,and un-classified Bacteroidales(Figure2D and Table S1).Of these67 discriminatory OTUs(relative abundance:13.3%±1.65%con-trol,15.93%±0.62%MIA),19are more abundant in the control samples and48are more abundant in MIA samples.Consistent with the PCoA results(Figures2A–2C),the majority of OTUs that discriminate MIA offspring from controls are assigned to theclasses Bacteroidia(45/67OTUs or67.2%;12.02%±1.62% control,13.48%±0.75%MIA)and Clostridia(14/67OTUs or 20.9%;1.00%±0.25%control,1.58%±0.34%MIA).Interest-ingly,Porphyromonadaceae,Prevotellaceae,unclassified Bac-teriodales(36/45discriminatory Bacteroidial OTUs or80%;4.46%±0.66%control,11.58%±0.86%MIA),and Lachnospir-iceae(8/14discriminatory Clostridial OTUs or57%;0.28%±0.06%control,1.13%±0.26%MIA)were more abundant in MIA offspring.Conversely,Ruminococcaceae(2OTUs),Erysipe-lotrichaceae(2OTUs),and the beta Proteobacteria family Alca-ligenaceae(2OTUs)were more abundant in control offspring (Figure2D and Table S1;0.95%±0.31%control,0.05%±0.01%MIA).This suggests that specific Lachnospiraceae,along with other Bacteroidial species,may play a role in MIA pathogen-esis,while other taxa may be protective.Importantly,there is no significant difference in the overall relative abundance of Clostridia(13.63%± 2.54%versus14.44%± 2.84%;p= 0.8340)and Bacteroidia(76.25%±3.22%versus76.22%±3.46%;p=0.9943)between MIA offspring and controls(Fig-ure2E,left),indicating that alterations in the membership of Clostridial and Bacteroidial OTUs drive major changes in the gut microbiota between experimental groups.Overall,wefind that MIA leads to dysbiosis of the gut microbiota,driven primarily by alterations in specific OTUs of the bacterial classes Clostridia and Bacteroidia.Changes in OTUs classified as Lachnospiraceae and Ruminococcaceae of the order Clostridiales parallel select reports of increased Clos-tridium species in the feces of subjects with ASD(Finegold et al.,2012).Altogether,modeling MIA in mice induces not only behavioral and neuropathological features of ASD(Malkova et al.,2012;Shi et al.,2009)but also microbiome changes as described in subsets of ASD individuals.Bacteroides fragilis Improves Gut Barrier Integrity in MIA OffspringGut microbes play an important role in the development,mainte-nance and repair of the intestinal epithelium(Turner,2009).To determine whether targeting the gut microbiota could impact MIA-associated GI abnormalities,we treated mice with the human commensal B.fragilis at weaning,and tested for GI abnormalities at8weeks of age.B.fragilis has previously been shown to ameliorate experimental colitis(Mazmanian et al., 2008;Round and Mazmanian,2010).Remarkably,B.fragilis treatment corrects intestinal permeability in MIA offspring(Fig-ure3A).In addition,B.fragilis treatment ameliorates MIA-associ-ated changes in expression of CLDNs8and15,but not TJP1, TJP2,or OCLN(Figure3B).Similar changes are observed in pro-tein levels of CLDNs8and15in the colon,with restoration by B.fragilis treatment(Figures3C and3D).No effects of B.fragilis on tight junction expression are observed in the small intestine(Figure S1B),consistent with the fact that Bacteroides species are predominantly found in the colon.Finally,the pres-ence of GI defects prior to probiotic administration(Figure1A, right)suggests that B.fragilis may treat,rather than prevent, this pathology in MIA offspring.B.fragilis treatment also restores MIA-associated increases in colon IL-6mRNA and protein levels(Figures3E and3F).Levels of other cytokines are altered in both colons and small intestines of Cell155,1451–1463,December19,2013ª2013Elsevier Inc.1453MIA offspring (Figures 1D and S1C),but these are not affected by B.fragilis treatment,revealing specificity for IL-6.We further find that recombinant IL-6treatment can modulate colon levels of both CLDN 8and 15in vivo and in in vitro colon organ cultures (data not shown),suggesting that B.fragilis -mediated restora-tion of colonic IL-6levels could underlie its effects on gut permeability.Collectively,these findings demonstrate that B.fragilis treatment of MIA offspring improves defects in GIbarrier integrity and corrects alterations in tight junction and cytokine expression.B.fragilis Treatment Restores Specific Microbiota Changes in MIA OffspringThe finding that B.fragilis ameliorates GI defects in MIA offspring prompted us to examine its effects on the intestinal microbiota.No significant differences are observedfollowingFigure 2.MIA Offspring Exhibit Dysbiosis of the Intestinal Microbiota(A)Unweighted UniFrac-based 3D PCoA plot based on all OTUs from feces of adult saline+vehicle (S)and poly(I:C)+vehicle (P)offspring.(B)Unweighted UniFrac-based 3D PCoA plot based on subsampling of Clostridia and Bacteroidia OTUs (2003reads per sample).(C)Unweighted UniFrac-based 3D PCoA plot based on subsampling of OTUs remaining after subtraction of Clostridia and Bacteroidia OTUs (47reads per sample).(D)Relative abundance of unique OTUs of the gut microbiota (bottom,x axis)for individual biological replicates (right,y axis),where red hues denote increasing relative abundance of a unique OTU.(E)Mean relative abundance of OTUs classified at the class level for the most (left)and least (right)abundant taxa.n =10/group.Data were simultaneously collected and analyzed for poly(I:C)+B.fragilis treatment group (See Figure 4).See also Figure S2and Table S1.1454Cell 155,1451–1463,December 19,2013ª2013Elsevier Inc.B.fragilis treatment of MIA offspring by PCoA (ANOSIM R =0.0060p =0.4470;Table S3),in microbiota richness (PD:p =0.2980,Observed Species:p =0.5440)and evenness (Gini:p =0.6110,Simpson Evenness:p =0.5600;Figures 4A,S2A and S2B),or in relative abundance at the class level (Fig-ure S2C).However,evaluation of key OTUs that discriminate adult MIA offspring from controls reveals that B.fragilis treatment significantly alters levels of 35OTUs (Table S2).Specifically,MIA offspring treated with B.fragilis display significant restoration in the relative abundance of 6out of the 67OTUs found to discriminate MIA from control offspring (Figure 4B and Table S2),which are taxonomically assigned as unclassified Bacteroidia and Clostridia of the family Lachnospiraceae (Figure 4B and Table S2).Notably,these alterations occur in the absence of persistent colonization of B.fragilis ,which remains undetectable in fecal and cecal samples isolated from treated MIA offspring (Figures S2D and S2E).Phylogenetic reconstruction of the OTUs that are altered by MIA and restored by B.fragilis treatment reveals that the Bacteroidia OTUs cluster together into a monophyletic group and the Lachnospiraceae OTUs cluster into two monophyletic groups (Figure 4C).This result suggests that,although treatment of MIA offspring with B.fragilis may not lead to persistent colonization,this probiotic corrects the relative abundance of specific groups of related microbes of the Lachnospiraceae family as well as unclassified Bacteriodales.Altogether,we demonstrate that treatment of MIA offspring with B.fragilis ameliorates particular MIA-associ-ated alterations in the commensalmicrobiota.C LD N 8/T U B B (f o l d c h a n g e )F I T C i n t e n s i t y /m l s e r u m (f o l d c h a n g e )*****CLDN8: -tubulin: CLDN15:-tubulin:I L 6 m R N A /A C T B (f o l d c h a n g e )**CLDN8T J PT J P O C L C L D N C L D N 1m R N A /A C T B (f o l d c h a n g e )n.s.n.s.*****P P+BFS I L -I L -12p 40/p 7I P -1M I M I P -1p g /m g p r o t e i n (f o l dc h a ng e )C LD N 15/T U B B (f o l d c h a n g e )ABCDEFFigure 3.B.fragilis Treatment Corrects GI Deficits in MIA Offspring(A)Intestinal permeability assay,measuring FITC intensity in serum after oral gavage of FITC-dextran.Data are normalized to saline controls.Data for DSS,saline +vehicle (S)and poly(I:C)+vehicle (P)are as in Figure 1.poly(I:C)+B.fragilis (P+BF):n =9/group.(B)Colon expression of tight junction components relative to b -actin.Data for each gene are normalized saline controls.Data for S and P are as in Figure 1.Asterisks directly above bars indicate significance compared to saline control (normalized to 1,as denoted by the black line),whereas asterisks at the top of the graph denote statistical significance between P and P+BF groups.n =8/group.(C)Immunofluorescence staining for claudin 8.Representative images for n =5.(D)Colon protein levels of claudin 8(left)and claudin 15(right).Representative signals are depicted below.Data are normalized to signal intensity in saline controls.n =3/group.(E)Colon expression of IL-6relative to b -actin.Data are normalized to saline controls.Data for S and P are as in Figure 1.P+BF:n =3/group.(F)Colon protein levels of cytokines and chemokines relative to total protein content.Data are normalized to saline controls.Data for S and P are as in Figure 1.n =10/group.See also Figure S1.Cell 155,1451–1463,December 19,2013ª2013Elsevier Inc.1455B.fragilis Treatment Corrects ASD-Related Behavioral AbnormalitiesStudies suggest that GI issues can contribute to the develop-ment,persistence,and/or severity of symptoms seen in ASD and related neurodevelopmental disorders (Buie et al.,2010;Coury et al.,2012).To explore the potential impact of GI dysfunc-tion on core ASD behavioral abnormalities,we tested whetherB.fragilis treatment impacts anxiety-like,sensorimotor,repeti-tive,communicative,and social behavior in MIA offspring.We replicated previous findings that adult MIA offspring display several core behavioral features of ASD (Malkova et al.,2012).Open field exploration involves mapping an animal’s movement in an open arena to measure locomotion and anxiety (Bourin et al.,2007).MIA offspring display decreased entries and time0.2LachnospiraceaeBFigure 4.B.fragilis Treatment Alters the Intestinal Microbiota and Corrects Species-Level Abnormalities in MIA Offspring(A)Unweighted UniFrac-based 3D PCoA plot based on all OTUs.Data for saline (S)and poly(I:C)(P)are as in Figure 2.(B)Relative abundance of key OTUs of the family Lachnospiraceae (top)and order Bacteroidales (bottom)that are significantly altered by MIA and restored by B.fragilis treatment.(C)Phylogenetic tree based on nearest-neighbor analysis of 16S rRNA gene sequences for key OTUs presented in (B).Red clades indicate OTUs of the family Lachnospiraceae and green clades indicate OTUs of the order Bacteriodales.Purple taxa indicate OTUs that are significantly elevated in P and corrected by B.fragilis (BF)treatment.n =10/group.See also Figure S2and Table S2.1456Cell 155,1451–1463,December 19,2013ª2013Elsevier Inc.spent in the center of the arena,which is indicative of anxiety-like behavior (Figure 5A;compare saline [S]to poly(I:C)[P]).The prepulse inhibition (PPI)task measures the ability of an animal to inhibit its startle in response to an acoustic tone (‘‘pulse’’)when it is preceded by a lower-intensity stimulus (‘‘prepulse’’).Deficiencies in PPI are a measure of impaired sensorimotor gating and are observed in several neurodevelopmental disor-ders,including autism (Perry et al.,2007).MIA offspring exhibit decreased PPI in response to 5or 15db prepulses (Figure 5B).The marble burying test measures the propensity of mice to engage repetitively in a natural digging behavior that is not confounded by anxiety (Thomas et al.,2009).MIA offspring display increased stereotyped marble burying compared tocontrols (Figure 5C).Ultrasonic vocalizations are used to mea-sure communication by mice,wherein calls of varying types and motifs are produced in different social paradigms (Grimsley et al.,2011).MIA offspring exhibit deficits in communication,as indicated by reduced number and duration of ultrasonic vocali-zations produced in response to a social encounter (Figure 5D).Finally,the three-chamber social test is used to measure ASD-P P I (%)5 db 15 dbS P P+BF**S P P+BF 0255075D u r a t i o n p e r c a l l (m s )****T o t a l c a l l d u r a t i o n (s )****S P P+BF200400600T o t a l n u m b e r o f c a l l s**S P P+BF ****S PP+BF 010203040D i s t a n c e (m )S o c i a l p r e f e r e n c e :c h a m b e r d u r a t i o n (%)***S o c i a b i l i t y :c h a m b e r d u r a t i o n (%)*Anxiety and locomotion: Open field exploration Sensorimotor gating: Pre-pulse inhibitionStereotyped behavior: Marble burying Communication:Ultrasonic vocalizationsSocial Interaction:SociabilitySocial Interaction: Social preference BDEF Figure 5.B.fragilis Treatment Ameliorates Autism-Related Behavioral Abnormalities in MIA Offspring(A)Anxiety-like and locomotor behavior in the open field exploration assay.n =35–75/group.(B)Sensorimotor gating in the PPI assay.n =35–75/group.(C)Repetitive marble burying assay.n =16–45/group.(D)Ultrasonic vocalizations produced by adult male mice during social encounter.n =10/group.S =saline+vehicle,p =poly(I:C)+vehicle,P+BF =poly(I:C)+B.fragilis .Data were collected simul-taneously for poly(I:C)+B.fragilis D PSA and poly(I:C)+B.thetaiotaomicron treatment groups (See also Figures S3and S4).related impairments in social interaction (Silverman et al.,2010).MIA offspringexhibit deficits in both sociability,or pref-erence to interact with a novel mouseover a novel object,and social preference (social novelty),or preference to interact with an unfamiliar versus a familiar mouse(Figures 5E and 5F).Altogether,MIA offspring demonstrate a number of be-havioral abnormalities associated with ASD as well as others such as anxiety and deficient sensorimotor gating.Remarkably,oral treatment with B.fragilis ameliorates many of these be-haviors.B.fragilis -treated MIA offspring do not exhibit anxiety-like behavior in theopen field (Figure 5A;compare poly(I:C)[P]to poly(I:C)+B.fragilis [P+BF]),as shown by restoration in the number of center entries and duration of time spent in the center of the arena.B.fragilis im-proves sensorimotor gating in MIA offspring,as indicated by increased combined PPI in response to 5and 15db prepulses (Figure 5B),with no significant effect on the intensity of startle to the acoustic stimulus (data not shown).B.fragilis -treated mice also exhibit decreased levels of stereotyped marble burying and restored communicative behavior (Figures 5C and 5D).Interestingly,B.fragilis raises the duration per call by MIA offspring to levels exceeding those observed in saline controls (Figure 5D),suggesting that despite normalization of the pro-pensity to communicate (number of calls),there is a qualitative difference in the types of calls generated with enrichment of longer syllables.Although B.fragilis -treated MIA offspring exhibit improved communicative,repetitive,anxiety-like,and sensorimotor behavior,they retain deficits in sociability and social preference (Figure 5E).Selective amelioration of ASD-related behaviors is also seen with risperidone treatment of CNTNAP2knockoutmice,a genetic mouse model for ASD (Pen˜agarikano et al.,2011),wherein communicative and repetitive,but not social,behavior is corrected.These data suggest that there may beCell 155,1451–1463,December 19,2013ª2013Elsevier Inc.1457。

2008年诺贝尔生理学或医学奖揭晓作者：佚名来源：生物秀时间：2008-10-8北京时间10月6日下午5点30分，2008年诺贝尔生理学或医学奖揭晓，德法三科学家分享该奖项。

德国癌症研究中心的科学家Harald zur Hausen 因发现人类乳突淋瘤病毒（HPV）导致子宫颈癌而获奖；法国两位科学家，巴斯德研究所病毒学系逆转录病毒感染调控小组的FrançoiseBarré-Sinoussi 和巴黎世界艾滋病研究与预防基金会的Luc Montagnier 因发现人类免疫缺陷病毒（HIV）而获奖。

今年的诺贝尔生理学或医学奖奖励的是导致严重人类疾病的两类病毒的发现。

Harald zur Hausen一反传统教条，假定瘤原性人类乳突淋瘤病毒（HPV）导致了宫颈癌——女性第二常见的癌症。

他认识到，HPV-DNA能够存在于肿瘤非生长性状态中，而且应该能够通过对病毒DNA的特定搜寻发现。

他发现，HPV是病毒的一种异质家族。

只有一些HPV种类会导致癌症。

他的发现引发了对HPV感染自然史的描述，以及对于HPV诱导致癌作用机制和抗HPV获得性预防疫苗发展的理解。

Françoise Barré-Sinoussi 和Luc Montagnier发现了人类免疫缺陷病毒（HIV）。

病毒产生分别在淋巴（来自在获得性免疫缺陷早期阶段带有增大淋巴结病人）和血液（来自晚期患者）中得到鉴别。

根据这一病毒的形态、生化及免疫学特性，他们将它确定为第一个已知的人类慢病毒。

HIV损伤免疫系统，因为病毒大量复制，对淋巴细胞造成损伤。

这一发现对于当前的艾滋病生物学及它的抗逆转录病毒治疗来说是一个先决条件。

德国科学家Harald zur Hausen 法国科学家Françoise Barré-Sinoussi法国科学家Luc Montagnier发现人类乳突淋瘤病毒导致宫颈癌与20世纪70年代流行的观点不同，Harald zur Hausen假定人类乳突淋瘤病毒在宫颈癌中扮演了一个角色。

Colloids and Surfaces B:Biointerfaces 65(2008)239–246Contents lists available at ScienceDirectColloids and Surfaces B:Biointerfacesj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c o l s u r fbPhysico-chemical properties and cytotoxicity assessment of PEG-modified liposomes containing human hemoglobinVal ´erie Centis a ,Patrick Vermette a ,b ,∗aLaboratoire de Bioing´e nierie et de Biophysique de l’Universit´e de Sherbrooke,Department of Chemical Engineering,Universit´e de Sherbrooke,2500,boul.de l’Universit´e ,Sherbrooke (QC),Canada J1K 2R1bResearch Centre on Aging,Institut universitaire de g´e riatrie de Sherbrooke,1036,rue Belv´e d`e re Sud,Sherbrooke (QC),Canada J1H 4C4a r t i c l e i n f o Article history:Received 5December 2007Received in revised form 14April 2008Accepted 15April 2008Available online 24April 2008Keywords:PEGylated liposomes encapsulating hemoglobin Oxygen carriers Cytotoxicity HUVECCharacterizationTransmission electron microscopy Zeta potentialParticle size distributiona b s t r a c tPEGylated liposomes encapsulating human hemoglobin as oxygen carriers were prepared frompurified carbonylhemoglobin (HbCO)solution and a lipid mixture composed of 1,2-dipalmitoyl-sn -glycero-3-phosphatidylcholine (DPPC),cholesterol,1,2-dimyristoyl-sn -glycero-3-phosphoethanolamine-N -[poly(ethylene glycol)2000](DMPE-PEG 2000)and palmitic acid.Hemoglobin was extracted and purified from human blood samples.SDS-PAGE was used to assess its purity.Diameter of liposomes containing hemoglobin was controlled to approximately 200nm using extrusion as measured by dynamic light scattering and transmission electron microscopy.Liposome size distributions were shown to remain unimodal over 14days,even at different storage temperatures.Zeta potential measurements revealed that liposome containing hemoglobin have a net surface charge of −7.16±0.33mV.Also,hemoglobin encapsulated in liposomes was able to perform several cycles of oxygen loading and unloading using oxygen (O 2)and carbon monoxide (CO).The hemoglobin vesicle dispersion showed some toxicity as revealed by three in vitro assays in which endothelial cell (HUVECs)monolayers were exposed to these dispersions.Cytotoxicity was function of the liposome concentration in the culture medium.©2008Elsevier B.V.All rights reserved.1.IntroductionSince Rabiner et al.in 1967and Savitsky et al.in 1978[1,2]stud-ied stroma-free hemoglobin solution as a possible substitute for human blood,research on the development of universal,safe and effective oxygen carriers has been an ongoing subject.Such a solu-tion,despite its purpose to be used in blood replacement therapy,could also be used to supply oxygen in high-density cell culture sys-tems,where oxygen is often the limiting factor.This is particularly flagrant in tissue engineering applications [3–7].Many perfluorocarbon emulsions and hemoglobin-based prepa-rations have been considered as oxygen carriers,with their loads of advantages and disadvantages.For a complete review on the dif-ferent types of oxygen carriers,their design and characterization,the reader is referred to Riess [8].Liposomes and biodegradable polymer capsules have been examined as confinement vehicles for tetrameric hemoglobin [9–12].Liposomes are double-layered phospholipid vesicles able∗Corresponding author.Tel.:+18198218000x62826;fax:+18198217955.E-mail address:Patrick.Vermette@USherbrooke.ca (P.Vermette).to encapsulate different drugs or molecules [13].They have been thoroughly studied for the past 40years and the reader is directed to the work of Lasic and colleagues for more information [13–15].Surface modification with PEG was reported to improve the cir-culation of liposomes in circulation [8,16].PEG is a biologically inert polymer extensively used in drug delivery to create,once conjugated,a steric barrier and/or a water structure around lipo-somes,with the aim to protect liposomes from plasma proteins in the body [17,18].The steric barrier created by PEG conjugation also prevents liposome fusion and aggregation and thus,stabilizes the dispersions during storage [17,18].It was shown by Sakai et al.[17]that PEGylated liposomes encapsulating hemoglobin stored in a deoxygenated state at 4◦C and 23◦C were stable for 1year,while dispersions stored at 40◦C were stable for 6months before liposome aggregation and hemoglobin leakage were observed [17].To be used in cell cultures,liposome dispersions should have limited cell toxicity and their formulations should be stable throughout the duration of the culture.Also,the liposome prepa-ration should be able to increase the oxygen concentration in the cultures and therefore,have a positive impact on the cell growth.Cytotoxicity of PEGylated liposomes encapsulating hemoglobin was assayed in different studies.For example,hemoglobin vesicles0927-7765/$–see front matter ©2008Elsevier B.V.All rights reserved.doi:10.1016/j.colsurfb.2008.04.009240V.Centis,P.Vermette/Colloids and Surfaces B:Biointerfaces65(2008)239–246were tested on human cord blood hematopoietic progenitor cells [19].Mild toxicity was observed on short-term exposures,while inhibition of proliferation was monitored at longer times[19].Also, the effect of liposome encapsulating hemoglobin(LEH)on human platelet function in plasma was studied.Wakamoto et al.observed neither aggregation,activation,nor adverse effects when human platelets were exposed to liposome dispersions[20].Similarly,the exposure of rat erythrocytes to PEG-LEH did not affect the struc-tural integrity of the blood structures[21].No signs of aggregation and deformation of the erythrocytes were observed,suggesting that liposomes exhibited low,if any toxicity[21].Another study examined the cytokine-induced adhesiveness of monocytic cells to HUVECs[22].The data suggested that,in this specific model,LEH did not induce leukocyte adhesion and could have a beneficial effect in preventing leukocyte adhesion to vascular endothelium caused by inflammatory cytokines[22].To our knowledge,in vitro long-term cytotoxicity of LEH formulations towards human cells have not been fully addressed and such testing is afirst and manda-tory step to investigate the applicability of these LEH in tissue engineering.Therefore,this work examines the preparation and characteri-zation of liposomes encapsulating human hemoglobin as potential oxygen carriers for tissue engineering purposes and presents a char-acterization of the LEH dispersions.To assess the feasibility of using LEH as oxygen transporters,size distribution,zeta potential,mor-phology and shape,and cytotoxicity of these LEH dispersions were examined.To our knowledge,no similar study has been reported.2.Materials and methods2.1.Materials1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine(DPPC,cat. #PCS-020)and1,2-dimyristoyl-sn-glycero-3-phosphoethanol-amine-N-[poly(ethylene glycol)2000](DMPE-PEG2000,cat.#PPE-010)were purchased from Northern Lipids(Vancouver,BC, Canada).Cholesterol(Chol,cat.#110525)was obtained from Avanti Polar Lipids(Alabaster,AL,USA).Palmitic acid(PA,cat.#P-0500),Medium199(M-199,cat.M-5017),Heparin(cat.#H-1027), Hank’s balances salt solution(HBSS,cat.#H-6648),fetal bovine serum(FBS,cat.#F-1051),endothelial cell growth supplement (ECGS,cat.#E-2759),pyridoxal5 -phosphate(PLP,cat.#P-3657) and dl-homocysteine(Hcy,cat.#H-4628)were purchased from Sigma–Aldrich(Oakville,ON,Canada).Chloroform,sodium dithionite,and dichloromethane used for hemoglobin purification and liposome preparation were of analyt-ical grade and were obtained from Fisher Chemicals.All the water used in these experiments was taken from a Milli-Q Gradient(Milli-pore,Billerca,MA,USA)ultra-pure deionization water system with a resistivity of18.2M cm.CellTiter96®AQueous One Solution Cell Proliferation Assay Kit(cat.#G3580)and CytoTox96®Non-Radioactive Cytotoxicity Assay(Lactate deshydrogenase,cat.#G1780)were obtained from Promega(Madison,WI,USA).CyQuant®NF Cell Proliferation Assay Kit(cat.#C35006)was purchased from Invitrogen(Burlington,ON, Canada).2.2.Hemoglobin purificationHemoglobin was obtained from human whole blood of healthy volunteers.All procedures were approved by the Ethics Committee of the Research Centre on Aging(Sherbrooke,Qc).After the subjects were thoroughly informed about the nature and goal of the study, they provided written consent.The protein was purified accord-ing to an adapted method previously published[23].In summary: (1)hemoglobin was stabilized by CO complexion(HbCO)(60min at room temperature);(2)the removal of membranes and stroma was done by solvent treatment(20%(v/v)CH2Cl2,shaken for3min with subsequent centrifugation at1900×g for15min);(3)traces of dichloromethane were removed by slowly heating the sample with aflow of inert gas(Ar)(heating from40◦C to60◦C,120min);(4)virus inactivation was done by heating the hemoglobin solu-tion(60◦C,overnight,with magnetic stirring agitation);(5)dialysis (MW cutoff of25kDa,against ultra-pure water,for24h,with mag-netic stirring agitation)was used to remove small molecules;(6) ultra-filtration(MW cutoff of30kDa,Millipore,USA)was used to filter out molecules with MW<30kDa.Solutions containing puri-fied HbCO were aliquoted and preserved in liquid nitrogen with PLP(18mM)and Hcy(15mM).SDS-PAGE was used to assess the presence of hemoglobin.2.2.1.SDS-PAGEThe purification of Hb was determined via SDS-PAGE using a FisherBiotech system(Fisher Scientific,cat.#FB200).All the sam-ples were prepared in a Laemmli buffer(Bio-rad,cat.#161–0158) with a4%acrylamide stacking gel and a15%acrylamide resolving gel(Bio-rad,cat.#161–0158).Human hemoglobin is known to have a MW of approximately 64kDa[8,24].The hemoglobin samples were run along with a pre-stained broad-range molecular weight marker(Bio-rad,cat. #161–0318),which consisted of proteins having molecular weights ranging between6.9and210kDa.10␮L of hemoglobin samples (2␮g/mL)and7␮L of the molecular weight marker(dilution ratio of1:75(v/v)i.e.,1part of the marker in75parts of a buffer)were deposited in each well.A purified commercial hemoglobin sam-ple(Sigma,cat.#H7379)was used as a control and was run at a concentration of0.1␮g/mL.All samples were run at115V until the migration front reached the bottom of the gel(±120min).The gels were then stained using Silver SNAP Stain kit II(Fisher Scientific, cat.#24612)to determine band intensities.2.3.Liposome preparationLiposomes were prepared in a slightly different way than the method previously reported[23].Briefly,the mixture of phos-pholipids DPPC,Chol,PA and DMPE-PEG2000in a molar ratio of 5/5/1/(0.3%[mole/total moles of lipids])was dissolved in chloro-form.The organic solvent was then evaporated using a rotary evaporator(B¨uchi,Switzerland)under reduced pressure to remove all traces of solvent.The resulting thinfilm was then hydrated with a NaOH solution(0.9mM)overnight to allow thefilm to dissolve to yield a lipid concentration of50mg/mL.The solution was then diluted with pure water to obtain a concentration of25mg/mL of lipids.Then,ten cycles of freeze-thawing(−196◦C and40◦C)were performed.The solution was then frozen for10min in liquid nitro-gen and lyophilized for48h.After dispersing the lipid mixture into a hemoglobin solution(4g/mL)for2h,the resulting multilamel-lar vesicles were extruded with the LiposoFast-Basic(Avestin Inc, Ottawa,ON,Canada)using11passes at54◦C through polycarbon-ate membranes with pore sizes of200nm.The resulting solution was then ultra-centrifuged at50,000×g for30min(Optima TLX, Beckman Coulter,Mississauga,On)to recover and eliminate the top layer containing non-encapsulated Hb.2.4.Liposome characterization2.4.1.Particle size distributionThe intensity mean diameter of the liposomes and the polydis-persity index(PI)of the distribution were determined by dynamicV.Centis,P.Vermette/Colloids and Surfaces B:Biointerfaces65(2008)239–246241light scattering(DLS)using a Zetasizer Nanoseries(Malvern Instru-ments,UK).Samples of liposomes stored at4◦C,22◦C and37◦C were analyzed.The refractive index and viscosity of pure water were used as calculation parameters and each sample was mea-sured3times for25runs using the unimodal model for size distribution.All samples were diluted to an appropriate counting rate prior to analysis.2.4.2.Zeta potentialThe zeta potential was measured by electrophoresis at25◦C (Zetasizer Nanoseries,Malvern Instruments,UK).The surface charge of the particles was obtained by measuring the velocity of the dispersion in an electricalfield.Samples were diluted and placed in an electrophoretic cell where a potential of±60mV was established.Actual values were calculated from the mean elec-trophoretic mobility using Smoluchowski’s equation.The viscosity and dielectric constant of pure water were used as calculation parameters and each sample was measured in quadruplicate.All samples were diluted to an appropriate counting rate prior to anal-ysis.2.4.3.Influence of pH and osmolaritySlight changes in pH and osmolarity can have deleterious effects on cells in culture.To ensure that seeded cells would not respond to a change in pH and osmolarity due to the addition of liposomes in culture media,these parameters were moni-tored over24h(6,12,and24h).HUVECs were seeded onto 24-well plates(6000cells/well)(Corning,cat.#3527)and let to adhere in normal incubator conditions.The next day,HUVECs were either exposed to:(1)LEH(2mg/mL),(2)“Empty”lipo-somes i.e.,liposomes with no hemoglobin(2mg/mL),or(3)M-199 media.After6,12and24h,250␮L were pipetted and used for osmolarity analyses using a pre-calibrated osmometer(Advanced Instruments,model3250,Norwood,MS,USA).The rest of the solution was used for pH monitoring(Orion Research,model 710A,Lausanne,Switzerland).Measurements were done in triplicate.2.5.Functionality of the LEH as oxygen carriersHemoglobin possesses characteristic bands throughout the vis-ible spectra.Q-band(around500–600nm)and Soret band(in the blue region,around400nm)are typical regions that provide infor-mation about the hemoglobin state.Therefore,functionality of hemoglobin entrapped in liposomes and its ability to shift from its oxygen-loaded position to unloaded position was assayed by spectrometry.LEH dispersions(2mg/mL,using HBSS buffer)were exposed to pure oxygen and halogen light(90min,400W)[25]and sub-sequently compared to carbon monoxide(CO)exposure(15min, in the dark).To obtain deoxygenated spectra of hemoglobin,solu-tions were equilibrated with inert gas(Ar)to remove most of the oxygen before treatment with dithionite[26].Wavelengths shifts of the Q-band and Soret band under these conditions were mea-sured using a micro-plate reader(Bio-Tek Instruments,Winooski, VT,USA).2.6.Transmission electron microscopy(TEM)analysesThe samples were diluted appropriately and were negatively stained with uranyl acetate and allowed to air-dry directly on TEM grids.Observations were made using an H-7500transmission elec-tron microscope(HITASHI,Pleasanton,CA,USA)at a voltage of 60kV.2.7.HUVEC cultureHuman Umbilical Vein Endothelial Cells(HUVEC,PromoCell, Heidelberg,Germany)were cultured in M-199supplemented with10%(v/v)de-complemented FBS.ECGS(20␮g/mL),heparin (90mg/L),and antibiotics(100U/mL penicillin G and100␮g/mL streptomycin)were also added.Culture media was replenished three times a week.All cells were maintained at37◦C in an incu-bator with a humidified atmosphere containing5%CO2.Cells of passages4and5were used throughout this study.2.8.Cytotoxicity of LEHFollowing cell exposure to LEH,cell proliferation assays were performed to evaluate the cytotoxicity of the LEH formulations.For this purpose,HUVECs were incubated with three concentrations of LEH dispersions(5,2and1mg/mL)for6,12,and24h.The M-199culture medium concentration was adjusted to compensate for the dilution caused by the addition of liposomes.Empty liposomes (5mg/mL)were used as controls.Three assays were performed to evaluate the cytotoxicity of the LEH dispersions.Different assays were used since it was reported that cellular metabolic processes vary greatly over time and that methodologies that rely only on measurements of ATP content can cause some problems in further cytotoxicity interpretation[27].LEH cytotoxicity wasfirst examined using the MTS assay,a colorimetric method optimized for adherent cells.Viable cells enzymatically reduce the colorless tetrazolium salt MTS to inten-sively colored MTS-formazan.Briefly,4000cells/well were seeded inflat bottom96-well plates(Corning Incorporated,Corning,NY, USA)and incubated for24h.After removing the culture medium, 100␮L of LEH dispersions were applied,and the plates were incu-bated at37◦C for a period of4h.The absorbance was read at490nm on a micro-plate reader(Bio-Tek Instruments,Winooski,VT,USA). Values were corrected for background absorbance.Secondly,cell number was assessed using the CyQuant®Cell Proliferation Assay Kit,a highly sensitive,fluorescence-based micro-plate assay[27].The CyQuant®assay measures the ability of CyQuant®dye to bind to the cellular nucleic acids of viable cells. Measurements of cellular proliferation provide a general measure of toxicity.Cell cultures were seeded at103cells/well in duplicate inflat bottom96-well plates.After exposure of the liposomes to adherent cells,cells were rinsed with HBSS buffer to remove dead cells no longer adhering to the plate,lysed,and the DNA was stained using the CyQuant®fluorescent dye solution as recommended by the manufacturer.Plates were incubated at37◦C for60min.Flu-orescence was measured using afluorescence micro-plate reader (Bio-Tek Instruments,Winooski,VT,USA).The excitation maximum was480nm and the emission maximum was530nm.Values were corrected for background absorbance.Finally,quantification of the release of the cytoplasmic enzyme, lactate deshydrogenase(LDH)in culture media was evaluated.LDH liberation is correlated with the number of lysed cells.Briefly,4000 cells/well were seeded inflat bottom96-well plates(Corning Incor-porated,Corning,NY,USA)and incubated for24h.After HUVEC exposure to the different concentrations of LEH,cells were lysed using a lysis buffer,as described by the manufacturer.LDH activ-ities in the culture media and in the corresponding cell lysates were measured at490nm on a micro-plate reader(Bio-Tek Instru-ments,Winooski,VT,USA).Values were corrected for background absorbance.2.8.1.Assessment of potential residual CH2Cl2cytotoxicityTo ensure that the dichloromethane used in the hemoglobin purification process was not responsible for any cell death,a sample242V.Centis,P.Vermette/Colloids and Surfaces B:Biointerfaces65(2008)239–246 of M-199culture media was treated in the same way.In a con-trol experiment,M-199was exposed to CH2Cl2with subsequentcentrifugation and evaporation procedures(steps2–3,Section2.2).Then,10%(v/v)of de-complemented FBS was added to the media.This“CH2Cl2treated”M-199sample was then added inflat bot-tom96-well plates seeded with4000cells/well.Cells were thenincubated for24h with this M-199and a standard MTS assay wasperformed after the exposure.Normal M-199media was used ascomparison.The absorbance was read at490nm on a micro-platereader(Bio-Tek Instruments,Winooski,VT,USA).Values were onceagain corrected for background absorbance.Results were done intriplicate.2.9.Statistical analysisAll the data collected throughout the study were expressed asmeans±standard deviations(S.D.s).Analysis of variance(ANOVA)was used to determine if data were significantly different usingp≤0.05.3.Results and discussion3.1.Hemoglobin purification3.1.1.SDS-PAGEFig.1shows SDS-PAGE results for the Hb purification proceduredescribed above.In this study,the SDS-PAGE technique was usedas a qualitative method to show the presence of hemoglobin in thesample.Proteins of interest were well defined on the ne Bshows the molecular weight marker containing proteins rangingfrom6.9to210kDa while lanes A and C show purified samples ofhuman Hb and a commercial hemoglobin standard,respectively.It is possible to observe the tetramer,dimer and monomer com-posing hemoglobin at values of,respectively,64,32and16kDa(black arrows on left side,respectively noted1,2and3).The sub-units are visible since the sample was heated prior to its run,thusdenaturing the protein.When purified Hb was compared with thecommercial sample,it was possible to observe the same bandsexcept for one(under the last arrow),which was present only ontheFig.1.SDS-PAGE nes A and C show a purified human hemoglobin sample and a commercial hemoglobin ne B presents broad-range molecular weight markers.Black arrows on left side(1,2,and3)show,respectively,the64,32 and16kDa subunits of the protein.purified sample.It was published by Pearson et al.that this band is not apparent on freshly prepared specimens[28],as opposed to outdated blood,as in our case.Also,the observed streaking in the obtained SDS-PAGE analyses can be caused by the presence of other proteins.Therefore,the effect of the presence of these other proteins on the functionality of Hb has been tested(see below).3.2.Hemoglobin functionalitySpectra of hemoglobin encapsulated in liposomes are presented in Fig.2.Fig.2A clearly shows the difference between oxygen-loaded and unloaded states of the hemoglobin.This difference is even more evident in Fig.2B,showing the characteristic Q-bands and the shift from538and569nm to540and577nm.Panel C of Fig.2summarizes the shifts of hemoglobin from oxygen-loaded states to unloaded ones.Also,Fig.2C shows that hemoglobin can be oxygen-loaded and unloaded several times.The ability of the pro-tein to shift back and forth from one position to another(in thiscase,Fig.2.(A)Complete visible spectra of oxygen-loaded and unloaded purified human hemoglobin,(B)zoom on Q-bands showing the shift between oxygen-loaded and unloaded positions of purified human hemoglobin,and(C)variation of Q-band wavelengths after exposure to carbon monoxide and oxygen.V.Centis,P.Vermette /Colloids and Surfaces B:Biointerfaces 65(2008)239–246243Table 1Mean particle diameter of empty liposomes and liposomes encapsulating hemoglobin stored at 4◦C,22◦C and 37◦C over 2weeks4◦C22◦C37◦CEmpty liposomesLiposomes encapsulating hemoglobin Empty liposomes Liposomes encapsulating hemoglobin Empty liposomes Liposomes encapsulating hemoglobin Day 1220(39.6)226(48.5)240(45.6)269(47.2)229(48.1)239(53.3)Day 7229(41.8)237(47.7)245(46.2)252(45.1)249(44.2)255(42.7)Day 14240(40.7)239(57.6)249(45.9)247(47.8)248(45.6)254(50.1)Data in parentheses indicate the %Polydispersity (%Pd).from oxygen to carbon monoxide)demonstrates the functionality of hemoglobin even after its encapsulation into liposomes.3.3.Liposome characterization3.3.1.Particle size distributionResults of size distribution of liposomes encapsulating hemoglobin are presented in Table 1.It is evident from the data that the size distributions were unimodal and relatively narrow for all samples.%Polydispersity (%Pd)values of all LEH disper-sions were higher than 40%(Table 1).Therefore,LEH dispersions can be considered to as polydispersed.The term polydispersity is derived from the polydispersity index (PdI).PdI is a number cal-culated from a simple two-parameter fit to the correlation data called a cumulants analysis.PdI is known as the relative variance,while %Pd is the coefficient of variation or relative polydisper-sity and can be expressed as (PdI)1/2×100(Malvern Instruments,UK).As a rule of thumb,samples with %Pd ≤20%are considered monodisperse.The size of a particle is an important factor in determining its use as an oxygen carrier.Size is of great importance when one wishes to use these particle-systems in a body to supply increased (or opti-mized)oxygen concentration.Rapid extravasion from the body by the reticulo-endothelial system (RES)and immunogenic effects are only a few of the problems foreign particles encounter in the body [8,19,20,29–34].For tissue engineering purposes,particle size is also an important issue to consider.Liposomes added to a culture medium should be able,for example,to circulate through porous scaffolds often used in tissue engineering.Also,as for LEH,their small size (±200nm)should enable to increase the surface area between the liposomes and the dispersing medium phase allowing a better oxygen transfer.The results in Table 1show that LEH sizes are within the expected range of the 200-nm pores of the extrusion membrane used.These results are comparable and consistent with the findings of Arifin and Palmer [35,36].Using asymmetric field flow fraction-ation coupled to a multi-angle static light scattering photometer,they found that liposomes extruded through membranes having 200-nm pore diameter exhibited a mean diameter that was close to that of the membrane.The dispersion observed was unimodal with distribution widths of 10–20nm.Mean LEH diameter does not vary significantly with storage tem-perature (p ≤0.01).Still,LEH samples should be kept at 4◦C prior to their use in order to limit possible particle size enlargement.Stor-age time of 14days affected to some extent particle size growth in the samples (p ≥0.05).On the other hand,all the preparations exhibited a unimodal distribution,allowing to conclude,that over a short period of time (≤7days),the particles should not aggregate or fuse (p ≤0.05).PEG grafted onto the surface of the vesicles cer-tainly help to stabilize LEH by creating a steric hindrance around liposomes [18].The molar ratio of PEG used in our study is fairly small compared to those of other studies in which PEG was used.But this PEG concentration was sufficient to stabilize the liposomes over 14days,a time corresponding to a culture period required to grow a network of vascular micro-vessels,for instance.The data presented in Table 1reveal that the encapsulation of hemoglobin molecules does not seem to affect the size distribution of the liposomes.Liposomes encapsulating hemoglobin exhibit the same size distribution behaviour as “empty”ones [37].3.3.2.Zeta potentialMeasurements of zeta potential can yield information about the colloidal stability of particle dispersions [36–39].On one hand,in a solution containing no proteins,no amino acids,and little to no multi-valent electrolytes,the larger the magnitude of the zeta potential the more stable the dispersion should be.Limited floccu-lation occurs between |5|and |15|mV [37].For “naked”liposomes (prepared in the same manner as LEH,but hydrated with M-199culture media instead of hemoglobin),it was shown that the zeta potential at the surface of liposomes was approximately −45mV [12,40].When PEG 2000was added to the liposome preparation,the zeta potential increased to reach a plateau around −5mV [12].ThisFig.3.TEM images revealing shape,structure,and sizes of liposomes encapsulating hemoglobin molecules.244V.Centis,P.Vermette/Colloids and Surfaces B:Biointerfaces65(2008)239–246increase of the surface charge was thought in part to be because of the drag caused by the presence of PEG chains on the liposome sur-face,decreasing the mobility of the liposomes,hence affecting the zeta potential[12,41].On the other hand,the presence of surface charge should be counter-balanced with a good steric hindrance in solutions containing proteins and amino acids(e.g.,culture media), as these often charged molecules can adsorb on the surface of the particles and enhance particle aggregation.For the PEGylated liposomes encapsulating Hb studied here,the mean zeta potential on the particle surface was−7.16±0.33mV. This indicates that limited aggregation of the particles should be observed and that stability over time can be,to some extent,con-trolled,as observed in Table1.In fact,over14days,the particle size did not vary for a given temperature and confirmed the stability of the distribution.The values obtained can also be compared with results obtained by Sakai et al.[37].They reported LEH,without PEG and PA,with a surface charge of−21mV.PA was reported to decrease surface charge proportionally to its increase in concentra-tion in the lipid bilayer[37,42].3.3.3.pH and osmolarity of culture media with LEHpH and osmolarity of culture media containing either“empty”liposomes or LEH were monitored over24h.All the solutions had a constant pH of7.4.As for osmolarity measurements,for both“empty”liposomes and M-199controls,the values remained constant at approximately330±3mOsm.The osmolarity of LEH dispersions was319±2mOsm.From these data,it can be concluded that the addition of dispersions of“empty”liposomes and LEH does not affect the osmolarity of the culture medium used in this study.3.4.Transmission electron microscopy(TEM)analysesTEM images of LEH are presented in Fig.3.In the TEM images, the characteristic aspects of large unilamellar vesicles(LUV)can be observed[14].Large unilamellar vesicles are liposomal structures exhibiting a rather empty core that can be used to encapsulate different molecules,such as drugs or proteins[15].LUV’s can be prepared from large“onion-like”structures of multilamellar vesi-cles(MLV)by different methods such as extrusion.The TEM images clearly show that the extrusion method was convenient to produce LEHs.The distinctively recognizable double-layered membrane and the empty core are good indications of a LUV structure[13,14].TEM images revealed that LEH(over62individual liposomes)have a mean diameter of170±50nm.These results are in good agreement with the ones obtained from dynamic light scattering analyses. 3.5.Cytotoxicity of liposomes encapsulating hemoglobin(LEH)Fig.4shows the behaviour of HUVECs in contact with disper-sions made of different concentrations of LEH.Similar cytotoxicity trends were observed when the same experiments were conducted using humanfibroblasts from foreskin(data not shown).Fig.4A and B illustrate the percentage of live cells when HUVECs were exposed to LEH dispersions,while Fig.4C shows the percent-age of cell death after similar exposure.Statistical analyses of the results of these three cytotoxicity assays reveal that exposure time had a significant effect on cell viability(p=0.05).The analyses also show that LEH concentration had an effect on the cell death rate.For example,a significant difference(p≤0.05)was observed between 5and1mg/mL for all tested exposure times.From Fig.4,it can be seen that approximately50%cell loss occurred following24-h cell exposure to the LEH dispersions.The results obtained from the three assays are consistent with one another(p≤0.05).Unfor-tunately,these results cannot be directly correlated with thoseof Fig.4.Cytotoxicity assays of HUVECs exposed to PEGylated liposomes containing hemoglobin for different exposure times.(A)AQueous One assay,(B)CyQuant®assay,and(C)lactate deshydrogenase assay.All data are significantly different with p≤0.05.other studies since very few long-term in vitro tests have been car-ried out.A recent study by Yamaguchi et al.[19]investigated the short and long-term effects of hemoglobin vesicles(HbVs)on the clono-genic and proliferative activity of human hematopoietic progenitor cells derived from umbilical cord blood.They found that,at a concentration of3%(v/v),continuous exposure of HbV signif-icantly decreased the number and size of mature-committed colonies[19].Moreover,HbVs also notably reduced the number of high-proliferative potential colony-forming cells and lead to the suppression of cellular proliferation and differentiation in liquid culture[19].On the other hand,the same study presents the effect of HbV exposure to cord blood for20h or3days.Yamaguchi et。

2008年8月份欧洲批准新产品获准产品
李晓明（摘）
【期刊名称】《国外药讯》
【年(卷),期】2008(000)010
【摘要】欧盟的EMEA已批准Tibotec／Johnson＆Johnson公司的非核苷逆转录酶抑制剂（NNRTI）Intelence（etravirine）与包括darunavir／利托那韦（ritonavir）在内的最佳基础治疗联用。

【总页数】2页(P6-7)
【作者】李晓明（摘）
【作者单位】无
【正文语种】中文
【中图分类】R978.7
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ORIGINAL PAPERPurification and characterization of a thermostable uricase from Microbacterium sp.strain ZZJ4-1Lei Kai ÆXiao-Hang Ma ÆXue-Lai Zhou ÆXiao-Ming Jia ÆXia Li ÆKang-Ping GuoReceived:10April 2007/Accepted:30June 2007/Published online:25July 2007ÓSpringer Science+Business Media B.V.2007Abstract In order to study the properties of a thermo-stable uricase produced by Microbacterium sp.strain ZZJ4-1,the enzyme was purified by ammonium sulfate precipitation and DEAE-cellulose ion exchange,hydro-phobic and molecular sieve chromatography.The molec-ular mass of the purified enzyme was estimated to be 34kDa by SDS-PAGE.The enzyme was stable between pH 7.0and 10.00.The optimal reaction temperature of the enzyme was 30°C at pH 8.5.The K m and K cat of the enzyme were 0.31mM and 3.01s –1,respectively.Fe 3+could enhance the enzyme activity,whereas Ag +,Hg 2+,o -phenanthroline and SDS inhibited the activity of the enzyme considerably.After purification,the enzyme was purified 19.7-fold with 31%yield.As compared with uri-cases from other microbial sources,the purified enzyme showed excellent thermostability and other unique char-acteristics.The results of this work showed that strains of Microbacterium could be candidates for the production of a thermostable uricase,which has the potential clinical application in measurement of uric acid.Keywords Uricase ÁThermostability ÁMicrobacterium .ÁPurification ÁCharacterization ÁUric acidIntroductionUricases (urate oxidoreductase;EC 1.7.3.3),which catalyse the oxidative breakdown of uric acid,belong to a group ofenzymes in the purine degradation pathway found in ani-mals (Keilin 1959;Wallrath and Friedman 1991),plants (Montalbini et al.1997),fungi (Montalbini et al.1999),yeasts (Adamek et al.1990;Hongoh et al.2000;Koyama et al.1996)and bacteria (Yamamoto et al.1996).Since Bongaert et al.(Bongaert et al.1978)found that Bacillus fastidiosus could produce uricase and use uric acid as the only carbon source for growth in 1978,many reports on bacterial and yeast uricases,such as those from Arthrob-acter globiformis (Nobutoshi et al.2000),Bacillus subtilis (Hunag and Wu 2004),Candida utilis (Liu et al.1994)and Pseudomonas aeruginosa (Ishikawa et al.2004),have been published.In the human body,uric acid is a final product of purine catabolism and is excreted out of the body by the kidney.When the level of uric acid in blood increases over the normal value,it can lead to a group of diseases,such as gout (Nakagawa et al.2006),idiopathic calcium urate nephrolithiasis (Masseoud et al.2005)and renal failure (Capasso et al.2005),and it was also reported that a high level of uric acid was related to leukemia in children (Larsen &Loghman-Adham 1996).Consequently,uric acid concentration is an important parameter monitored in urine and blood samples in routine clinical examinations.At present,the colorimetric method that employs uricase and peroxidase is widely accepted as a simple,sensitive and highly specific test for uric acid examination.In this enzymatic system of uric acid analysis,uricase plays an important role:uric acid À!uricase5Àhydroxyisourate þH 2O 2þCO 2By determining the amount of peroxide formed in the reaction,the concentration of uric acid can be estimated.For the best result in this examination,the properties of theL.Kai ÁX.-H.Ma (&)ÁX.-L.Zhou ÁX.-M.Jia ÁX.Li ÁK.-P.GuoCollege of Life Sciences,Zhejiang University,Yuhangtang Road 388#,Hangzhou,Chinae-mail:maxiaohong@World J Microbiol Biotechnol (2008)24:401–406DOI 10.1007/s11274-007-9489-1uricase in this reaction system are important,especially its stability,which will determine the precision of the mea-surement.At present,most enzymes applied in the clinical test are used in solution,and most proteins,including this enzyme,are relatively unstable when dissolved in aqueous solution.Therefore,for the enzymatic examination appli-cation,research was undertaken to search for a thermostable enzyme(Guo et al.2006;Huang et al.1998;Zhou et al.2005).As mentioned above,there were many uricases that have been isolated from microorganisms,but the thermostability of the published uricases were relatively low(Suzuki et al. 2004).The most thermostable uricase was reported by Suzuki,but it would lose its activity after a short period of treatment at60°C(Suzuki et al.2004).This low stability is a disadvantage in clinical applications.In a previous study,we isolated a bacterium Micro-bacterium sp.strain ZZJ4-1that produced a thermostable uricase.The enzyme was stable at65°C and its solution retained its original activity even after storage at37°C for 40days(Zhou et al.2005).Considering that this enzyme has a potential value in practical application,the present study was undertaken to purify and study the properties of this new enzyme.Materials and methodsMaterials and chemicalsThe culture of Microbaterium sp.(strain ZZJ4-1)was maintained in our laboratory(Zhou et al.2005).The protein standards for SDS-PAGE were purchased from Invitrogen(Shanghai,China).All other chemicals used were of reagent or molecular biology grade and pur-chased from Hangzhou Huadong Medicine Group Co.,Ltd (Hangzhou,China).Cultivation conditionsThe fermentation medium consisted of3.0g of uric acid, 10.0g of maize milk,0.5g of MgSO4Á7H2O,0.5g of KH2PO4,2.0g of K2HPO4Á3H2O,0.1g of NaCl,1.0l of tap water and the pH was adjusted to7.5(Zhou et al. 2005).To cultivate the strain for production of the uricase, a loop of bacteria from a slant was inoculated into a500ml flask containing100ml liquid medium and incubated at 30°C for30h with a rotary shaker at120rev/min.Enzyme assay and protein measurementThe principle of enzyme measurement was as follows: uricase can catalyse the oxidation of uric acid to form 5-hydroxyisourate and H2O2,which is then measured using a reaction system containing4-aminoantipyrine,phenol and peroxidase as chromogens.In practical analysis, 0.10ml enzyme solution was incubated with a mixture of 0.6ml0.1M sodium borate buffer(pH8.5)containing 2mM uric acid,0.15ml of30mM4-aminoantipyrine, 0.1ml of1.5%phenol and0.05ml peroxidase(15U/ml) at37°C for20min(Masaru1981).The reaction was stopped by addition of1.0ml ethanol and the absorbance at540nm was read against the blank in a spectropho-tometer.One unit of enzyme was defined as the amount of enzyme that produces1.0l mole of H2O2per minute under the standard assay conditions.The protein was measured by the Folin-phenol method (Lowry et al.1951).Enzyme purificationUnless otherwise stated,all of the enzyme purification processes were performed with0.1M phosphate buffer at pH7.0(buffer A)at4°C.Cells from10.0l of media were harvested by centrifu-gation and washed twice with50mM phosphate buffer(pH 7.5),re-suspended in buffer A and disrupted by ultrasonic oscillation(120W oscillating for3s with6s intervals, repeated100times).After the cell debris had been sepa-rated by centrifugation,solid ammonium sulfate was added to the enzyme solution and the precipitate of the fractions from55%to80%saturation was collected by centrifuga-tion(8000rpm,40min,4°C).The enzyme was then dis-solved in a small amount of buffer(2:1,volume of the buffer/weight of the precipitate),dialyzed against0.01M phosphate buffer until the ammonium sulfate was removed.The dialyzed enzyme solution was put onto a DEAE-Cellulose column(5.5·50cm)previously equilibrated with buffer A and it was then eluted with2.0l of buffer A with a linear gradient of0–1.5M KCl(flow rate:2ml/ min).The fractions containing enzyme activity were col-lected.After the ammonium sulfate had been added to65% saturation,the enzyme solution was loaded onto a Toyo-pearl HW-65column(5.5·85cm)equilibrated previ-ously with buffer A containing65%ammonium sulfate. The enzyme was eluted with2.0l of the same buffer with a linear gradient of ammonium sulfate from65%to0%(flow rate:2ml/min).When the fractions containing enzyme activity had been collected,the ammonium sulfate was added to get85%saturation.The precipitate of the enzyme was then collected by centrifugation,dissolved in a small amount of buffer A,dialysed with the same buffer con-taining0.1M KCl and applied to a Sephadex G-75column (5.5·100cm)equilibrated with the same buffer.The enzyme was then eluted with2.0l of the same buffer(flow rate:2ml/min)and the fractions containing the highest specific activity were collected for further study.Characterization of enzymeTo study the effect of pH on the activity of uricase,the enzyme was assayed at different pHs in the range from4.0 to11.0with intervals of0.5.The following buffers were used:100mM citrate for pH4.0–6.0,100mM phosphate for pH6.0–8.5and100mM borate for pH8.5–11.The pH stability was studied by incubating the purified enzyme solution in the corresponding buffers in the range from4.0 to11.0at25°C for18h and measuring the residual activity.To study the effect of temperature on the uricase activity,the standard enzyme reaction solution was pre-incubated at temperatures of20–60°C with5°C intervals for5min and the enzyme solution was then added and incubated for20min at the same temperature to measure its activity.For thermostability testing,the purified uricase solution was incubated at temperatures in the range from 20°C to80°C for30min and the remaining activity was then measured.The apparent K m of the uricase was estimated by the double reciprocal plot method.At different concentrations of uric acid,the enzyme activity was assayed and the Km was calculated by the Lineweaver-Burk plotting according to the Michaelis-Menten equation.To study the effects of chemicals on the uricase activity, the enzyme solution was pre-incubated with the chemicals for30min at room temperature in phosphate buffer and the remaining uricase activity was assayed with the standard reaction system containing the corresponding chemical.The relative molecular mass of the enzyme was deter-mined by SDS-PAGE with10%polyacrylamide gels (Laemmli1970).The purity was determined by the specific activity of the enzyme and PAGE with10%polyacryl-amide gels.The protein was stained with0.1%Coomassie brilliant blue R250in4:1:5methanol/acetic acid/water (vol/vol/vol)solution and destained in the same solution without dye.ResultsPurification of the enzymeIn thefirst step of the purification,ammonium sulfate precipitation was applied.The amount of protein and the enzyme activity of each fraction were measured.The fractions with ammonium sulfate concentrations from65% to80%had the highest enzyme specific activity(0.43U/ mg),while fractions from35–55,55–65to80–85had the specific activity of0.02U/mg,0.09U/mg and0.07U/mg, respectively.The fractions with concentrations from65% to80%were collected,dialyzed and loaded onto a DEAE-cellulose column.The enzyme was eluted with the same buffer containing a linear concentration gradient of KCl from0M to1.5M.Enzyme activity was found in fractions from75to130and fractions from80to105were pooled, solid ammonium sulfate was added to65%saturation and the solution was loaded onto a Toyopearl HW65-C col-umn.The enzyme was then eluted with the same buffer with a decreasing linear concentration of ammonium sul-fate from65%to0%.The enzyme activity was found in the fractions from100to160and fractions from110to135 were pooled,concentrated and applied to the Sephadex G-75column.After being washed with buffer A containing 0.1M NaCl,the enzyme activity was observed in the fractions from30to80.The fractions from50to60were pooled as purified enzyme for further study.The purity of the enzyme after each purification step was examined by SDS-PAGE and is shown in Figure1. The results of the purification process are summarized in Table1.During the purification,the enzyme was purified 19.7-fold with a recovery of31%and the purified enzyme had a specific activity of5.32U mg-1.Molecular mass determinationThe purified enzyme showed a single protein band in SDS-PAGE and its molecular mass was estimated to be34kDa (Fig.2).Optimum reaction temperature and thermostability ofthe purified enzymeThe purified enzyme was stable at a relative high temper-ature.As shown in Fig.3,it was stable at65°C and it retained64%of its original activity even after beingtreated Fig.1SDS-PAGE pattern of uricase samples from different steps of purification.(A)crude extract;(B)ammonium sulfate precipitation;(C)DEAE-cellulose chromatography;(D)Toyopearl HW-65chro-matography;(E)Sephadex G-75chromatographyat70°C for30min.By comparison between the optimal reaction and stable temperature,it was shown that although the enzyme was stable at65°C,its optimum temperature was30°C,which was relatively low,and it only showed 21%relative activity at60°C.Optimum pH and the stability of the enzymeat different pHsThe activity of the enzyme was measured in different buffers with a pH range from4.0to11.0.The results showed that the enzyme had low activity at pH below5.5 or over10.5and had relatively high activity in the range from7.0to10.0,with the optimal reaction pH at8.5 (Fig.4).As shown in Fig.4,after being incubated in dif-ferent buffers at25°C for18h,the uricase was stable in the pH range from5.5to9.5.Kinetics and effect of chemicals on the activityof the enzymeThe K m of the purified enzyme for uric acid was estimated to be0.31mM,which was calculated from the slopes and intercepts of the regression lines of the Lineweaver-Burk plot by determining the enzyme activity at37°C and the K cat of the enzyme was estimated to be3.01s–1.The effects of different chemicals on the activity of the enzyme are summarized in Table2.It was shown that among the metal ions,Li+,Ag+and Hg+greatly inhibitedTable1Summary of the uricase purification process Purification steps Activity(U)Total protein(mg)Specific activity(U mg–1)Purification(fold)Yeild(%)Crude enzyme6012220400.271100 Ammoniumsulfate488595190.51 1.9081 DEAE-cellulose338540840.83 3.0756 Toyopearl HW-6528451530 1.86 6.8947 Sephdax G-751866351 5.3219.7031 Fig.2SDS-PAGE electrophoregram of uricase.M:Standard ProteinMarker;U:Uricasethe enzyme activity.The strongest suppression was ob-served in the case of Hg+,which suppressed almost all of the activity of this enzyme.The chelating reagents had different effects;EDTA had no inhibitory effect,whereas o-phenanthroline(OPT)could inhibit the activity of uricase considerably.DiscussionAt present,the uricases from many microorganisms have been studied and some gene sequences of uricases have been cloned and studied.It was shown that uricases belong to a group of enzymes that have the same catalytic char-acter,but a great diversity of molecular structures.Uricases from different sources may have different molecular mas-ses and amino acid sequences.In this study,the molecular mass of the uricase from strain ZZJ4-1was estimated to be 34kDa by SDS-PAGE,whereas uricases produced by Candida utilis(Koyama et al.1996)and Pseudomonas aeruginosa(Ishikawa et al.2004)had the molecular mas-ses of34and54kDa,respectively.The apparent K m value of this uricase was0.31mM,while uricases from Ar-throbacter,Bacillus sp.and Candida sp.had K m values of 75,75,46l M,respectively(Suzuki et al.2004).Some relatively thermostable uricases,such as those produced by Arthrobacter globiformis FERM BP-360, Bacillus sp.TB-90and Candida utili s,have been studied and these were stable at55or60°C(Suzuki et al.2004). In this work,the uricase produced by strain ZZJ4-1showed good thermostability.After incubation at65°C for30min, the enzyme from strain ZZJ4-1still retained98.9%of its original activity,and even after incubation at70°C for 30min,the remaining activity was64%.Additionally,the pH stability of the enzyme from strain ZZJ4-1was a little broader than that of the uricases from the above three species(Suzuki et al.2004).The effects of metal ions on the enzyme activity from strain ZZJ4-1were also compared with the above three enzymes(Suzuki et al.2004).Considering that some uri-cases require metal ion cofactors for activity(Wu et al. 1989;Chu et al.1996)and whether or not an enzyme is inhibited by certain metals is an important characteristic, experiments were carried out to examine the effect of metals on the activity of this enzyme.It was shown that the enzyme from strain ZZJ4-1was not inhibited by Cu2+,Fe3+ or Zn2+,while uricases from the other three strains were strongly inhibited by these ions.Ag+is a strong inhibitor of the uricases from strain ZZJ4-1,Candida utilis and Bacillus sp.TB-90,while it had no such effect on the uricase from Arthrobacter globiformis FERM BP-360. When EDTA,a chelating reagent,was added to the enzyme solution at afinal concentration of20mM,the activity of the uricase was only slightly inhibited.When the stronger metal-ion-chelating reagent o-phenanthroline was added to the enzyme solution,the activity of the enzyme was strongly inhibited.This phenomenon indicates that some yet unidentified metal ion is strongly bound in the enzyme and forms part of the uricase structure,which is very important to keep its catalytic activity.This property is also different from the uricase of Arthrobacter globiformis FERM BP-360(Suzuki et al.2004).It was shown that although the uricase from the strain ZZJ4-1was stable at65°C,the enzyme was at its optimal activity at30°C and it only showed21%of its maximal activity at60°C.This implied that the molecular structure of enzyme had a reversible change at a temperature be-tween30°and60°C,which had a negative effect on its catalysis activity.But considering that most clinical enzy-matic examinations are undertaken at25to37°C,this property will not affect its application in clinical exami-nation.ReferencesAdamek V,Suchova M,Demnerova K et al(1990)Fermentation of Candida utilis for uricase production.J Indu Microbiol6:85–90 Bongaerts GP,Uitzetter J,Brouns R et al(1978)Uricase of Bacillus fastidiosus.Properties and regulation of synthesis.Biochim Biophys Acta527:348–358Capasso G,Jaeger Ph,Robertson WG et al(2005)Uric acid and the kidney:urate transport,S tone disease and progressive renal failure.Curr Pharm Des11:4153–4159Table2Effects of chemicals on uricase activityChemicals Final Concentration Residual activity(%)Blank–100Fe3+1mM118.5Ca2+1mM107.7Ba2+1mM103.1Mn2+1mM101.5Zn2+1mM100.7Cu2+1mM98.9Li+1mM81.5Ag+1mM7.5Hg2+1mM 1.8NaN320mM101.2EDTA20mM99.8o-Phenanthroline2mM 1.2Tween200.10%(w/v)99.7Tween800.10%(w/v)80.5Triton X-1000.10%(w/v)99.7SDS0.50%(w/v)51.3Chu R,Lin Y,Usuda N,Rao MS et al(1996)Mutational analysis of the putative copperbinding site of rat urate oxidase.Ann NY Acad Sci804:777–780Guo K,Ma X,Sun G et al(2006)Expression and characterization of a thermostable sarcosine oxidase(SOX)from Bacillus sp.in Escherichia coli.Appl Microbiol Biotechnol73:559–566 Hongoh Y,Sasaki T,Ishikawa H(2000)Cloning,sequence analysis and expression in Escherichia coli of the gene encoding a uricase from the yeast-like symbiont of the brown planthopper, Nilaparvata lugens.Insect Biochem Mol Biol30:173–182 Huang HS,Kabashima T,Ito K et al(1998)Thermostable glycerol kinase from Thermusflavus:cloning,sequencing,and expression of the enzyme gene.Biochim Biophys Acta1382:186–190 Hunag S,Wu T(2004)Modified colorimetric assay for uricase activity and a screen for mutant Bacillus subtilis uricase genes following StEP mutagenesis.Eur J Biochem271:517–523 Ishikawa J,Yamashita A,Mikami Y et al(2004)The complete genomic sequence of Nocardia farcinica IFM10152.Proc Natl Acad Sci USA101:14925–14930Keilin J(1959)The biological significance of uric acid and guanine excretion.Biol Rev34:265–296Koyama Y,Ichikawa T,Nakano E(1996)Cloning,sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis urate oxidase(uricase).J Biochem(Tokyo), 120:969–973Laemmli UK(1970)Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature227:680–685 Larsen G,Loghman-Adham M(1996)Acute renal failure with hyperuricemia as initial presentation of leukemia in children.J Pediatr Hematol Oncol18:191–194Liu J,Li G,Liu H et al(1994)Purification and properties of uricase from Candida sp.and its application in uric acid analysis in serum.Appl Biochem Biotechnol47:57–63Lowry OH,Rosebrough NJ,Farr AL et al(1951)Protein measure-ment with the Folin Phenol reagent.J Biol Chem193:265–275Masaru P(1981)Purification and some properties of Sarcosine Oxidase from Corynebacterium sp.U-96.J Biochem89:599–607 Masseoud D,Rott K,Liu-Bryan R et al(2005)Overview of hyperuricaemia and gout.Curr Pharm Des11:4117–4124 Montalbini P,Aguilar M,Ineda M(1999)Isolation and character-ization of uricase from bean leaves and its comparison with uredospore enzyme.Plant Sci147:139–147Montalbini P,Redondo J,Caballero JL(1997)Uricase from leaves: its purification and characterization from three different higher plants.Planta202:277–283Nakagawa T,Mazzali M,Kang DH et al(2006)Uric acid-a uremic toxin?Blood Purif24:67–70Nobutoshi K,Keisuke S,Takao M et al(2000)Determination of uric acid in plasma by closed-loopfia with a coimmobilized enzyme flow cell.Anal Sci16:1203–1205Suzuki K,Sakasegawa SI,Misaki H et al(2004)Molecular cloning and expression of uricase gene from Arthrobacter globiformis in Escherichia coli and characterization of the gene product.J Biosci Bioeng98:153–158Wallrath LL,Friedman TB(1991)Species differences in the temporal pattern of Drosophila urate oxidase gene expression are attrib-uted to trans-acting regulatory changes.Proc Natl Acad Sci USA 88:5489–5493Wu XW,Lee CC,Muzny DM et al(1989)Urate oxidase:primary structure and 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Mutation in acetylcholinesterase1associated with triazophos resistance in rice stem borer,Chilo suppressalis (Lepidoptera:Pyralidae)Xiaojing Jiang a,1,Mingjing Qu b,1,Ian Denholm c ,Jichao Fang d ,Weihua Jiang a ,Zhaojun Han a,*aKey Laboratory of Monitoring and Management of Plant Diseases and Insects,Ministry of Agriculture,Nanjing Agricultural University,Weigang No.1,Nanjing 210095,China bShandong Peanut Research Institute,Qingdao 266100,China cPlant and Invertebrate Ecology Division,Rothamsted Research,Harpenden,Hertfordshire AL52JQ,UK dInstitute of Plant Protection,Jiangsu Academy of Agricultural Science,Nanjing 210014,Chinaa r t i c l e i n f o Article history:Received 5November 2008Available online 24November 2008Keywords:Chilo suppressalis Acetylcholinesterase Gene mutationInsecticide resistance Triazophosa b s t r a c tTwo full-length genes encoding different acetylcholinesterases (AChEs),designated as Ch-ace1and Ch-ace2,were cloned from strains of the rice stem borer (Chilo suppressalis )susceptible and resistant to the organophosphate insecticide triazophos.Sequence analysis found an amino acid mutation A314S in Ch-ace1(corresponding to A201in Torpedo californica AChE)that was consistently associated with the occurrence of resistance.This mutation removed an MspA1I restriction site from the wild type allele.An assay based on restriction fragment length polymorphism (RFLP)analysis was developed to diagnose A314S genotypes in field populations.Results showed a strong correlation between frequencies of the mutation and phenotypic levels of resistance to triazophos.The assay offers a prospect for rapid monitoring of resistance and assisting with the appropriate choice of insecticide for combating damage caused by C.suppressalis .Ó2008Elsevier Inc.All rights reserved.Although one of the most important insect pests in Asia,the rice stem borer,Chilo suppressalis (Walker),is difficult to control because most of its life-cycle is spent inside rice stems.Only insec-ticides that combine good penetration or systemicity,low toxicity to fish,and affordability to farmers are appropriate for stem borer control.In the past,hexachlorocyolohexane (BHC),chlordimeform and monosultap were used extensively in China.However,the first two have been banned for ecotoxicological reasons,and monosul-tap has lost its effectiveness due to resistance.The organophos-phate triazophos initially proved an excellent replacement for monosultap,but recently its effectiveness has declined.A previous publication confirmed resistance to triazophos in C.suppressalis from some locations in China,and suggested reduced sensitivity of acetylcholinesterase –the target site of organophosphates –to be the primary resistance mechanism [1].Acetylcholinesterase (AChE,EC3.1.1.7)is a key enzyme in neu-rotransmission.It regulates nerve impulses at cholinergic synapses by rapidly catalyzing the hydrolysis of the neurotransmitter acetyl-choline.Aside from some species of Diptera,insects have two dif-ferent AChE genes [2],but it is believed that only one of these (ace1)plays a key role in insecticide toxicology [3–5].Inhibition of AChE by insecticides causes excessive excitement in nerves,blockage of neurotransmission and subsequent death.Mutationsmaking AChE less sensitive to insecticides can confer potent resis-tance and have been described in a range of insect species includ-ing Drosophila melanogaster ,Musca domestica (housefly),Bactrocera oleae (olive fruit fly),Leptinotarsa decemlineata (Colorado beetle),Anopheles gambiae and Culex pipiens (mosquitoes),Myzus persicae (peach-potato aphid),Cydia pomonella (codling moth)and Putella xylostella (diamondback moth)[6–13].In the present work,AChE genes were cloned from triazophos-resistant and -susceptible C.suppressalis to investigate possible mutations responsible for resistance in this species.The results demonstrated a mutation in Ch-AChE1consistently associated with resistance.Furthermore,the frequency of the mutation was confirmed to indicate well the level of triazophos resistance in field populations.Materials and methodsInsects.The susceptible strain (Gy-S)of C.suppressalis was collected from Ganyu County in Jiangsu Province in 2006,where little triazophos had been used for rice pest control.The standard resistant strain (Tp-R)with 1172-fold resistance to triazophos was produced by eight generations of laboratory selection from a field strain with 203-fold resistance collected from Cangnan County in Zhejiang Province,where triazophos had been exten-sively used for over 7years.Additional field strains were collected from Cangnan County in Wenzhou in 2008(WZCN2008),Ruian County in Wenzhou in 2007and 2008(WZRA2007and WZRA2008,0006-291X/$-see front matter Ó2008Elsevier Inc.All rights reserved.doi:10.1016/j.bbrc.2008.11.046*Corresponding author.Fax:+862584395245.E-mail address:zjhan@ (Z.Han).1These authors contributed equally.Biochemical and Biophysical Research Communications 378(2009)269–272Contents lists available at ScienceDirectBiochemical and Biophysical Research Communicationsjournal homepage:www.elsev i e r.c o m /l o c a t e /y b b rcrespectively),Gaochun County in Nanjing in2008(NJGC2008), Jiande County in Hangzhou in2007(HZJD2007),Guilin County in Guangxi in2008(GXGL2008),and Ganyu County in northern Jiang-su in2008(JSGY2008).All insects were reared in the laboratory on rice seedlings[14]at28±1°C under a16h photoperiod and>80% relative humidity.Total RNA extraction and cDNA synthesis.Total RNAs were extracted with TRIzolÒreagent(Invitrogen Co.,USA)from individ-ual fourth-instar larva according to the manufacturer’s specifica-tions.First-strand cDNA was synthesized from the total RNA using ThermoScript TM reverse transcriptase(Invitrogen).The reac-tion vessel contained3l g of total RNA,1l M of OligodT-Adaptor primer(50-AAGCAGTGGTATCAACGCAGAGTAC(T30)VN),1l l of reverse transcriptase and the reaction mix in afinal volume of 20l l.The following cycling parameters were used:65°C for 50min to synthesize cDNA and37°C for20min to remove RNA by adding RNaseH(Invitrogen).Cloning of cDNA fragments of AChE genes.Two sets of primers (Table S1)based on known differences between the two AChE genes found in insects(Ch-ace1and Ch-ace2)were designed to clone cDNA fragments from C.suppressalis.PCR for cloning Ch-ace1fragments was performed by Taq polymerase(TaKaRa Co.,Japan)with following parameters:94°C for2min followed by40cycles at94°C for30s,45°C for30s,and72°C for40s, and one additional cycle at72°C for6min.For Ch-ace2fragments, only the annealing temperature was adjusted to43°C.Rapid amplification of cDNA end(RACE)was used to obtain the complete gene.The downstream region to the30-end of Ch-ace1was ampli-fied by LATaq polymerase and10ÂLA PCR Buffer II(Takara)with specific and adaptor primers(Table S1).The PCR reaction was per-formed as follows:94°C for2min followed by40cycles at94°C for30s,60°C for30s,and72°C for2min,and afinal extension at72°C for6min.50-RACE was performed with cDNA synthesized with GeneRacers TM kit(Invitrogen).LATaq polymerase and2ÂGC Buffer II(Takara)were used to amplify the50region with the fol-lowing parameters:94°C for2min followed by40cycles at 94°C for30s,61°C for30s,and72°C for1min and afinal exten-sion at72°C for6min.For Ch-ace2,LATaq polymerase and2ÂGC Buffer I(Takara)were used to amplify the30region.The50-RACE was performed with the BD SMARTTM RACE kit(Clontech,USA) and UPM primers in the kit were used as sense and antisense prim-ers.RACE PCR was performed under manufacturer’s recommended conditions.Amplification of the full length of two AChE genes.All primers were designed with primer5.0software(Table S1).All PCR reactions were performed with2ÂGC Buffer and LATaq polymerase(Takara)in a MyCycler TM Thermal Cycler(Bio-RAD Inc.,USA)under the condi-tions:25l l reaction mixture containing12.5l l2ÂGC buffer I or II,0.4mM dNTPs,0.4l M of each primer,1l l cDNA template,and 1.25U LATaq polymerase.The following parameters were used: 94°C for2min followed by40cycles at94°C for30s,54°C for 30s,72°C for2.5min,and afinal extension step at72°C for6min.DNA sequencing.PCR products were separated by agarose gel electrophoresis,purified with AxyPrep TM DNA Gel Extraction Kit (Axygen Biosciences,USA),and then cloned into pGEM-T easy vec-tor(Promega,USA).The ligation reactions were used for transfor-mations with the DH5a competent cells.Successful stains were screened with blue/white and standard ampicillin selection. Recombinant plasmids were fully sequenced on an Applied Biosys-tems377automated sequencer.RFLP-PCR diagnostic test for detection of the specific point muta-tion.A C/T nucleotide transition caused an alanine/serine amino acid substitution(A314S)in the insecticide-insensitive Ch-ace1. To detect this mutation at the genomic level,a method was devel-oped based on the restriction fragment length polymorphism anal-ysis of an appropriate PCR product(RFLP-PCR)encompassing this nucleotide position.As template, C.suppressalis genomic DNA was extracted from single fourth-instar larvae with Universal Genomic DNA Extraction Kit V.3.0(Takara).PCR was performed by LATaq polymerase and2ÂGC Buffer II(Takara)with the primers (Table S1)for thirty cycles(94°C for30s,59°C for30s and72°C for50s).The PCR product fragments were digested with MspA1I restriction enzyme(New England BioLabs Inc.,USA)according to the manufacturer’s instructions and fractionated on a2%agaroseFig.1.The deduced amino acid sequences of Ch-ace1(GenBank:EF453724)and Ch-ace2(GenBank:EF470245)from a susceptible strain of Chilo suppressalis.The putative signal peptide in the N-terminal region is underlined.The putative hydrophobic amino acid tail in the C-terminal region is double-underlined.The characteristic‘FGESAG’motif surrounding the active serine is shown in boxes.Acyl pocket residues are encircled.Anionic subsites are indicated by black triangles.The residues identified with black squares shape the oxianion holes.The double-underlined Cys residues indicate the interfaces for intra-and inter-molecular disulfide bonds.The shaded residues of the catalytic triads are boxed.270X.Jiang et al./Biochemical and Biophysical Research Communications378(2009)269–272gel.In order to check the identity of the amplified fragments, sequences were also done directly on PCR products.ResultsIdentification and characterization of AChE genes from C.suppressalis Two full-length AChE genes were successfully cloned and desig-nated as Ch-ace1and Ch-ace2(Fig.1).Ch-ace1(GenBank: EF453724)contained an open reading frame(ORF)of2082bp encoding694amino acids,235bp of50untranslated region(UTR) and204bp of30UTR containing a polyadenylation signal. Ch-ace2(GenBank:EF470245)contained an ORF of1914bp encod-ing638amino acids,321bp of50UTR and277bp of30UTR con-taining a polyadenylation signal.Both were predicted to contain the sequence of a signal peptide by SignalP V.3.0software (http://www.cbs.dtu.dk/services/SignalP/).The alignment of the deduced amino acid sequences did not show very high homology between Ch-AChE1and Ch-AChE2.Nevertheless,both had the fea-tures shared by members of AChE family(Fig.1).Compared with AChE in Torpedo californica[15],the functional motifs were highlyconserved:the catalytic triads(S313,E439and H553in Ch-AChE1, and S266,E395and H509in Ch-AChE2),the characteristic‘FGE-SAG’motif surrounding the active serine,3intra-chain disulfide bridges(C181–C208,C367–380and C515–C637in Ch-AChE1,and C115–C143,C320–C335and C471–C590in Ch-AChE2),a anionic choline-binding site(W198in Ch-AChE1and W133in Ch-AChE2), acyl pocket residues(W346,F402and F443in Ch-AChE1,and W299,F358and F399in Ch-AChE2)that accommodate the acyl moiety of the active site,and the residues that form the oxyanion hole(G232,G233and A314in Ch-AChE1,and G179,G180and A267in Ch-AChE2)that helps to stabilize the tetrahedral molecule during catalysis[16].The C-terminal Cys residues that contributed to an intermolecular dimerization were Cys660and Cys608, respectively.Following these Cys residues,both genes had amino acid tails enriched in hydrophobic residues as predicted using ProtScale(/cgi-bin/protscale.pl).Sequence blast also showed that the two cloned genes were similar to previously reported insect AChE genes,especially those from Lepidoptera (Table1).Thus,these two genes were concluded to be the relevant AChE genes in C.suppressalis.Difference in AChE sequence between susceptible and resistant strains When the cDNA sequences of AChE genes amplified from resis-tant and susceptible individuals were aligned,considerable non-si-lent single nucleotide polymorphisms(SNPs)and deduced amino acid replacements were found(Table S2).However,only one of these–GCG to TCG in Ch-ace1–was exclusively associated with the resistant phenotype.This change accounted for an A314S mutation in the oxyanion hole of the enzyme and involved an ami-no acid residue homologous to A201in T.californica AChE.Frequency of the mutation infield populations with different resistanceThe mutation GCG to TCG in Ch-ace1removes an MspA1I restriction site from the wild type allele.An RFLP-PCR assay was therefore designed using primers able to amplify specifically a 758-bp fragment encompassing the mutation site in Ch-ace1from genomic DNA,and by checking the digestion of the resulting frag-ment with MspA1I.As shown in Fig.2,the PCR product amplified from the wild type allele was completely cut into two pieces(534 and224bp long).That amplified from the heterozygote was par-tially cut and presented as three bands(758,534and224bp long), whereas that amplified from the mutated allele remained intact.With this method,individual larvae of C.suppressalis collected from differentfield sites were analysed,and results showed a good correlation between mutation frequencies and levels of triazophos resistance(Table2).DiscussionPrevious work suggested that target-site insensitivity was a ma-jor mechanism of triazophos resistance in C.suppressalis[1].We have now cloned two full-length AChE genes in this species that show very high identity to insect AChE genes reported in other spe-cies[17,18].We found a single amino acid substitution,A314S in Ch-AChE1,which associates consistently with the resistance phe-notype.This alanine residue is located in the characteristic‘FGE-SAG’motif surrounding the active serine,and lies in the oxyanion hole that contributes to stabilizing the tetrahedral mole-cule during catalysis.The alanine to serine substitution changes the side group from–CH3to–CH2OH,which is believed to alter the conformation of the adjacent serine of the catalytic triad and to affect the interaction between AChE and both substrates and inhibitors.This site corresponds to A201in T.californica AChE, and the same amino acid substitution has been reported as a resis-tance mutation in cotton aphid,Aphis gossypii[19–21],and dia-mondback moth,P.xylostella[22].The frequency of this mutation correlated well with levels of triazophos resistance,known to vary substantially between geographical regions[23].Thus,A314S in Ch-AChE1is proposed as a widespread and functional mutation contributing to triazaphos resistance in C.suppressalis.Table1Identity of cloned genes with some other insect AChE genes previously reported.Genes types Host insects Genbankaccession no.Identity%Ch-AChE1Ch-AChE2AChE1Plutella xylostella AAY347438130 AChE1Cydia pomonella ABB766668631 AChE1Helicoverpa armigera AAY595307734 AChE1Helicoverpa assulta AAY421368630 AChE1Bombyx mori NP0010373808331 AChE2Plutella xylostella AAL338203193 AChE2Cydia pomonella ABB766653192 AChE2Helicoverpa armigera AAM903333294 AChE2Helicoverpa assulta AAV656383294 AChE2Bombyx mori NP0010373663194Fig. 2.RFLP-PCR assay to detect the presence of the A314S mutation in Chilo suppressalis.Genomic DNA amplification produces a758bp fragment,which is cut into two fragments(224and534bp)by MspA1I restriction enzyme for susceptible homozygotes(SS),but is undigested for resistant homozygotes(RR).Heterozygous individuals(RS)display all three bands.X.Jiang et al./Biochemical and Biophysical Research Communications378(2009)269–272271Ch-ace2did not show any changes associated with resistance, which supports a view that in those insects with two different AChE genes,only ace1plays a major role in insecticide toxicology. The discovery of two different AChE genes in most insects[17,24] prompted work to identify their relative importance as insecticide targets.In P.xylostella,the bollworm Helicoverpa assulta and the cockroach Blattella germanica,expression studies using quantita-tive real time-PCR showed that transcription levels of ace1were significantly higher than those of ace2in all tissues examined [4,13,25].Furthermore,functional expression studies with Culex tritaeniorhynchus and A.gossypii showed susceptible ace1to have much higher affinity to acetylcholine than ace2[26,27].Thesefind-ings lead to an expectation that mutations conferring resistance to organophosphate and carbamate insecticides will reside in ace1 rather than the ace2counterpart.The RFLP-PCR assay developed to diagnose the A314S mutation infield populations provides a potentially valuable way of moni-toring resistance and investigating the relative frequencies of resis-tance genotypes.It overcomes many of the difficulties encountered with time-consuming bioassays against C.suppressalis,and offers the prospect of a rapid and high throughput kit to assist with choosing insecticides and combating resistance in this species. AcknowledgmentsThis work was supported by the National Basic Research Pro-gramme(Grant No.2006CB102003),Bumper Harvest Programme (Grant No.2006BAD02a16),National Natural Science Foundation of China(Grant No.30471145),National Science&Technology Pillar Programme(Grant No.2006BAD08a03)and Jiangsu Key Research Project(Grant No.BE2006303).Appendix A.Supplementary dataSupplementary data associated with this article can be found,in the online version,at doi:10.1016/j.bbrc.2008.11.046. 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Review articleMolecular and clinical pharmacology of intranasal corticosteroids: clinical and therapeutic implicationsIntranasal corticosteroids(INSs)are the most effective treatments available for allergic rhinitis(AR).They have been shown to be superior to oral H1receptor antagonists for AR(1)and have been designated as the treatment of choice for this condition(2–4).The development of INSs is one of the best examples of molecular modification of drug structure and delivery to achieve an almost ideal therapeutic index.Systemic corticosteroids,which were developed in the 1950s,are effective in treating AR,but the high risk of serious toxicity with long-term administration has hin-dered their usefulness(5).Initial attempts to deliver compounds such as hydrocortisone and dexamethasone directly into the airways were only partially successful(6). Thefirst successful use of beclomethasone as a pressur-ized aerosol with no apparent evidence of systemic toxicity was published in1972(6).In the years since, corticosteroid molecules have been refined to create more potent agents with lower bioavailability and enhanced safety profiles.Currently,eight INS compounds are approved for the management of AR in the United States:triamcinolone acetonide,flunisolide,budesonide, beclomethasone dipropionate,ciclesonide,fluticasone propionate,mometasone furoate,andfluticasone furoate. The furoate moiety in the later compound is the same as that found in the mometasone furoate molecule,i.e.fluticasone furoate isfluticasone propionate with the furoate ester of mometasone furoate.Ciclesonide is essentially a prodrug,requiring conversion by esterases to an active metabolite,desisobutyryl-ciclesonide(des-ciclesonide)at the site of desired action(e.g.lung/nasal tissue).Subsequent intracellular esterification of des-ciclesonide with endogenous fatty acids produces revers-ible lipid conjugates of des-ciclesonide that become a reservoir of the drug in the lung or nasal mucosa(7,8). Each corticosteroid has characteristics that determine its pharmacokinetic profile and pharmacodynamic activ-ity.This article will provide an overview of corticosteroid pharmacology in the allergic inflammatory process, review the characteristics of individual compounds,and evaluate these differences from a clinical perspective. The allergic inflammatory processAllergic rhinitis stems from localized type I hypersensi-tivity reactions in atopic individuals,who have IgE antibodies that bind to mast cell receptor sites.When crossbound by allergen,degranulation and release of inflammatory mediators occurs(Fig.1).These mediatorsIntranasal corticosteroids(INSs)are effective treatments for allergic rhinitis, rhinosinusitis,and nasal polyposis.In recent years,increased understanding of corticosteroid and glucocorticoid receptor pharmacology has enabled the development of molecules designed specifically to achieve potent,localized activity with minimal risk of systemic exposure.Pharmacologic potency studies using affinity and other assessments have produced similar rank orders of po-tency,with the most potent being mometasone furoate,fluticasone propionate, and its modification,fluticasone furoate.The furoate and propionate ester side chains render these agents highly lipophilic,which may facilitate their absorp-tion through nasal mucosa and uptake across phospholipid cell membranes. These compounds demonstrate negligible systemic absorption.Systemic absorption rates are higher among the older corticosteroids(flunisolide,beclo-methasone dipropionate,triamcinolone acetonide,and budesonide),which have bioavailabilities in the range of34–49%.Studies,including1-year studies with mometasone furoate,fluticasone propionate,and budesonide that evaluated potential systemic effects of INSs in children have generally found no adverse effects on hypothalamic–pituitary–adrenal axis function or growth.Clinical data suggest no significant differences in efficacy between the INSs.Theoretically, newer agents with lower systemic availability may be preferable,and may come closer to the pharmacokinetic/pharmacologic criteria for the ideal therapeutic choice.H.Derendorf1,E.O.Meltzer21Department of Pharmaceutics,University of Florida, Gainesville,FL;2Allergy and Asthma Medical Group &Research Center,San Diego,CA,USAKey words:intranasal corticosteroids;pharmacology; systemic safety.H.Derendorf,PhDDepartment of PharmaceuticsUniversity of FloridaPO Box100494GainesvilleFL32610-0494USAAccepted for publication2March2008Allergy2008:63:1292–1300Ó2008The AuthorsJournal compilationÓ2008Blackwell MunksgaardDOI:10.1111/j.1398-9995.2008.01750.x1292act quickly on nasal end-organs to elicit the well-known symptoms of sneezing,congestion,rhinorrhea,and pru-ritus.This acute phase of the allergic reaction develops within minutes of allergen exposure and is caused primarily by histamine and arachidonic acid metabolites (leukotrienes,prostaglandins,and thromboxanes)(9,10). The late-phase allergic reaction,which occurs within 6–12h of allergen exposure,is characterized by an influx of monocytes,T lymphocytes,basophils,and eosinophils to the nasal mucosa.In fact,the presence of infiltrating eosinophils in the nasal mucosa is considered an impor-tant indicator of an allergic basis for the rhinitis.The release of toxic proteins(such as major basic protein and eosinophilic cationic protein)from eosinophils and solu-ble mediators from these recruited cells(primarily inter-leukins,tumor necrosis factor alpha,interferon-c),along with continued release of leukotrienes from eosinophils and mast cells,facilitates the prolongation of symptoms (9,10).Intranasal corticosteroids downregulate the recruit-ment and influx of inflammatory cells,and inhibit the secretion of pro-inflammatory mediators during the late phase of the inflammatory response(9).This process is evidenced by reduced levels of histamine,leukotrienes, and mast cells recovered in the nasalfluid and mucosa of AR patients treated with INSs(9,11–13).The glucocorticoid receptorCorticosteroid activity is mediated by intracellular acti-vation of the glucocorticoid receptor(GR)(14,15).In its inactive state,the GR exists as a cytosolic protein bound to two heat shock protein90chaperonin molecules. Binding to the corticosteroid ligand results in a confor-mational change that allows dissociation of the GR from the protein complex,and a quick translocation into the cell nucleus.The ligand-bound GR can modulate gene expression in the nucleus by binding to glucocorticoid response elements in promoter regions of responsive genes.The GR binds to the glucocorticoid response elements as a homodimer and acts as a transcription factor.It has recently become evident that the GR can also regulate gene expression unfacilitated by glucocor-ticoid response elements through direct interaction with transcription factors such as nuclear factor-j B and activating protein(14,16,17).The inhibition of these two factors leads to downregulation of the production of cytokines and other inflammatory molecules and is thought to be among the primary mechanisms for the anti-inflammatory effects of corticosteroids(14,15). Corticosteroid structure-activity relationshipsCorticosteroid molecules are derived from the parent molecule,cortisol(10).The carbon framework of each corticosteroid is made up of three6-carbon rings(rings A, B,and C)and one5-carbon ring(ring D)(Fig.2).All anti-inflammatory corticosteroids have features in com-mon with cortisol and with each other:a ketone oxygen at position3;an unsaturated bond between carbons4and5;a hydroxyl group at position11;and a ketone oxygen group on carbon20.The variations occurring offring D at positions16,17,and21are the greatest differentiatingRelevance of pharmacology of intranasal steroidsÓ2008The AuthorsJournal compilationÓ2008Blackwell Munksgaard Allergy2008:63:1292–13001293factors between the individual molecules.Structure–activity relationship studies of this region led to the identification of chemical groups that enhance topical activity and reduce systemic adverse events (5).For example,the furoate group of mometasone furoate was found to enhance molecular affinity for the GR binding site.Other modifications have improved the activity of corticosteroid compounds.The 21-chloro-17(2¢-furoate)group on the mometasone furoate structure improves anti-inflammatory activity,while the chloride at position 21provides the additional benefit of inferring resistance to degradation by esterases (5).Halogen substitutions at positions 6and 9are thought to increase potency,as are side-chain substitutions at position 17(18).Pharmacodynamic properties of intranasal corticosteroids PotencyGlucocorticoid potency can be measured in various ways,but is thought to be closely related to GR binding affinity.Fig.3illustrates the relative GR binding affinities for most intranasal compounds,as well as fluticasone furoate and des-ciclesonide,which is the active metabolite of the prodrug ciclesonide (19).The Valotis study was based on kinetic methods and was performed on cytosolicGRFigure 2.Chemical structures of intranasalcorticosteroids.Derendorf and MeltzerÓ2008The Authors1294Journal compilation Ó2008Blackwell Munksgaard Allergy 2008:63:1292–1300extracts from pooled human lung tissue.Receptor asso-ciation and dissociation rate constants were determined, and relative receptor affinities calculated based on the equilibrium dissociation rate constants.The highest relative receptor affinities were associated with some of the newest compounds,fluticasone furoate,mometasone furoate,andfluticasone propionate,reflecting the improved understanding of the GR complex that has led to the design of more potent corticosteroid molecules in recent years.Relative receptor affinity studies are highly dependent on assay methodology and prone to error(20).Although specific values vary between studies,most have shown a similar order of potency.An earlier study of INSs that used competition assay methodology reported a similar rank order of relative binding potency:mometasone furoate>fluticasone propionate>budesonide>tri-amcinolone acetonide>dexamethasone(15).Studies conducted before the development of mometasone furo-ate consistently described the same relative binding affinity order(15,21–24);the binding affinity of des-ciclesonide(the active metabolite of the prodrug cicleso-nide)is slightly less than that offluticasone propionate (7,19).Corticosteroid potency also has been evaluated using the McKenzie assay,which compares the relative cuta-neous vasoconstrictor and skin blanching responses of individual compounds(25).Using this methodology,the rank order of potency was:fluticasone propionate>mo-metasone furoate>budesonide>flunisolide>triam-cinolone acetonide(26).Results of studies using another marker of cortico-steroid potency,transactivation potency,correlate with receptor binding affinity results.One study found mometasone furoate the most potent GR ligand,requir-ing the lowest concentration to affect50%of the maximum level of transcription activation of a gluco-corticoid response element reporter gene in cells(15). Overall rank of potency using this methodology was: mometasone furoate>fluticasone propionate>triam-cinolone acetonide>budesonide>dexamethasone. There is no evidence of a linear association between glucocorticoid potency and clinical response,nor is there a knownÔplateauÕbeyond which greater potency does not add additional benefit.Likewise,it is not evident that the compound with the highest receptor affinity will have superior clinical efficacy.Although increased potency at intranasal sites would seem desirable,the possibility of greater potency at other sites could theoretically increase the risk of systemic adverse effects,as GRs are similar throughout the body.Pharmacokinetic propertiesThe pharmacokinetic properties of an INS determine the concentration and disposition of drug at the receptor site,as well as the potential for the drug to reach the systemic circulation.Because the goals of INS therapy are to deposit drug at the site of action,have it remain there as long as possible,and limit the amount that leaves the site and enters the systemic circulation,the pharmacokinetic features of particular interest are lipophilicity and systemic availability.LipophilicityThe lipophilicity of a corticosteroid,as expressed in its log P(log of the octanol/water portion coefficient), provides an index of its lipid-partitioning potential(27). The more highly lipophilic compounds are absorbed more quickly and thoroughly by the nasal mucosa and retained longer in nasal tissue,increasing exposure to the GR(27).The addition of lipophilic side chain groups to a corticosteroid molecule facilitates its uptake across phos-pholipid cell membranes to the cytoplasmic interior, where it can interface with GRs(28).Lipophilicity also contributes to increased plasma protein binding.In this sense,lipophilicity is a desired characteristic.In the event of systemic absorption,lipophilicity may contribute to the accumulation of drug in other tissues,possibly contributing to unwanted side effects.Thus,the ideal combination of features would include a high degree of lipophilicity coupled with low systemic absorption and rapid clearance.The order of lipid solubility for corticosteroids has been reported as:mometasone furoate>fluticasone propionate>beclomethasone dipropionate>budeso-nide>triamcinolone acetonide>flunisolide(27).Ci-clesonide and des-ciclesonide have greater lipophilicity thanfluticasone propionate(28).The furoate(mometa-sone)and propionate(fluticasone)ester side chains are pharmacologically similar moieties that contribute greatly to the lipophilicity of their associated compounds (29).The lipophilicity offluticasone propionate has been reported as three times higher than beclomethasone dipropionate,300times more than budesonide,and at least1000-fold greater thanflunisolide or triamcinolone acetonide(26).Lipophilicity is an important determinant of drug solubility.Only dissolved corticosteroid can cross the membrane and be absorbed systemically.Any solid undissolved compound may be removed from the absorption site by mechanical physiological clearing mechanisms before the steroid can cross the membrane and become bioavailable.Absorption/bioavailabilityIn addition to degree of lipophilicity,systemic exposure of an INS is also dependent on oral and local bioavail-ability and hepaticfirst-pass effect.Following intranasal administration,a drug can enter the systemic circulation through direct local absorption in the nasal mucosa or oral absorption of swallowed material.As illustrated in Relevance of pharmacology of intranasal steroidsÓ2008The AuthorsJournal compilationÓ2008Blackwell Munksgaard Allergy2008:63:1292–13001295Fig.4,a large proportion of intranasally administered drug is quickly cleared from the nose into the throat and swallowed,becoming available for absorption from the gastrointestinal tract.A high rate offirst-pass metabolism will inactivate the absorbed drug,but direct absorption into the systemic circulation through the nasal tissues bypasses the protective hepaticfirst-pass mechanism. Therefore,careful attention needs to be paid to the potential for direct systemic drug exposure of potent corticosteroid agents(30).Systemic absorption rates are highest among the relatively older compounds,flunisolide,beclomethasone, and budesonide(Fig.5).One-third to half of an intrana-sally administered dose of the older agents may reach the systemic circulation.For budesonide,systemic exposure is primarily due to absorption through the nasal mucosa (31,32).The newer compounds—fluticasone propionate and mometasone furoate—are more lipophilic(5)and undergo rapid and extensivefirst-pass metabolism fol-lowing oral administration,contributing to their negligi-ble systemic absorption(29).While published data on fluticasone furoate are still sparse,in vitro data presented as a poster at the Congress of the European Academy of Allergology and Clinical Immunology in2006reported very high tissue binding affinity to human nasal tissue and,hence,low systemic absorption(19).A pharmaco-kinetic study of ciclesonide in both healthy subjects and patients with seasonal AR(SAR)found serum levels of ciclesonide and the active metabolite des-ciclesonide were below the lower limits of quantification(25and10pg/ml, respectively)in the majority of subjects(33).The systemic bioavailability of des-ciclesonide following oral adminis-tration of ciclesonide was<1%(34).While detecting the physical presence of corticosteroid molecules in blood samples is a relatively straightforward task,identifying potential physiologic consequences of their absorption is more difficult and necessitates the use of highly sensitive tests(35).One of the most sensitive measures of systemic corticosteroid bioactivity is sup-pression of endogenous cortisol secretion from the adrenal cortex.These determinations measure basal adrenocorticoid secretion(morning plasma cortisol, 24-h urinary-free cortisol,or overnight urinary cortisol) or dynamic function of the hypothalamic–pituitary–adrenal(HPA)axis to determine adrenal reserve(ACTH cosyntropin stimulation or corticotropin releasing factor test)(35,36).Adrenocorticoid secretion is subject to a normal circadian rhythm,with highest plasma levels occurring between5and9am.ÔSpotÕsampling of early morning plasma cortisol,often used in clinical trials,can introduce an unacceptable degree of inter-and intrasub-ject variability(36).A more sensitive assessment of basal adrenocortical function involves overnight or24-h eval-uations of cortisol output via urine or plasma sampling (37).Urinary cortisol measurements are often corrected for creatinine excretion and expressed in terms of urinary:creatinine ratio(35).Clinical trials evaluating the effects of INSs on HPA axis function and growth parameters in children have generally found that INSs do not affect growth.In one exception,mean urine cortisol:creatinine ratio was significantly lower during treatment withfluticasone propionate200l g/day than with triamcinolone acetonide 110or220l g/day or placebo(38).Aside from this report, the use of INSs at recommended doses does not appear to have a significant effect on the HPA axis(36).The effects of these agents on bone growth and height have been evaluated in numerous studies,with knemometry consid-ered the most reliable and sensitive indicator(38).One of the earliest studies(1993)reported slower lower leg growth velocity among11children receiving high-dose intranasal budesonide for6weeks compared with a pre-treatment run-in period(39).Other studies with budes-onide,mometasone furoate,andfluticasone propionate utilizing knemometry have not demonstrated significantDerendorf and MeltzerÓ2008The Authors 1296Journal compilationÓ2008Blackwell Munksgaard Allergy2008:63:1292–1300changes in lower leg growth velocity(38,40,41).Among studies based upon stadiometry measurements,only one reported a smaller mean increase in height among children treated with high-dose intranasal beclometha-sone for1year compared with placebo(42).One-year studies involving mometasone furoate,fluticasone propi-onate,budesonide,and ciclesonide did not report any significant negative effects on height(43–46).A12-month double-blind study of ciclesonide in children found no significant difference in standing height between treated children and those using placebo(46).Clinical implicationsOnset of actionIt was traditionally accepted that INSs should be used for days or weeks to achieve significant benefits,and because these agents were used primarily to control chronic symptoms,few studies focused on onset of action(27). Symptom improvement has been noted within1–2days following administration of most newer agents(27).A clinical study of ciclesonide nasal spray in patients with SAR reported a significantly greater effect of treatment on total nasal symptom scores compared with placebo as early as hour12(47).In subjects with SAR,mometasone furoate nasal spray significantly improved nasal symptom scores compared with placebo in as little as7h after a single200l g dose and total symptom scores as soon as 5h(48).It is not uncommon for patients to use intranasal medications intermittently on anÔas-neededÕbasis(27), and such use may be justified given the potential for a more rapid onset of action than was previously ascribed to these medications.Clinical efficacy and safetyThe design of topically active INS formulations has provided a much better therapeutic ratio than oral corticosteroids(35).The pharmacodynamic and phar-macokinetic properties of these agents play an important role in facilitating local anti-inflammatory activity with a low rate of side effects.However,it remains to be seen whether the often subtle pharmacodynamic and phar-macokinetic differences between the compounds distin-guish them in a clinical setting.Table1lists the INSs and their approved indications for AR in the United States.Based on currently available data,there is no clear evidence that any INS is superior to any other for AR symptom relief(27,49),despite the pharmacologic differences between many of them.It is possible that the degree of anti-inflammatory activity required for AR symptom relief is low enough that relief is easily achieved by agents of varying potencies,or that GR saturation or near saturation occurs with all of the preparations (27,50).Differences in safety between INSs are more theoret-ical than evidence-based,with the greatest concern being systemic exposure and effects on adrenal function and growth in children.Pharmacokinetic studies confirm that the newer agents–mometasone furoate,fluticasone propionate,fluticasone furoate,and ciclesonide–exhibit negligible systemic absorption and would be expected to pose fewer risks.It could also be argued that their dramatically greater potency could pose safety risks, even if they were absorbed to only a small degree.To date,these concerns have not been realized.Special caution is warranted in treating individuals who may beTable1.Intranasal corticosteroids:recommended dosages for allergic rhinitis(32,57,59–64)Generic(proprietary)name Recommended dosageBeclomethasone dipropionate(Beconase AQ)Adults and children>12years of age:1or2sprays(42–84l g)per nostril b.i.d.(total dose168–336l g/day)Children6–12years:1spray(42l g)per nostril b.i.d.for total of168l g/day up to2sprays per nostril b.i.d.for total of336l g/dayBudesonide(Rhinocort Aqua)*Adults and children‡6years of age:1spray(32l g/spray)per nostril q.i.d.up to a maximum of256l g/day(‡12yearsof age)or128l g/day(6to<12years of age)Ciclesonide(Omnaris)Adults and children‡12years of age:2sprays(50l g/spray)per nostril q.i.d.Flunisolide(Nasarel)Adults:2sprays(58l g)per nostril b.i.d.,not to exceed8sprays per nostril per day(464l g)Children6–14years of age:1spray(29l g)per nostril t.i.d.or2sprays(58l g)per nostril b.i.d.,not to exceed4spraysper nostril per day(232l g)Fluticasone furoate(Veramyst)Adults and children‡12years of age:2sprays(55l g)per nostril q.i.d.Children2–11years of age:1spray(27.5l g)per nostril q.i.d.up to2sprays(55l g)per nostril q.i.d.Fluticasone propionate(Flonase)Adults:2sprays(100l g)per nostril q.i.d.or1spray(50l g)b.i.d.Adolescents and children‡4years of age:1spray(50l g)per nostril per day up to,but not in excess of,2sprays(100l g)per nostril per dayMometasone furoate(Nasonex)Adults and children‡12years of age:2sprays(100l g)per nostril q.i.d.Children2–11years of age:1spray(50l g)per nostril q.i.d.Triamcinolone acetonide(Nasacort AQ)Adults and children‡12years of age:2sprays(110l g)per nostril q.i.d.Children6–12years of age:1spray(55l g)per nostril or110l g q.i.d.,up to2sprays(110l g each)per nostril or220l g q.i.d.*Available in two strengths:32and64l g per spray.Relevance of pharmacology of intranasal steroidsÓ2008The AuthorsJournal compilationÓ2008Blackwell Munksgaard Allergy2008:63:1292–13001297using concomitant corticosteroid therapy for other medical conditions,such as asthma.The safety of overlapping corticosteroid therapy has not been well studied,but combined treatments may be a concern because of the worsening of total systemic corticosteroid load.A recent open-label study with intranasal mo-metasone furoate did not identify any increased inci-dence of adverse events when the agent was coadministered with treatments(the antihistamines desloratadine and levocetirizine and the corticosteroid betamethasone)for asthma,chronic rhinosinusitis,and nasal polyposis(51).Common local side effects of INSs include dryness, stinging,burning,and epistaxis,the frequencies of which are similar in the various compounds.Nasal mucosal atrophy is a concern with chronic topical steroid use.A long-term study with mometasone furoate found no evidence of atrophy or metaplasia following12months of intranasal use(13).Similar studies withfluticasone propionate did not identify indications of atrophy and noted only a nonsignificant increase in metaplasia in one patient(52).Lipid conjugation,or fatty acid esterification,is a process through which a corticosteroid molecule forms a reversible chemical bond with fatty acids in the nasal tissues.These drug-lipid complexes serve as a slow-release drug reservoir that holds the corticosteroid within the target area,increasing the local residence time.As the binding process is reversible,the retained corticosteroid remains available for binding to local GRs.This binding process may be partly responsible for the once-daily efficacy of many INSs.Lipid conjugation has been reported for budesonide(53)and des-cicleso-nide(54).Binding to proteins such as albumin and other bio-logical material can occur at the local site of action,and also in general circulation if a corticosteroid is systemi-cally absorbed.Most INSs have a high degree of protein binding,ranging from71%(triamcinolone acetonide)to 99%(ciclesonide,des-ciclesonide).Only free drug is pharmacologically active,therefore a high degree of serum protein binding for these compounds is desirable to limit potential systemic adverse events(55).The measurement of binding at the local site of action is very difficult,and assessment of therapeutic efficacy is the best way to ensure that sufficient unbound drug is available at the site of action.ConclusionsAn increasing body of scientific knowledge continues to accumulate about corticosteroids and their effects on the GR and cellular transcription processes.Despite this information,clinical distinctions between the individual compounds are not readily apparent.As a class,the INSs demonstrate comparable efficacy in treating AR condi-tions.It could be argued that the newer agents,flutica-sone propionate,mometasone furoate,ciclesonide,and fluticasone furoate,come remarkably close to the phar-macokinetic/pharmacodynamic criteria for the ideal INS because they have:1)a high degree of GR affinity, potency,and specificity;2)low systemic availability;3) high rate of hepaticfirst-pass clearance and rapid systemic elimination;and4)once-daily dosing.Even so, there continues to be a scientific pursuit of even more pharmacologicallyfine-tuned molecules.Whether such endeavors will produce new compounds with significantly distinguishing characteristics remains to be seen. AcknowledgmentsResearch for this article was funded through a grant from Schering-Plough Corporation.Neither author received any payment for this article.Dr Derendorf reports being a consultant(ad hoc)for Altana, GlaxoSmithKline,Sanofi-Aventis,and Schering-Plough.Dr Meltzer reports receiving grant/research support from Alcon, Allux,Altana,AstraZeneca,Capnia,Clay-Park,Critical Therapeu-tics,Genentech,GlaxoSmithKline,Hoffmann-LaRoche,KOS, Medicinova,MedPointe,Merck,Novartis,Pharmaxis,Rigel, Sanofi-Aventis,Schering-Plough,Teva,and Wyeth;being a con-sultant for Abbott,Adelphi,Alcon,Allux,Altana,Amgen,Astra-Zeneca,Capnia,Critical Therapeutics,Dey,Evolutec,Genentech, GlaxoSmithKline,Greer,Inspire,KOS,MedPointe,Merck,Nov-artis,Pfizer,Rigel,Sanofi-Aventis,Schering-Plough,Shionogi, Verus,and Wyeth;and being a speaker for AstraZeneca,Alcon, Altana,Genentech,Genesis,GlaxoSmithKline,MedPointe,Merck, Pfizer,Sanofi-Aventis,Schering-Plough,and Verus.Editorial assistance was provided by Adelphi Eden Health Communications.References1.Weiner JM,Abramson MJ,Puy RM.Intranasal corticosteroids versus oral H1 receptor antagonists in allergic rhinitis: systematic review of randomisedcontrolled trials.BMJ1998;317:1624–1629.2.Bousquet J,van Cauwenberge P,Khaltaev N,Aria Workshop Group;World Health Organization.Allergicrhinitis and its impact on asthma.JAllergy Clin Immunol2001;108(5Sup-pl):S147–S336.3.Dykewicz MS,Fineman S,Skoner DP,Nicklas R,Lee R,Blessing-Moore J et al.Diagnosis and management of rhinitis:complete guidelines of the Joint TaskForce on Practice Parameters in Allergy,Asthma,and Immunology.Ann AllergyAsthma Immunol1998;81:478–518.Derendorf and MeltzerÓ2008The Authors 1298Journal compilationÓ2008Blackwell Munksgaard Allergy2008:63:1292–1300。