深圳市福田区2004~2009年风疹流行病学论文

Vaccine(2008)26,460—468Intranasal administration of a recombinant adenovirus expressing the norovirus capsidprotein stimulates specific humoral,mucosal,and cellular immune responses in miceLi Guo a,1,Jianwei Wang a,b,∗,1,Hongli Zhou a,b,Hongli Si a,c,Min Wang a, Jingdong Song b,Bingjuan Han b,Yi Shu a,Lili Ren b,Jianguo Qu b,Tao Hung a,ba National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing100052,Chinab State Key Laboratory of Molecular Virology and Genetic Engineering,Institute of Pathogen Biology,Chinese Academy of Medical Sciences,9#Dong Dan San Tiao,Beijing100730,Chinac North-western A&F University,Yangling712100,ChinaReceived18May2007;received in revised form19October2007;accepted18November2007Available online4December2007KEYWORDSNorovirus capsidprotein;Adenovirus vector;Immune responseSummary Norovirus(NV)is a major cause of acute,epidemic nonbacterial gastroenteritis inindividuals of all ages.The immunological mechanism of NV infection and the approaches usedto prevent infection remain to be elucidated.In this study,the specific immune responses ofBALB/c mice were assessed following intranasal immunization with a recombinant adenovirusvector expressing the genogroup II4(GGII/4)norovirus capsid protein.Analysis of IgM,IgG,andIgA antibodies specific for the recombinant virus-like particles(VLPs)of NV demonstrated that ahigh level of humoral immunity developed following immunization.Mucosal immune responseswere also detectable in stool,intestinal homogenates,lung homogenates,and lung lavage sam-ples.Specific cellular immune responses were observed in NV VLPs-restimulated splenocytesby ELISPOT and Th1/Th2cytokine cytometric array(CBA).Serum IgG subclass analysis showedthat a balanced Th1-and Th2-like cellular immune response was induced in BALB/c mice follow-ing immunization with recombinant adenovirus.Thesefindings demonstrate that the intranasalimmunization of a recombinant adenovirus expressing the NV capsid protein is an efficient strat-egy to stimulate systemic,mucosal,and cellular Th1/Th2immune responses in mice,and couldserve as a novel approach for designing NV vaccines.©2007Elsevier Ltd.All rights reserved.∗Corresponding author at:State Key Laboratory of Molecular Virology and Genetic Engineering,Institute of Pathogen Biology,Chinese Academy of Medical Sciences,9#Dong Dan San Tiao,Beijing100730,China.T el.:+861065105188;fax:+861065105188.E-mail address:wangjw28@(J.Wang).1These authors contributed equally to this work.0264-410X/$—see front matter©2007Elsevier Ltd.All rights reserved.doi:10.1016/j.vaccine.2007.11.039Immunity against norovirus induced by recombinant adenovirus461IntroductionNorovirus(NV),or Norwalk-like virus,is a genus of the Caliciviridae family and is recognized as a major cause of acute,epidemic nonbacterial gastroenteritis in individuals of all ages[1,2].NV-related gastroenteritis outbreaks usu-ally occur in families,schools,nursing homes,hospitals,and military facilities through contact with contaminated food and water[2—4].While NV gastroenteritis is a mild self-limiting illness,it is associated with a high incidence of morbidity and hospitalization even in industrialized coun-tries.Recent studies have illustrated that∼90%of acute nonbacterial gastroenteritis outbreaks in the United States were caused by NV[5—7].In England and Wales,NV is responsible for almost50%of gastroenteritis outbreaks[8]. Finland,Sweden,Netherlands,Germany,and Japan have similar infectivity rates[9—12].In developing countries,NV-associated disease also takes an economic toll,especially in the young and very old[13,14].For these reasons,it is important to develop an effective NV vaccine especially for these at-risk populations[15].NV is composed of a7.4—8.3kb positive-sense,single-stranded RNA genome with three open reading frames (ORFs)[16,17].ORF1encodes a non-structural polyprotein that contains the3D RNA-dependent RNA polymerase region, ORF2encodes the viral capsid(VP-1),and ORF3encodes a minor basic virion protein(VP-2)[18].ORF2expres-sion by recombinant baculovirus in insect cells[19]or by the Venezuelan equine encephalitis virus replicon vector in mammalian cells[20,21]yields self-assembled virus-like particles(VLPs).These recombinant NV VLPs are morpho-logically and antigenically similar to the wild-type virus [20,21].Since cell culture systems and animal models of NVs are lacking[2],little is known about the details of the anti-NV immune response and few approaches have been designed to stimulate long-term,stable Th1/Th2-balanced immunity that would be required for a reliable prophylaxis. Recombinant VLPs may thus serve as an important source of antigen to study anti-NV immunity.Recent studies sug-gest that rNV VLPs can elicit systemic and mucosal immune responses in CD1and BALB/c mice,indicating that they may serve as a potential mucosal vaccine against NV[21—24]. However,these responses were only achieved by the addi-tion of mucosal adjuvants such as cholera toxin(CT)or the mutant E.coli heat-labile toxin,LT(R192G)[21—23].Since such adjuvants cannot be used in humans as a result of their potential toxicity,it is important to develop other immu-nization approaches to vaccine development.In this report,we determined whether intranasal immu-nization of mice with a recombinant adenovirus,rvAdGGII4, expressing the NV capsid,could elicit a strong anti-NV immune response,and illustrated a potential new approach for NV vaccine development.Materials and methodsPreparation of the recombinant adenovirus expressing NV capsid proteinThe GGII/4NV capsid protein gene from Lordsdale virus (Genbank X86557)was codon-optimized and artificially syn-thesized by adopting codons that were used in insect cells as described previously[25].The recombinant adenovirus, rvAdGGII4,was generated in HEK293cells using the AdEasy adenoviral vector system(Stratagene,Cedar Creek,TX) according to the manufacturer’s protocol.NV capsid pro-tein expression was confirmed by Western blot using an anti-NV capsid monoclonal antibody(Guo L.et al.,unpub-lished data).The recombinant adenovirus was purified by cesium chloride density gradient centrifugation[26],titered with an Adeno-X TM Rapid Titer Kit(BD Biosciences Clon-tech,Mountain View,CA)and stored at−70◦C prior to use.Preparation of NV-like particlesNV VLPs were prepared and purified as previously described, with minor modifications[27].The optimized GGII/4NV cap-sid gene was cloned into the baculovirus transfer vector, pFastBac1(Invitrogen,Carlsbad,CA)and a recombinant baculovirus was generated in Sf9cells using the Bac-to-Bac®Baculovirus Expression System(Invitrogen,Carlsbad,CA) protocol provided by the manufacturer.Recombinant bac-ulovirus infected Sf9cell supernatants were collected5days post-infection and cellular debris was removed by centrifu-gation(20min at10,000rpm).VLPs were precipitated from the clarified supernatants using PEG6000(6%),and the pel-lets were sonicated and centrifuged at35,000rpm through a40%sucrose cushion for3h.Purified VLPs were confirmed by Western blot using an anti-NV capsid monoclonal antibody [25].Protein concentrations were determined using the BCA Protein Assay Reagent Kit(Pierce,Rockford,IL)and stored at−70◦C prior to use.Animal immunization and sample collectionSix-to8-week-old female BALB/c mice,purchased from the Institute of Laboratory Animal Sciences at the Chinese Academy of Medical Sciences,were used for all immuniza-tions and maintained in Animal Biosafety Level-2facilities. After intranasal anesthetization with ether,mice were intranasally(i.n.)inoculated with1×106ifu(infectious units)(in0.1ml)of the recombinant adenovirus,rvAdG-GII4(rvAdGGII4group),1×106ifu(in0.1ml)of the empty recombinant adenovirus serotype5vector,which contained no insert(rvAd5-empty group,negative control),or0.1ml phosphate-buffered saline(PBS;PBS group,blank control) at day0.All mice were boosted at days14and28.Serum and fecal samples from all groups were col-lected at days0,14,28,and35post-inoculation(dpi),and stored at−20◦C prior to testing.At35dpi,the mice were sacrificed,and lung lavages,lung homogenates,and intesti-nal homogenates were used to measure mucosal immune responses as previously described[28]with minor modifica-tions.In brief,the lungs and20-cm sections of the small intestines excluding the Peyer’s patches were ground into homogenates using a high-speed tissue grinder,suspended in PBS(pH7.2)to produce a10%(w/v)suspension,and cen-trifuged at3000rpm for5min.The supernatants and lung lavages were stored at−70◦C prior to use.Splenocytes were also isolated and restimulated in order to measure cellular immune responses.462L.Guo et al.Analysis of NV-specific antibody production Antibodies in the sera,fecal matter,intestinal homogenates, lung homogenates,and lung lavages were measured by indi-rect ELISA.Microtiter plates(Costar,Bethesda,MD)were coated with0.1␮g VLPs from GGII/4NV per well at4◦C overnight.The plates were blocked with1%(w/v)BSA in PBS at37◦C for2h.The fecal samples were diluted into a10%(w/v)suspension in PBS.Serum and fecal extracts were twofold serially diluted using0.1%BSA in PBS.Intesti-nal homogenates,lung homogenates,and lung lavages were diluted1:10.Samples were added to the wells and incubated for1h at37◦C.After washingfive times with0.05%T ween 20in PBS(PBS-T),the plates were incubated at37◦C for1h using a1:5000dilution of horseradish peroxidase-conjugated goat anti-mouse IgG,IgA,IgM,IgG1,or IgG2a(Sigma, St.Louis,MO).Plates were washed and developed with 100␮l/well of0.1mg/ml3,3 ,5,5 -tetramethylbenzidine (TMB;Sigma,St.Louis,MO)peroxidase substrate containing 0.03%hydrogen peroxide.Color development was stopped by the addition of50␮l/well of2M sulfuric acid.The absorbency at450nm(A450)was determined using an ELISA plate reader(BioRad550,Hercules,CA).IFN-␥ELISPOT assayMurine spleens were removed and ground under sterile conditions using a5-ml syringe plunger,and splenocytes were isolated with Mouse Lymphoprep(Dakewe,Shenzhen, China).The freshly made splenocytes were washed twice and resuspended in R10medium[RPMI1640supplemented with10%fetal bovine serum(FBS),100IU/ml penicillin, and100␮g/ml streptomycin(Hyclone,Logan,UT)].IFN-␥ELISPOT assays were performed using an ELISPOT Set(BD Biosciences,San Diego,CA)as recommended by the manu-facturer.In brief,5×105freshly isolated splenocytes were added to each of two replicate wells coated with anti-mouse IFN-␥monoclonal antibody(MAb)(BD Biosciences,Catalog No.51-2525kc)and stimulated with20␮g/ml VLPs at37◦C in a5%CO2incubator for24h.Unstimulated splenocytes were used to measure background cytokine production.The cells were then lysed with deionized(DI)water,and the plates were incubated at room temperature with biotinylated IFN-␥antibody(BD Biosciences,Catalog No.51-1818kz)for2h, and peroxidase-labeled streptavidin(BD Biosciences,Cata-log No.51-9000209)for1h.After washing with PBS,100␮l of thefinal substrate solution(BD Biosciences,Catalog No. 551951)was added to each well and spot development was monitored.The plates were washed with distilled water to stop the reaction.IFN-␥spot-forming cells(SFC)were counted using an automated ELISPOT plate reader system. Results were expressed as the number of SFC/106cells. Assay for multi-cytokine productionFreshly isolated murine splenocytes were cultured in a96-well,round-bottom tissue culture plate at5×105cells/well in R10medium and stimulated with NV VLPs for48h.Super-natants were collected and IL-2,IL-4,IL-5,TNF-␣,and IFN-␥levels were quantitated using the mouse Th1/Th2Cytokine Cytometric Array Bead(CBA)Kit(BD PharMingen,San Diego,CA)according to the manufacturer’s protocol.The data were acquired with a FACSCalibur®flow cytometer(BD Bio-sciences,San Jose,CA)using2-color detection and analyzed using CBA software(BD PharMingen).Analysis of anti-adenoviral serotype5(Ad5) immunityThe anti-Ad5serum IgG,IgM,and IgA antibodies were ana-lyzed by indirect ELISA as described in Section of Analysis of NV-specific antibody production.The microtiter plates (Costar,Bethesda,MD)were coated with1×105ifu(in 0.1ml)of wild serotype5adenovirus,which was puri-fied using the CsCl2cushion ultra-centrifuge[26],at4◦C overnight.Statistical analysisSerum and fecal antibody titers were log10transformed, expressed as geometric mean titers(GMT s),and com-pared using the Student’s t-test.The Wilcoxon rank sum test was used to compare intestinal homogenates,lung homogenates,and lung lavage antibody levels between groups.ELISPOT and CBA results between groups were compared using the Student’s t-test.Nonresponders were included in all calculations.The lowest serum and fecal sample dilution(1:50)was divided by two and used as the negative sample titer(i.e.,negative samples were assigned a titer of25).All tests were two-tailed,and a value of p<0.05was considered statistically significant.ResultsHumoral immune responses in miceT o determine whether the recombinant adenovirus,rvAdG-GII4,could stimulate specific immune responses following i.n.administration,BALB/c mice were immunized three times on days0,14,and28,respectively.The control groups were treated with rvAd5-empty or PBS accord-ing to the same scheme.All preimmune serum titers of IgM,IgA,and IgG antibodies against norovirus VLPs or adenovirus were negative(<50)(data not shown).In addi-tion,each of the control mice remained negative for VLP antibodies at35dpi.After thefirst i.n.administra-tion of rvAdGGII4,however,100%of the mice had strong specific serum IgM titers(GMT=315.9).The GMT of IgM peaked(GMT=348.2)after the second immunization(28dpi) and decreased(GMT=219.1)after the third immunization (35dpi)(Fig.1A).Serum IgG responses were mounted by allfive mice at day14post-immunization with rvAdGGII4 (GMT=3816,ranging from3200to10,000).After the sec-ond(28dpi)and the third immunizations(35dpi),IgG titers continued to increase,with GMT s of155,261and632,978, respectively(Fig.1B).No mice had detectable levels of serum IgA antibodies by14dpi.Serum-specific IgA anti-body titers were detected in all groups after the second immunization(GMT=377.6,ranging from300to800)and increased after the third immunization(GMT=2009.5,rang-ing from1600to3200)(Fig.1C).IgM,IgG,and IgA responsesImmunity against norovirus induced by recombinant adenovirus463Figure 1Humoral responses in mice sera.BALB/c mice were immunized as described in Section 2.Blood samples were col-lected at 14,28,and 35dpi,respectively .Serum-specific IgM (A),IgG (B),and IgA (C)antibodies from individual mice were examined by ELISA and used to calculate the GMT for each group.For the IgM-negative,IgG-negative,and IgA-negative (<50)control samples,titers of 25were used to calculate the GMT .Days post-immunization schedule are shown on the X-axis.Error bars represent the standard errors of the means.Above each bar is the number of responders over the total number of mice tested.were undetectable in mice treated with the rvAd5-empty vector or PBS.These data indicate that rvAdGGII4stimu-lates a strong NV specific humoral response and that serum antibodies were substantially enhanced following rvAdGGII4immunization.Figure 2Fecal IgA and IgG responses in BALB/c mice.Murine fecal samples were collected at 35dpi.Levels of specific IgG and IgA antibodies in the feces were measured by ELISA and used to calculate the GMTs.The error bars show the standard errors of the mean.The number above each bar depicts the number of responders over the total number of animals tested.Serum IgG subclass analysisAnti-VLPs IgG1and IgG2a antibodies were measured in NV VLP-specific IgG positive sera (T able 1).The increase in IgG1and IgG2a levels coincided with the rise in serum IgG.Sig-nificantly high titers of both IgG1and IgG2a were observed following administration of rvAdGGII4,and there was no significant difference between these class types (p >0.05,Student’s t -test).Thus,the Th1-(IgG2a)and Th2-like (IgG1)immune responses were roughly balanced in rvAdGGII4-immunized mice.Mucosal responses in miceT o evaluate whether recombinant adenovirus could stimu-late specific mucosal responses,fecal suspensions,intestinal homogenates,lung homogenates,and lung lavages were prepared from immunized mice.Mucosal immune responses to NV VLPs were observed in all vaccinated mice fol-lowing primary i.n.administration of 1×106ifu rvAdGGII4and after each rvAdGGII4boost immunization.All immu-nized mice (5/5)had NV VLP-specific fecal IgG and IgA responses (GMT =470.43and 819.07,respectively),while the rvAd5-empty and PBS groups remained nega-tive (titers <50)(Fig.2).IgA and IgG levels in intestinal homogenates,lung homogenates,and lung lavages were evaluated at a single dilution (1:10),and results were expressed as A 450values.Strong IgA and IgG responses were induced in the intestinal homogenates (p <0.05for IgA,p <0.001for IgG,respectively),lung homogenates (p <0.001for IgA and IgG,respectively)and lung lavage samples (p <0.001for IgA and IgG,respectively)(Fig.3).TheseTable 1Analysis of serum Ig antibody isotypes to norovirus VLPsImmunogenDays post-immunizationGeometric mean titer IgG1/IgG2aIgG1IgG2a rvAdGGII435201189.380546.30.539rvAd5-empty 35<50<50PBS 35<50<50464L.Guo etal.Figure3Levels of specific IgA(A)and IgG(B)antibodies in the intestines and lungs of BALB/c mice immunized with rvAdGGII4. At days14and28following the primary immunization,each group of mice was administered the same amount of antigen, and intestinal homogenates,lung homogenates,and lung lavage were obtained from each animal1week later.Levels of specific IgA and IgG antibodies were examined by ELISA.The results are represented as the mean OD(450nm)that was detected in individual mice at a1:10dilution.Error bars represent the standard errors of the mean.data suggest that rvAdGGII4elicits strong mucosal immune responses.IFN-␥ELISPOT responses and cytokine assaysT o measure the cellular immune responses elicited by i.n. administration of rvAdGGII4,the specific T-cell responses against NV VLPs were monitored in immunized mice by IFN-␥ELISPOT.The mean number of SFC/106in the rvAdGGII4 group was108±31.No spots were detected in unstimulated splenocytes from the rvAdGGII4group.The level of SFC/106 in the rvAd5-empty and PBS control groups were14±2and 9±7,respectively.The differences between the rvAdGGII4 group and the rvAd5-empty or PBS groups was statistically significant(p<0.01and0.001,respectively)(Fig.4).These findings suggest that mice receiving the rvAdGGII4vector developed cellular immune responses to the NV capsid pro-tein.T o further characterize the cellular immune response, rvAdGGII4immunized splenocytes were isolated and T-cell responses were assessed using a Mouse Th1/Th2Cytokine Cytometric Bead Array system.This allows simultaneous measurement of TNF-␣,IFN-␥,IL-5,IL-4,and IL-2lev-els in antigen-stimulated T-cell supernatants.After the mice were immunized with rvAdGGII4and stimulated with NV VLPs,IFN-␥,TNF-␣,and IL-5production were notably high(217.71pg/ml,156.5pg/ml and265.27pg/ml,respec-tively),while IL-2and IL4levels were only moderately increased(32.2pg/ml and27.59pg/ml,respectively).TNF-Figure4IFN-␥ELISPOT assays of recombinant norovirus VLP-stimulated splenocytes following rvAdGGII4immunization. Splenocytes were isolated7days after thefinal immunization and restimulated with20␮g/ml VLP for24h.IFN-␥-producing cells were quantitated using an ELISPOT reader.Results were expressed as the average number of SFC per million input splenocytes.Error bars represent the standard errors of the mean.␣,IFN-␥,IL-5,IL-4,and IL-2levels were significantly higher in splenocyte supernatants from rvAdGGII4immu-nized mice than from rvAd5-empty vector(33.63pg/ml, p≤0.001;2.43pg/ml,p<0.001;0pg/ml,p<0.001;0pg/ml, p<0.001and 3.09pg/ml,p<0.05,respectively)or PBS-treated mice(36.22pg/ml,p<0.05;1.22pg/ml,p<0.001;0.42pg/ml,p<0.001;0pg/ml,p<0.001and 1.19pg/ml, p<0.01,respectively)(Fig.5).Since IFN-␥,TNF-␣,and IL-2 are markers of a Th1response,while IL-5and IL-4are mark-ers of a Th2response,these results indicated that the Th1-and Th2-like cellular immune responses were both stimu-lated by i.n.administration of rvAdGGII4.Anti-Ad5antibodies in miceT o determine whether the recombinant adenovirus,rvAdG-GII4,could elicit anti-Ad5immune responses following i.n. administration,anti-Ad5serum antibody levels were mea-sured by indirect ELISA.All immunized mice hadanti-Ad5 Figure5Cytokines detected in splenocyte cultures from mice immunized with rvAdGGII4.Splenocytes were isolated7days after thefinal immunization and stimulated with NV VLPs for 48h.TNF-␣,IFN-␥,IL-5,IL-4,and IL-2levels were measured in the culture supernatant using the Cytometric Bead Array. Significant differences were observed between the rvAdGGII4 and rvAd5-empty or PBS group.Error bars indicate the standard errors of the mean.Immunity against norovirus induced by recombinant adenovirus465Figure6Anti-Ad5immunity in mice after rvAdGGII4immu-nization.Blood samples were collected at14,28,and35dpi, respectively.Anti-Ad5antibodies were assessed by ELISA and used to calculate the GMT s.The days post-immunization are shown on the X-axis,and the error bars reveal the standard errors of the mean.The number above each bar depicts the number of responders over the total number of animals tested.serum IgG responses following the primary i.n.rvAdGGII4 administration and boost(GMT=299.3,1912.7and6964.4, respectively)(Fig.6).The IgM responses were not observed in mice after thefirst administration.The GMT of IgM was 65.9after the second immunization(28dpi),and decreased to<50after the third immunization(35dpi)(Fig.6).While no detectable anti-Ad5IgA antibodies were found in the sera after thefirst and second immunizations,anti-Ad5serum-specific IgA antibody titers could still be detected in mice after the third immunization(GMT=269.9)(Fig.6).DiscussionSince NV infections are localized in the gastrointestinal tract,local mucosal immunity is believed to play an impor-tant role in protection against infection.It is accepted that an ideal NV vaccine would induce strong mucosal immu-nity[29].In previous studies,anti-NV immunity using VLPs primarily relied on the adjuvants like enterotoxin-based LT or CT,since VLPs are too weak to stimulate immune responses,especially within the mucosa.However,CT and LT are potentially toxic to the human central nervous sys-tem[30].An intranasal influenza vaccine containing LT as a mucosal adjuvant,which resulted in46cases of Bell’s palsy in Switzerland,was recently removed from the mar-ket[31],indicating that CT or LT adjuvants are not suitable for humans.Thus,it is important to develop a novel immu-nization strategy that does not require the use of mucosal adjuvants.The replication-defective adenovirus serotype5vector is recognized as an attractive vector in both gene ther-apy and novel genetic vaccine development[32].Its safety and efficacy is shown in numerous animal experiments and human clinical trials,and two adenovirus-based gene ther-apy drugs are licensed.Adenovirus vectors have been used in studies of potential Ebola virus,human immunodefi-ciency virus type1(HIV-1),influenzavirus,and tuberculosis vaccines[33—39].Clinical trials using adenoviral-vectored vaccines against influenza virus,HIV-1,some malignant tumors,and other pathogens are currently in progress [40—43].These clinical trials show that adenoviral vector-based vaccines are safe,effective,and have no known toxicity or inflammatory properties.Adenovirus vectors are also shown to boost CTL responses against heterologous gene products[44—47].However,existing anti-adenoviral immune responses impair foreign antigen expression after repeated intramuscular immunizations.Since humans are widely infected by adenoviruses and usually possess high anti-adenovirus antibody levels,existing immunity often limits application in cases where repeated immunization is required.Recently,intranasal immunization has become an attractive method for inducing multiple immunities.Thus, we tested the immunogenicity of the NV capsid protein after repeated i.n.administration.We illustrated that sig-nificant serum,mucosal,and cellular immune responses against NV VLPs were elicited after mice were immu-nized with the recombinant adenovirus.Serum IgM and IgG responses against NV VLPs rose remarkably after the first rvAdGGII4inoculation,and IgM,IgG,and IgA antibody titers increased to high levels after the third immunization. IgA and IgG responses were also observed in fecal sam-ples,intestinal homogenates,lung homogenates,and lung samples.Thesefindings suggest that the i.n.route is an effi-cient pathway to evoke NV capsid-specific immunity by an adenovirus vector and the regionalization of the mucosal immune system allows immunizations in the gut by deliver-ing a vaccine through the intranasal route.The results also show that repeated i.n.administration of the adenovirus vector effectively boosted NV capsid-specific serum antibod-ies,demonstrating that existing anti-adenoviral immunity did not impair the recombinant adenovirus-specific immune response.Thesefindings are similar to our results using an adenovirus vectored rotavirus antigen[48].Unexpectedly,intestinal homogenates from the rvAd5-empty and PBS control-treated mice produced low SIgA levels.We speculate that there may have been low SIgA levels in the mouse intestine.The intestine is the largest immunological organ,housing70—80%of all immunoglobulin-producing cells and producing large amounts of SIgA(50—100mg/kg body weight)[49].High background on the ELISA assay may be attributed to the interference of intestinal SIgA or other intestinal contents.Previous studies showed that cytotoxic T lymphocytes (CTLs)play an important role in preventing viral diseases and clearing viruses like hepatitis C[50].Cellular immune response may also be crucial for disease prevention,as well as control and clearance of NV infection.Lindesmith et al.first characterized the Th1and Th2cellular immune responses to a challenge with Snow Mountain virus[51]. Recent studies showed that BALB/c mice that were i.n. immunized with VLPs and the mucosal adjuvants,LT or LT (R192G),induced specific Th1/Th2-like immune responses [27].However,there has been little information regarding the anti-NV cellular immune response.T o measure the cel-lular immune responses in rvAdGGII4-immunized mice,we measured NV VLP-specific T-cell responses using the IFN-␥ELISPOT assay.Our results indicate that the recombinant adenovirus,rvAdGGII4,was able to induce a strong cellular immune response.Importantly,both Th1-and Th2-like cel-lular immune responses were induced in rvAdGGII4-treated mice.It is well known that Th1-associated cytokines help to regulate antiviral cellular responses,while Th2-associated cytokines enhance humoral immune responses.In our study, TNF-␣and IFN-␥secretion were significantly increased,and IL-2levels were moderately increased.At the same time,466L.Guo et al.the Th2cytokine,IL-5,which promotes IgA production, was significantly elevated,afinding consistent with high serum and mucosal IgA antibody titers.IL-4levels were also bined with analysis of serum IgG1/IgG2a lev-els,these data suggest that a roughly balanced Th1/Th2 immune response is induced by the adenovirus vectored NV capsid.Due to the unavailability of an animal model of viral chal-lenge,it remains unknown whether the immunity evoked by i.n.adenovirus immunization is capable of protecting against NV infection.However,we may anticipate that it would have a protective effect based on knowledge obtained from studies of other viral infections like rotavirus,which also infect the intestinal surface.Evidence suggests that high local mucosal immune responses against rotavirus are critical for resisting viral infection.Previous studies showed that serum and fecal IgA antibodies are associated with protection against rotavirus reinfection[52,53].Moreover, about a recent study showed that30—40%of volunteers developed mucosal anti-VLP IgA after oral NV VLP immuniza-tion[54].Thus,it is reasonable to speculate that high levels of immunity stimulated by recombinant adenoviruses play an important role in preventing NV infection[21].However, the precise level of protection will require further challenge experiments using volunteer or surrogate challenge models, such as recombinant vaccinia virus,that have been used to study hepatitis C virus(HCV)[55]and hantaan virus infec-tion[56].Further studies will also be required to determine the duration of immunity,and the relationships between the three immune responses,including their cross-protective role with other noroviruses.Although the regionalization of the mucosal immune sys-tem permits gut immunizations through intranasal delivery of a vaccine,swallowing of the inoculum by the vaccinated animals cannot be excluded.Since NV attacks and repli-cates in the small intestine,local immunization of the gut may help to protect the host from NV infection.The experi-ence of Adenovirus4and7in the US Army showed that oral immunization is safe and can evoke strong immune responses over the past four decades[57].Thus,oral immunization may be a potential way to stimulate NV-specific immunities using recombinant adenovirus immunization.In addition, oral immunization is easier to manage and may be better accepted by children.A recent study showed that oral immu-nization of NV VLPs can produce cross-reactive monoclonal antibodies[58].Thus,the feasibility of oral immunization using the NV capsid protein-expressing recombinant aden-ovirus should be addressed.AcknowledgementsWe are grateful to Dr.Zhendong Zhao(Peking University)for critical reading of the manuscript,Drs.Li Ruan and Xian-grong Qi(National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Preven-tion)for their assistance in ELISPOT assay,and Ms.Shan Mei and Li Li(Institute of Pathogen Biology,Chinese Academy of Medical Sciences)for their assistance in FACS assays.This research was supported by grants from the Chinese Key T ech-nologies R&D Program(No.2003BA712A03—04)and China Nature Science Foundation(No.30700741).References[1]Hedberg CW,Osterholm MT.Outbreaks of food-borneand waterborne viral gastroenteritis.Clin Microbiol Rev 1993;6(3):199—210.[2]Kapikian AZ,Estes MK,Chanock RM.Norwalk group of viruses.In:Fields B,editor.Fields Virology,vol.2,3rd ed.1996.p.783—810.[3]Arness MK,Feighner BH,Canham ML,T aylor DN,Monroe SS,Cieslak TJ,et al.Norwalk-like viral gastroenteritis outbreak in U.S.Army trainees.Emerg Infect Dis2000;6(2):204—7.[4]Koopmans M,Vinjeˇıde Wit M,Leenen I,van der Poel W,van Duynhoven Y.Molecular epidemiology of human enteric caliciviruses in The Netherlands.J Infect Dis2000;181(Suppl2):S262—9.[5]Fankhauser RL,Noel JS,Monroe SS,Ando T,Glass RI.Molecular epidemiology of‘‘Norwalk-like viruses’’in out-breaks of gastroenteritis in the United States.J Infect Dis 1998;178(6):1571—8.[6]Glass RI,Noel J,Ando T,Fankhauser R,Belliot G,MountsA,et al.The 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DOI: 10.1126/science.1094786, 441 (2004);304Science et al.Mitchell S. Abrahamsen,Cryptosporidium parvum Complete Genome Sequence of the Apicomplexan, (this information is current as of October 7, 2009 ):The following resources related to this article are available online at/cgi/content/full/304/5669/441version of this article at:including high-resolution figures, can be found in the online Updated information and services,/cgi/content/full/1094786/DC1 can be found at:Supporting Online Material/cgi/content/full/304/5669/441#otherarticles , 9 of which can be accessed for free: cites 25 articles This article 239 article(s) on the ISI Web of Science. cited by This article has been /cgi/content/full/304/5669/441#otherarticles 53 articles hosted by HighWire Press; see: cited by This article has been/cgi/collection/genetics Genetics: subject collections This article appears in the following/about/permissions.dtl in whole or in part can be found at: this article permission to reproduce of this article or about obtaining reprints Information about obtaining registered trademark of AAAS.is a Science 2004 by the American Association for the Advancement of Science; all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m3.R.Jackendoff,Foundations of Language:Brain,Gram-mar,Evolution(Oxford Univ.Press,Oxford,2003).4.Although for Frege(1),reference was established rela-tive to objects in the world,here we follow Jackendoff’s suggestion(3)that this is done relative to objects and the state of affairs as mentally represented.5.S.Zola-Morgan,L.R.Squire,in The Development andNeural Bases of Higher Cognitive Functions(New York Academy of Sciences,New York,1990),pp.434–456.6.N.Chomsky,Reflections on Language(Pantheon,New York,1975).7.J.Katz,Semantic Theory(Harper&Row,New York,1972).8.D.Sperber,D.Wilson,Relevance(Harvard Univ.Press,Cambridge,MA,1986).9.K.I.Forster,in Sentence Processing,W.E.Cooper,C.T.Walker,Eds.(Erlbaum,Hillsdale,NJ,1989),pp.27–85.10.H.H.Clark,Using Language(Cambridge Univ.Press,Cambridge,1996).11.Often word meanings can only be fully determined byinvokingworld knowledg e.For instance,the meaningof “flat”in a“flat road”implies the absence of holes.However,in the expression“aflat tire,”it indicates the presence of a hole.The meaningof“finish”in the phrase “Billfinished the book”implies that Bill completed readingthe book.However,the phrase“the g oatfin-ished the book”can only be interpreted as the goat eatingor destroyingthe book.The examples illustrate that word meaningis often underdetermined and nec-essarily intertwined with general world knowledge.In such cases,it is hard to see how the integration of lexical meaning and general world knowledge could be strictly separated(3,31).12.W.Marslen-Wilson,C.M.Brown,L.K.Tyler,Lang.Cognit.Process.3,1(1988).13.ERPs for30subjects were averaged time-locked to theonset of the critical words,with40items per condition.Sentences were presented word by word on the centerof a computer screen,with a stimulus onset 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critical word.This was done in order tonormalize for individual differences in EEG power anddifferences in baseline power between different fre-quency bands.Two relevant time-frequency compo-nents were identified:(i)a theta component,rangingfrom4to7Hz and from300to800ms after wordonset,and(ii)a gamma component,ranging from35to45Hz and from400to600ms after word onset.20.C.Tallon-Baudry,O.Bertrand,Trends Cognit.Sci.3,151(1999).tner et al.,Nature397,434(1999).22.M.Bastiaansen,P.Hagoort,Cortex39(2003).23.O.Jensen,C.D.Tesche,Eur.J.Neurosci.15,1395(2002).24.Whole brain T2*-weighted echo planar imaging bloodoxygen level–dependent(EPI-BOLD)fMRI data wereacquired with a Siemens Sonata1.5-T magnetic reso-nance scanner with interleaved slice ordering,a volumerepetition time of2.48s,an echo time of40ms,a90°flip angle,31horizontal slices,a64ϫ64slice matrix,and isotropic voxel size of3.5ϫ3.5ϫ3.5mm.For thestructural magnetic resonance image,we used a high-resolution(isotropic voxels of1mm3)T1-weightedmagnetization-prepared rapid gradient-echo pulse se-quence.The fMRI data were preprocessed and analyzedby statistical parametric mappingwith SPM99software(http://www.fi/spm99).25.S.E.Petersen et al.,Nature331,585(1988).26.B.T.Gold,R.L.Buckner,Neuron35,803(2002).27.E.Halgren et al.,J.Psychophysiol.88,1(1994).28.E.Halgren et al.,Neuroimage17,1101(2002).29.M.K.Tanenhaus et al.,Science268,1632(1995).30.J.J.A.van Berkum et al.,J.Cognit.Neurosci.11,657(1999).31.P.A.M.Seuren,Discourse Semantics(Basil Blackwell,Oxford,1985).32.We thank P.Indefrey,P.Fries,P.A.M.Seuren,and M.van Turennout for helpful discussions.Supported bythe Netherlands Organization for Scientific Research,grant no.400-56-384(P.H.).Supporting Online Material/cgi/content/full/1095455/DC1Materials and MethodsFig.S1References and Notes8January2004;accepted9March2004Published online18March2004;10.1126/science.1095455Include this information when citingthis paper.Complete Genome Sequence ofthe Apicomplexan,Cryptosporidium parvumMitchell S.Abrahamsen,1,2*†Thomas J.Templeton,3†Shinichiro Enomoto,1Juan E.Abrahante,1Guan Zhu,4 Cheryl ncto,1Mingqi Deng,1Chang Liu,1‡Giovanni Widmer,5Saul Tzipori,5GregoryA.Buck,6Ping Xu,6 Alan T.Bankier,7Paul H.Dear,7Bernard A.Konfortov,7 Helen F.Spriggs,7Lakshminarayan Iyer,8Vivek Anantharaman,8L.Aravind,8Vivek Kapur2,9The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals,and causes an unrelenting infection in immuno-compromised individuals such as AIDS patients.We report the complete ge-nome sequence of C.parvum,type II isolate.Genome analysis identifies ex-tremely streamlined metabolic pathways and a reliance on the host for nu-trients.In contrast to Plasmodium and Toxoplasma,the parasite lacks an api-coplast and its genome,and possesses a degenerate mitochondrion that has lost its genome.Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected.Elu-cidation of the core metabolism,including enzymes with high similarities to bacterial and plant counterparts,opens new avenues for drug development.Cryptosporidium parvum is a globally impor-tant intracellular pathogen of humans and animals.The duration of infection and patho-genesis of cryptosporidiosis depends on host immune status,ranging from a severe but self-limiting diarrhea in immunocompetent individuals to a life-threatening,prolonged infection in immunocompromised patients.Asubstantial degree of morbidity and mortalityis associated with infections in AIDS pa-tients.Despite intensive efforts over the past20years,there is currently no effective ther-apy for treating or preventing C.parvuminfection in humans.Cryptosporidium belongs to the phylumApicomplexa,whose members share a com-mon apical secretory apparatus mediating lo-comotion and tissue or cellular invasion.Many apicomplexans are of medical or vet-erinary importance,including Plasmodium,Babesia,Toxoplasma,Neosprora,Sarcocys-tis,Cyclospora,and Eimeria.The life cycle ofC.parvum is similar to that of other cyst-forming apicomplexans(e.g.,Eimeria and Tox-oplasma),resulting in the formation of oocysts1Department of Veterinary and Biomedical Science,College of Veterinary Medicine,2Biomedical Genom-ics Center,University of Minnesota,St.Paul,MN55108,USA.3Department of Microbiology and Immu-nology,Weill Medical College and Program in Immu-nology,Weill Graduate School of Medical Sciences ofCornell University,New York,NY10021,USA.4De-partment of Veterinary Pathobiology,College of Vet-erinary Medicine,Texas A&M University,College Sta-tion,TX77843,USA.5Division of Infectious Diseases,Tufts University School of Veterinary Medicine,NorthGrafton,MA01536,USA.6Center for the Study ofBiological Complexity and Department of Microbiol-ogy and Immunology,Virginia Commonwealth Uni-versity,Richmond,VA23198,USA.7MRC Laboratoryof Molecular Biology,Hills Road,Cambridge CB22QH,UK.8National Center for Biotechnology Infor-mation,National Library of Medicine,National Insti-tutes of Health,Bethesda,MD20894,USA.9Depart-ment of Microbiology,University of Minnesota,Min-neapolis,MN55455,USA.*To whom correspondence should be addressed.E-mail:abe@†These authors contributed equally to this work.‡Present address:Bioinformatics Division,Genetic Re-search,GlaxoSmithKline Pharmaceuticals,5MooreDrive,Research Triangle Park,NC27009,USA.R E P O R T S SCIENCE VOL30416APRIL2004441o n O c t o b e r 7 , 2 0 0 9 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mthat are shed in the feces of infected hosts.C.parvum oocysts are highly resistant to environ-mental stresses,including chlorine treatment of community water supplies;hence,the parasite is an important water-and food-borne pathogen (1).The obligate intracellular nature of the par-asite ’s life cycle and the inability to culture the parasite continuously in vitro greatly impair researchers ’ability to obtain purified samples of the different developmental stages.The par-asite cannot be genetically manipulated,and transformation methodologies are currently un-available.To begin to address these limitations,we have obtained the complete C.parvum ge-nome sequence and its predicted protein com-plement.(This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the project accession AAEE00000000.The version described in this paper is the first version,AAEE01000000.)The random shotgun approach was used to obtain the complete DNA sequence (2)of the Iowa “type II ”isolate of C.parvum .This isolate readily transmits disease among numerous mammals,including humans.The resulting ge-nome sequence has roughly 13ϫgenome cov-erage containing five gaps and 9.1Mb of totalDNA sequence within eight chromosomes.The C.parvum genome is thus quite compact rela-tive to the 23-Mb,14-chromosome genome of Plasmodium falciparum (3);this size difference is predominantly the result of shorter intergenic regions,fewer introns,and a smaller number of genes (Table 1).Comparison of the assembled sequence of chromosome VI to that of the recently published sequence of chromosome VI (4)revealed that our assembly contains an ad-ditional 160kb of sequence and a single gap versus two,with the common sequences dis-playing a 99.993%sequence identity (2).The relative paucity of introns greatly simplified gene predictions and facilitated an-notation (2)of predicted open reading frames (ORFs).These analyses provided an estimate of 3807protein-encoding genes for the C.parvum genome,far fewer than the estimated 5300genes predicted for the Plasmodium genome (3).This difference is primarily due to the absence of an apicoplast and mitochondrial genome,as well as the pres-ence of fewer genes encoding metabolic functions and variant surface proteins,such as the P.falciparum var and rifin molecules (Table 2).An analysis of the encoded pro-tein sequences with the program SEG (5)shows that these protein-encoding genes are not enriched in low-complexity se-quences (34%)to the extent observed in the proteins from Plasmodium (70%).Our sequence analysis indicates that Cryptosporidium ,unlike Plasmodium and Toxoplasma ,lacks both mitochondrion and apicoplast genomes.The overall complete-ness of the genome sequence,together with the fact that similar DNA extraction proce-dures used to isolate total genomic DNA from C.parvum efficiently yielded mito-chondrion and apicoplast genomes from Ei-meria sp.and Toxoplasma (6,7),indicates that the absence of organellar genomes was unlikely to have been the result of method-ological error.These conclusions are con-sistent with the absence of nuclear genes for the DNA replication and translation machinery characteristic of mitochondria and apicoplasts,and with the lack of mito-chondrial or apicoplast targeting signals for tRNA synthetases.A number of putative mitochondrial pro-teins were identified,including components of a mitochondrial protein import apparatus,chaperones,uncoupling proteins,and solute translocators (table S1).However,the ge-nome does not encode any Krebs cycle en-zymes,nor the components constituting the mitochondrial complexes I to IV;this finding indicates that the parasite does not rely on complete oxidation and respiratory chains for synthesizing adenosine triphosphate (ATP).Similar to Plasmodium ,no orthologs for the ␥,␦,or εsubunits or the c subunit of the F 0proton channel were detected (whereas all subunits were found for a V-type ATPase).Cryptosporidium ,like Eimeria (8)and Plas-modium ,possesses a pyridine nucleotide tran-shydrogenase integral membrane protein that may couple reduced nicotinamide adenine dinucleotide (NADH)and reduced nico-tinamide adenine dinucleotide phosphate (NADPH)redox to proton translocation across the inner mitochondrial membrane.Unlike Plasmodium ,the parasite has two copies of the pyridine nucleotide transhydrogenase gene.Also present is a likely mitochondrial membrane –associated,cyanide-resistant alter-native oxidase (AOX )that catalyzes the reduction of molecular oxygen by ubiquinol to produce H 2O,but not superoxide or H 2O 2.Several genes were identified as involved in biogenesis of iron-sulfur [Fe-S]complexes with potential mitochondrial targeting signals (e.g.,nifS,nifU,frataxin,and ferredoxin),supporting the presence of a limited electron flux in the mitochondrial remnant (table S2).Our sequence analysis confirms the absence of a plastid genome (7)and,additionally,the loss of plastid-associated metabolic pathways including the type II fatty acid synthases (FASs)and isoprenoid synthetic enzymes thatTable 1.General features of the C.parvum genome and comparison with other single-celled eukaryotes.Values are derived from respective genome project summaries (3,26–28).ND,not determined.FeatureC.parvum P.falciparum S.pombe S.cerevisiae E.cuniculiSize (Mbp)9.122.912.512.5 2.5(G ϩC)content (%)3019.43638.347No.of genes 38075268492957701997Mean gene length (bp)excluding introns 1795228314261424ND Gene density (bp per gene)23824338252820881256Percent coding75.352.657.570.590Genes with introns (%)553.9435ND Intergenic regions (G ϩC)content %23.913.632.435.145Mean length (bp)5661694952515129RNAsNo.of tRNA genes 454317429944No.of 5S rRNA genes 6330100–2003No.of 5.8S ,18S ,and 28S rRNA units 57200–400100–20022Table parison between predicted C.parvum and P.falciparum proteins.FeatureC.parvum P.falciparum *Common †Total predicted proteins380752681883Mitochondrial targeted/encoded 17(0.45%)246(4.7%)15Apicoplast targeted/encoded 0581(11.0%)0var/rif/stevor ‡0236(4.5%)0Annotated as protease §50(1.3%)31(0.59%)27Annotated as transporter ࿣69(1.8%)34(0.65%)34Assigned EC function ¶167(4.4%)389(7.4%)113Hypothetical proteins925(24.3%)3208(60.9%)126*Values indicated for P.falciparum are as reported (3)with the exception of those for proteins annotated as protease or transporter.†TBLASTN hits (e Ͻ–5)between C.parvum and P.falciparum .‡As reported in (3).§Pre-dicted proteins annotated as “protease or peptidase”for C.parvum (CryptoGenome database,)and P.falciparum (PlasmoDB database,).࿣Predicted proteins annotated as “trans-porter,permease of P-type ATPase”for C.parvum (CryptoGenome)and P.falciparum (PlasmoDB).¶Bidirectional BLAST hit (e Ͻ–15)to orthologs with assigned Enzyme Commission (EC)numbers.Does not include EC assignment numbers for protein kinases or protein phosphatases (due to inconsistent annotation across genomes),or DNA polymerases or RNA polymerases,as a result of issues related to subunit inclusion.(For consistency,46proteins were excluded from the reported P.falciparum values.)R E P O R T S16APRIL 2004VOL 304SCIENCE 442 o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mare otherwise localized to the plastid in other apicomplexans.C.parvum fatty acid biosynthe-sis appears to be cytoplasmic,conducted by a large(8252amino acids)modular type I FAS (9)and possibly by another large enzyme that is related to the multidomain bacterial polyketide synthase(10).Comprehensive screening of the C.parvum genome sequence also did not detect orthologs of Plasmodium nuclear-encoded genes that contain apicoplast-targeting and transit sequences(11).C.parvum metabolism is greatly stream-lined relative to that of Plasmodium,and in certain ways it is reminiscent of that of another obligate eukaryotic parasite,the microsporidian Encephalitozoon.The degeneration of the mi-tochondrion and associated metabolic capabili-ties suggests that the parasite largely relies on glycolysis for energy production.The parasite is capable of uptake and catabolism of mono-sugars(e.g.,glucose and fructose)as well as synthesis,storage,and catabolism of polysac-charides such as trehalose and amylopectin. Like many anaerobic organisms,it economizes ATP through the use of pyrophosphate-dependent phosphofructokinases.The conver-sion of pyruvate to acetyl–coenzyme A(CoA) is catalyzed by an atypical pyruvate-NADPH oxidoreductase(Cp PNO)that contains an N-terminal pyruvate–ferredoxin oxidoreductase (PFO)domain fused with a C-terminal NADPH–cytochrome P450reductase domain (CPR).Such a PFO-CPR fusion has previously been observed only in the euglenozoan protist Euglena gracilis(12).Acetyl-CoA can be con-verted to malonyl-CoA,an important precursor for fatty acid and polyketide biosynthesis.Gly-colysis leads to several possible organic end products,including lactate,acetate,and ethanol. The production of acetate from acetyl-CoA may be economically beneficial to the parasite via coupling with ATP production.Ethanol is potentially produced via two in-dependent pathways:(i)from the combination of pyruvate decarboxylase and alcohol dehy-drogenase,or(ii)from acetyl-CoA by means of a bifunctional dehydrogenase(adhE)with ac-etaldehyde and alcohol dehydrogenase activi-ties;adhE first converts acetyl-CoA to acetal-dehyde and then reduces the latter to ethanol. AdhE predominantly occurs in bacteria but has recently been identified in several protozoans, including vertebrate gut parasites such as Enta-moeba and Giardia(13,14).Adjacent to the adhE gene resides a second gene encoding only the AdhE C-terminal Fe-dependent alcohol de-hydrogenase domain.This gene product may form a multisubunit complex with AdhE,or it may function as an alternative alcohol dehydro-genase that is specific to certain growth condi-tions.C.parvum has a glycerol3-phosphate dehydrogenase similar to those of plants,fungi, and the kinetoplastid Trypanosoma,but(unlike trypanosomes)the parasite lacks an ortholog of glycerol kinase and thus this pathway does not yield glycerol production.In addition to themodular fatty acid synthase(Cp FAS1)andpolyketide synthase homolog(Cp PKS1), C.parvum possesses several fatty acyl–CoA syn-thases and a fatty acyl elongase that may partici-pate in fatty acid metabolism.Further,enzymesfor the metabolism of complex lipids(e.g.,glyc-erolipid and inositol phosphate)were identified inthe genome.Fatty acids are apparently not anenergy source,because enzymes of the fatty acidoxidative pathway are absent,with the exceptionof a3-hydroxyacyl-CoA dehydrogenase.C.parvum purine metabolism is greatlysimplified,retaining only an adenosine ki-nase and enzymes catalyzing conversionsof adenosine5Ј-monophosphate(AMP)toinosine,xanthosine,and guanosine5Ј-monophosphates(IMP,XMP,and GMP).Among these enzymes,IMP dehydrogenase(IMPDH)is phylogenetically related toε-proteobacterial IMPDH and is strikinglydifferent from its counterparts in both thehost and other apicomplexans(15).In con-trast to other apicomplexans such as Toxo-plasma gondii and P.falciparum,no geneencoding hypoxanthine-xanthineguaninephosphoribosyltransferase(HXGPRT)is de-tected,in contrast to a previous report on theactivity of this enzyme in C.parvum sporo-zoites(16).The absence of HXGPRT sug-gests that the parasite may rely solely on asingle enzyme system including IMPDH toproduce GMP from AMP.In contrast to otherapicomplexans,the parasite appears to relyon adenosine for purine salvage,a modelsupported by the identification of an adeno-sine transporter.Unlike other apicomplexansand many parasitic protists that can synthe-size pyrimidines de novo,C.parvum relies onpyrimidine salvage and retains the ability forinterconversions among uridine and cytidine5Ј-monophosphates(UMP and CMP),theirdeoxy forms(dUMP and dCMP),and dAMP,as well as their corresponding di-and triphos-phonucleotides.The parasite has also largelyshed the ability to synthesize amino acids denovo,although it retains the ability to convertselect amino acids,and instead appears torely on amino acid uptake from the host bymeans of a set of at least11amino acidtransporters(table S2).Most of the Cryptosporidium core pro-cesses involved in DNA replication,repair,transcription,and translation conform to thebasic eukaryotic blueprint(2).The transcrip-tional apparatus resembles Plasmodium interms of basal transcription machinery.How-ever,a striking numerical difference is seenin the complements of two RNA bindingdomains,Sm and RRM,between P.falcipa-rum(17and71domains,respectively)and C.parvum(9and51domains).This reductionresults in part from the loss of conservedproteins belonging to the spliceosomal ma-chinery,including all genes encoding Smdomain proteins belonging to the U6spliceo-somal particle,which suggests that this par-ticle activity is degenerate or entirely lost.This reduction in spliceosomal machinery isconsistent with the reduced number of pre-dicted introns in Cryptosporidium(5%)rela-tive to Plasmodium(Ͼ50%).In addition,keycomponents of the small RNA–mediatedposttranscriptional gene silencing system aremissing,such as the RNA-dependent RNApolymerase,Argonaute,and Dicer orthologs;hence,RNA interference–related technolo-gies are unlikely to be of much value intargeted disruption of genes in C.parvum.Cryptosporidium invasion of columnarbrush border epithelial cells has been de-scribed as“intracellular,but extracytoplas-mic,”as the parasite resides on the surface ofthe intestinal epithelium but lies underneaththe host cell membrane.This niche may al-low the parasite to evade immune surveil-lance but take advantage of solute transportacross the host microvillus membrane or theextensively convoluted parasitophorous vac-uole.Indeed,Cryptosporidium has numerousgenes(table S2)encoding families of putativesugar transporters(up to9genes)and aminoacid transporters(11genes).This is in starkcontrast to Plasmodium,which has fewersugar transporters and only one putative ami-no acid transporter(GenBank identificationnumber23612372).As a first step toward identification ofmulti–drug-resistant pumps,the genome se-quence was analyzed for all occurrences ofgenes encoding multitransmembrane proteins.Notable are a set of four paralogous proteinsthat belong to the sbmA family(table S2)thatare involved in the transport of peptide antibi-otics in bacteria.A putative ortholog of thePlasmodium chloroquine resistance–linkedgene Pf CRT(17)was also identified,althoughthe parasite does not possess a food vacuole likethe one seen in Plasmodium.Unlike Plasmodium,C.parvum does notpossess extensive subtelomeric clusters of anti-genically variant proteins(exemplified by thelarge families of var and rif/stevor genes)thatare involved in immune evasion.In contrast,more than20genes were identified that encodemucin-like proteins(18,19)having hallmarksof extensive Thr or Ser stretches suggestive ofglycosylation and signal peptide sequences sug-gesting secretion(table S2).One notable exam-ple is an11,700–amino acid protein with anuninterrupted stretch of308Thr residues(cgd3_720).Although large families of secretedproteins analogous to the Plasmodium multi-gene families were not found,several smallermultigene clusters were observed that encodepredicted secreted proteins,with no detectablesimilarity to proteins from other organisms(Fig.1,A and B).Within this group,at leastfour distinct families appear to have emergedthrough gene expansions specific to the Cryp-R E P O R T S SCIENCE VOL30416APRIL2004443o n O c t o b e r 7 , 2 0 0 9 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mtosporidium clade.These families —SKSR,MEDLE,WYLE,FGLN,and GGC —were named after well-conserved sequence motifs (table S2).Reverse transcription polymerase chain reaction (RT-PCR)expression analysis (20)of one cluster,a locus of seven adjacent CpLSP genes (Fig.1B),shows coexpression during the course of in vitro development (Fig.1C).An additional eight genes were identified that encode proteins having a periodic cysteine structure similar to the Cryptosporidium oocyst wall protein;these eight genes are similarly expressed during the onset of oocyst formation and likely participate in the formation of the coccidian rigid oocyst wall in both Cryptospo-ridium and Toxoplasma (21).Whereas the extracellular proteins described above are of apparent apicomplexan or lineage-specific in-vention,Cryptosporidium possesses many genesencodingsecretedproteinshavinglineage-specific multidomain architectures composed of animal-and bacterial-like extracellular adhe-sive domains (fig.S1).Lineage-specific expansions were ob-served for several proteases (table S2),in-cluding an aspartyl protease (six genes),a subtilisin-like protease,a cryptopain-like cys-teine protease (five genes),and a Plas-modium falcilysin-like (insulin degrading enzyme –like)protease (19genes).Nine of the Cryptosporidium falcilysin genes lack the Zn-chelating “HXXEH ”active site motif and are likely to be catalytically inactive copies that may have been reused for specific protein-protein interactions on the cell sur-face.In contrast to the Plasmodium falcilysin,the Cryptosporidium genes possess signal peptide sequences and are likely trafficked to a secretory pathway.The expansion of this family suggests either that the proteins have distinct cleavage specificities or that their diversity may be related to evasion of a host immune response.Completion of the C.parvum genome se-quence has highlighted the lack of conven-tional drug targets currently pursued for the control and treatment of other parasitic protists.On the basis of molecular and bio-chemical studies and drug screening of other apicomplexans,several putative Cryptospo-ridium metabolic pathways or enzymes have been erroneously proposed to be potential drug targets (22),including the apicoplast and its associated metabolic pathways,the shikimate pathway,the mannitol cycle,the electron transport chain,and HXGPRT.Nonetheless,complete genome sequence analysis identifies a number of classic and novel molecular candidates for drug explora-tion,including numerous plant-like and bacterial-like enzymes (tables S3and S4).Although the C.parvum genome lacks HXGPRT,a potent drug target in other api-complexans,it has only the single pathway dependent on IMPDH to convert AMP to GMP.The bacterial-type IMPDH may be a promising target because it differs substan-tially from that of eukaryotic enzymes (15).Because of the lack of de novo biosynthetic capacity for purines,pyrimidines,and amino acids,C.parvum relies solely on scavenge from the host via a series of transporters,which may be exploited for chemotherapy.C.parvum possesses a bacterial-type thymidine kinase,and the role of this enzyme in pyrim-idine metabolism and its drug target candida-cy should be pursued.The presence of an alternative oxidase,likely targeted to the remnant mitochondrion,gives promise to the study of salicylhydroxamic acid (SHAM),as-cofuranone,and their analogs as inhibitors of energy metabolism in the parasite (23).Cryptosporidium possesses at least 15“plant-like ”enzymes that are either absent in or highly divergent from those typically found in mammals (table S3).Within the glycolytic pathway,the plant-like PPi-PFK has been shown to be a potential target in other parasites including T.gondii ,and PEPCL and PGI ap-pear to be plant-type enzymes in C.parvum .Another example is a trehalose-6-phosphate synthase/phosphatase catalyzing trehalose bio-synthesis from glucose-6-phosphate and uridine diphosphate –glucose.Trehalose may serve as a sugar storage source or may function as an antidesiccant,antioxidant,or protein stability agent in oocysts,playing a role similar to that of mannitol in Eimeria oocysts (24).Orthologs of putative Eimeria mannitol synthesis enzymes were not found.However,two oxidoreductases (table S2)were identified in C.parvum ,one of which belongs to the same families as the plant mannose dehydrogenases (25)and the other to the plant cinnamyl alcohol dehydrogenases.In principle,these enzymes could synthesize protective polyol compounds,and the former enzyme could use host-derived mannose to syn-thesize mannitol.References and Notes1.D.G.Korich et al .,Appl.Environ.Microbiol.56,1423(1990).2.See supportingdata on Science Online.3.M.J.Gardner et al .,Nature 419,498(2002).4.A.T.Bankier et al .,Genome Res.13,1787(2003).5.J.C.Wootton,Comput.Chem.18,269(1994).Fig.1.(A )Schematic showing the chromosomal locations of clusters of potentially secreted proteins.Numbers of adjacent genes are indicated in paren-theses.Arrows indicate direc-tion of clusters containinguni-directional genes (encoded on the same strand);squares indi-cate clusters containingg enes encoded on both strands.Non-paralogous genes are indicated by solid gray squares or direc-tional triangles;SKSR (green triangles),FGLN (red trian-gles),and MEDLE (blue trian-gles)indicate three C.parvum –specific families of paralogous genes predominantly located at telomeres.Insl (yellow tri-angles)indicates an insulinase/falcilysin-like paralogous gene family.Cp LSP (white square)indicates the location of a clus-ter of adjacent large secreted proteins (table S2)that are cotranscriptionally regulated.Identified anchored telomeric repeat sequences are indicated by circles.(B )Schematic show-inga select locus containinga cluster of coexpressed large secreted proteins (Cp LSP).Genes and intergenic regions (regions between identified genes)are drawn to scale at the nucleotide level.The length of the intergenic re-gions is indicated above or be-low the locus.(C )Relative ex-pression levels of CpLSP (red lines)and,as a control,C.parvum Hedgehog-type HINT domain gene (blue line)duringin vitro development,as determined by semiquantitative RT-PCR usingg ene-specific primers correspondingto the seven adjacent g enes within the CpLSP locus as shown in (B).Expression levels from three independent time-course experiments are represented as the ratio of the expression of each gene to that of C.parvum 18S rRNA present in each of the infected samples (20).R E P O R T S16APRIL 2004VOL 304SCIENCE 444 o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m。

DNA Methylation Markers Predict Outcome in Node-Positive,Estrogen Receptor-Positive Breast Cancer with Adjuvant Anthracycline-Based ChemotherapyOliver Hartmann,1Frede rique Spyratos,4Nadia Harbeck,2Dimo Dietrich,1Anne Fassbender,1Manfred Schmitt,2Serenella Eppenberger-Castori,3VincentVuaroqueaux,3Florence Lerebours,4KatrinWelzel,1Sabine Maier,1Achim Plum,1Stephan Niemann,1John A.Foekens,5Ralf Lesche,1and JohnW.M.Martens 5AbstractPurpose:We have shown that DNAmethylation of the PI TX2gene predicts risk of distant recurrence in steroid hormone receptor-positive,node-negative breast cancer.Here,we present results from a multicenter study investigating whether PI TX2and other candidate DNAmeth-ylation markers predict outcome in node-positive,estrogen receptor-positive,HER-2-negative breast cancer patients who received adjuvant anthracycline-based chemotherapy.Experimental Design:Using a microarray platform,we analyzed DNAmethylation in regu-latory regions of PITX2and 60additional candidate genes in 241breast cancer ing Cox regression analysis,we assessed the predictive power of the individual marker/marker panel candidates.Clinical endpoints were time to distant metastasis,disease-free sur-vival,and overall survival.Anested bootstrap/cross-validation strategy was applied to identify and validate marker panels.Results:DNAmethylation of PI TX2and 14other genes was correlated with clinical outcome.In multivariate models,each methylation marker added significant information to established clinical factors.Afour-marker panel including PI TX2,BMP4,FGF4,and C20orf55was identified that resulted in improvement of outcome prediction compared with PITX2alone.Conclusions:This study provides further evidence for the PITX2biomarker,which has now been successfully confirmed to predict outcome among different breast cancer patient populations.We further identify new DNAmethylation biomarkers,three of which can be combined into a panel with PITX2to increase the outcome prediction performance in our anthracycline-treated primary breast cancer population.Our results show that a well-defined panel of DNAmethylation markers enables outcome prediction in lymph node-positive,HER-2-negative breast cancer patients treated with anthracycline-based chemotherapy.I n breast cancer,currently available methods are inadequate todetermine precisely the aggressiveness of the disease and the likelihood of response to a certain treatment in individual patients.Therefore,biomarkers predicting the tumor’s meta-static potential and its response to a specific treatment are urgently needed.This is particularly true for patients for whom current guidelines (1)recommend anthracycline-based chemo-therapy.These patients comprise a clinically distinct interme-diate-to high-risk subgroup,for which in view of the short and long-term toxicity as well as the availability of new effective anthracycline-free chemotherapy options (2),the widespread use of adjuvant anthracyclines is currently under debate (3).Unfortunately,reliable biomarkers for benefit from anthracy-cline therapy are still lacking.DNA methylation of CpG dinucleotides within gene regulatory regions,associated with suppression of gene expression,is a common and early event in cancer (4–6).Specific DNA methylation patterns for tumor subtypes including breast cancer have been reported and associated with clinical outcome (7–16).Cancer Therapy:ClinicalAuthors’Affiliations:1Epigenomics AG,Berlin,Germany;2Department of Obstetrics and Gynecology,T echnical University Munich,Munich,Germany;3Stiftung T umorbank,Basel,Switzerland;4Centre ReneHuguenin,St.Cloud,France;and 5Erasmus Medical Center,Rotterdam,The Netherlands Received 1/22/08;revised 8/25/08;accepted 9/2/08.Grant support:Epigenomics and European Union Sixth Framework Program grant LSHC-CT-2003-504586(J.W.M.Martens,J.A.Foekens,S.Eppenberger-Castori,F.Spyratos,M.Schmitt,and N.Harbeck).The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with 18U.S.C.Section 1734solely to indicate this fact.Note:Supplementary data for this article are available at Clinical Cancer Research Online (/).Preliminary results of this study were presented as a poster presentation at 29th San Antonio Breast Cancer Symposium,December 14-17,2006[Lesche et al.,Breast Cancer ResT reat 2006;100:A6009].Requests for reprints:John W.M.Martens,Department of Medical Oncology,Erasmus Medical Center,P.O.Box 2040,3000CARotterdam,The Netherlands.Phone:31-10-7044372;Fax:31-10-7044377;E-mail:j.martens @erasmusmc.nl.F 2009American Association for Cancer Research.doi:10.1158/R-08-0166www Clin Cancer Res 2009;15(1)January 1,2009315We have recently found and validated that DNA hyper-methylation of the PITX2promoter is associated with a high risk of recurrence in node-negative,steroid hormone receptor-positive breast cancer who received tamoxifen as their only systemic adjuvant therapy(13,17).These studies further showed that DNA methylation can be successfully measured in formalin-fixed,paraffin-embedded breast cancer specimens, which is one of the strengths of DNA methylation technologies and considered a prerequisite for routine clinical application (17).A follow-up study revealed that PITX2methylation is a strong and pure prognostic factor in patients with node-negative,steroid hormone receptor-positive breast cancer who did not receive any adjuvant systemic treatment(15).In the present study,we have used a microarray technology to study whether methylation of PITX2and60other candidate genes could be predictors for outcome in breast cancer patients with node-positive,steroid hormone receptor-positive,and HER-2-negative tumors receiving adjuvant anthracycline-based chemotherapy.In our study,we focused on patients with lymph node-positive,estrogen receptor(ER)-positive,and HER-2-negative breast cancer,as these patients comprise a clinically distinct subgroup for which anthracycline-based adjuvant chemotherapy is currently recommended(1).Our study suggests that reliable prediction of clinical outcome for these breast cancer patients may be possible using a well-defined panel of DNA methylation markers.Materials and MethodsPatients and samplesThe study cohort comprised384node-positive breast cancer patients whose tumor samples were recruited from four clinical centers:Erasmus Medical Center;Centre Rene´Huguenin;Stiftung Tumorbank;and Department of Obstetrics and Gynecology,Technical University of Munich.Appropriate consent,according to institutional requirements, has been obtained for all patients.The study protocol was approved by the local ethics committees.To be eligible for this study,patients had to fulfill all of the following criteria:(a)histologically confirmed invasive breast cancer,(b)primary tumor stage pT1to pT3,(c)histologically confirmed lymph node involvement(pN>1),(d)surgery before2002(potential follow-up of at least5years),(e)standard adjuvant anthracycline-based chemother-apy[no dose-dense therapy,no other primary systemic chemotherapy (except hormonal therapy),and no additional taxane],and(f) availability of clinical follow-up data.Among the384patients recruited for the study,284were reported as ER-positive.HER-2status was determined in all samples using the Light Cycler HER-2/neu DNA quantification Kit(Roche Applied Science). HER-2-positive patients(n=43)who nowadays would receive trastuzumab treatment were then excluded,leaving a total of241 node-positive,ER-positive,and HER-2-negative breast cancer patients available for analysis.For the whole cohort,the percentages of patients with distant metastasis at5and10years were estimated as70.1% (64.3-76.3%)and56.1%(48.5-65.0%),respectively;5-and10-year disease-free survival(DFS)were64.5%(58.6-71.0%)and47.4%(40.0-64.2%),respectively;and5-and10-year overall survival(OS)were 85.8%(81.4-90.5%)and67.7%(59.8-79.1%),respectively.The median follow-up81.5months.DNA extraction and bisulfite treatmentGenomic DNA was extracted from snap-frozen tumor tissue or tumor cell nuclei pelleted at100,000Âg as described(14).Bisulfite conversion of the DNA was done using the EpiTect Kit(Qiagen).Candidate gene selectionThe61candidate genes(listed in the Supplementary Material)to be analyzed on the microarray were selected based on(a)genome-wide screens for prognostic markers in ER-positive and ER-negative breast cancer,6(b)P TX2pathway analyses,and(c)literature.PCR amplification and microarray hybridizationPCR amplification and microarray hybridization were done as described previously(7,14).In total,64PCR amplificates representing 61genes were pooled and hybridized to the microarray on which detection oligonucleotides for methylated(CG)and nonmethylated (TG)gene copies were spotted.This allowed for simultaneous quantitative measurement of unmethylated and methylated copies of the genes.Microarrays included4oligonucleotide pairs for each of the 64PCR amplificates(total of256pairs).Each probe pair covered between one and three CpG dinucleotides in the regulatory regions of the respective candidate gene.The methylation score for each CpG site was calculated from the fluorescence intensity values of the methylated (FI m)and unmethylated(F I u)oligonucleotides.To stabilize the variance,the score was transformed using the generalized log transformation(g LOG):methylation score=g LOG(FI m/FI u)(18). For statistical analysis,methylation scores for each amplificate were determined by averaging measurements from all probe pairs belonging to one amplificate using the median.Multiple amplificates from the same candidate gene entered data analysis independently.Valid microarray results were obtained for all samples.Clinical endpointsThe primary clinical endpoint in this study was time to distant metastasis(TDM).Secondary endpoints were DFS and OS.For TDM, only distant recurrences were considered.Ipsilateral and locoregional recurrences were not considered as events or censoring events. Contralateral breast cancer,development of other primary tumors, death(from any cause)without observed recurrence,and loss for follow-up were considered censoring events.For DFS,all recurrences were considered as events,whereas death(from any cause)before recurrence,contralateral breast cancer,and development of other 6Unpublisheddata.Cancer Therapy:Clinical Clin Cancer Res2009;15(1)January1,2009316primary tumors or loss for follow-up were considered censoring events. For OS,only death(from any cause)was considered as event and patients were censored only when lost for follow-up.Statistical analysesThe relation between clinical endpoints and DNA methylation score was analyzed for each amplificate by linear univariate Cox proportional hazards models(19,20).Here,likelihood ratio tests(LRT)were done to test for a significant relationship of methylation scores of each amplificate with clinical endpoints.Hazard ratios(HR)for continuous variables were calculated relative to an increment of the interquartile range of that variable(for the increment from the25%quantile to the 75%quantile of the measurements;ref.20).Multivariate regression analysis,testing the association between clinical endpoint and multiple methylation scores and/or clinical variables,was done by linear Cox proportional hazards models.In that case,LRT was done to test for a significant association of the outcome variables(TDM,DFS,and OS) with the derived model consisting of clinical variables and/or methylation scores.In addition,Wald tests(testing the hypothesis that the variable in question provides significant information to the multivariate model)were calculated.Survival curves were plotted according to Kaplan-Meier(21).Log-rank tests were used to test for differences between survival curves.To describe and compare the predictive performance of each variable and each multivariate model, the concordance index(C index;refs.20,22)was calculated.To penalize overfitting by entering multiple factors,the bootstrap-corrected version of the C index is given(20,22).To correct for multiple testing in univariate testing(64amplificates), significance levels were adjusted using false discovery rate correction using the linear step-up procedure proposed in ref.23.Marker panel feature selection.A nested cross-validation/bootstrap procedure was applied to perform feature selection and panel validation (24,25).A schematic illustration of the methodology is shown in the Supplementary Material.To identify a marker panel for outcome prediction,the63amplificates[all but the amplificate designed in the promoter of transcripts A and B for PITX2(PITX2P2)]were first ranked by univariate performance(Cox proportional hazards model,LRT). Then,marker panels containing PITX2P2and increasing numbers of the best19single markers were evaluated with respect to their prognostic predictability,which was estimated using the bootstrap-corrected C index with B=200bootstrap runs(20).A gene was selected for the final predictor model if the C index of the model including this marker was larger than that of the model excluding the marker.A C index was considered‘‘larger’’if at least an increase of1%over the model containing PITX2P2was observed.The extensive search was limited to a maximum of20amplificates altogether,balancing the critical sample size to build a reasonable regression model(number of distant metastases)on the one side and the request to find markers complementing previously included ones(and therefore not necessarily good univariate markers)on the other.Marker panel validation.The cross-validated prediction score is an unbiased estimate of the performance of the marker panel in a future data set.It corrects for the optimism introduced due to feature selection within the same data set(24,25).A schematic illustration of the methodology is presented in the Supplementary Material.In brief,the generalization error for the marker panel selection was estimated by repeating the feature selection procedure using a20-fold cross-validation,starting with all64amplificates in each cross-validation run.We trained on95%of the samples and computed the cross-validated predictive index for the samples in the remaining5%of samples using the Cox model developed for the training set.To combine results from all runs,the predictive indices for all samples were transformed into risk percentiles within each run.The nested cross-validation procedure itself was replicated100times using random permutations of the data set to determine the estimation error of the cross-validated C index.As cross-validated risk percentiles are correlated among cases,the usual null distributions of the test statistics are not valid.To derive P values of both the likelihood ratio and Wald tests (Cox model)as well as the log-rank test(Kaplan-Meier analysis)for the cross-validated prediction score,the null distribution was estimated using100repetitions of the entire procedure for permutations of survival times and censoring indicators.To further illustrate the performance of the cross-validated prediction score,the score was analyzed in a similar way as described above for our individual amplificates.All results using a single cross-validated prediction score were generated using results of the first replicate of the nested cross-validation procedure.Statistical analysis was done using R version2.4.17and the R package‘‘Design’’version2.1. ResultsPatient and tumor characteristics.Patient and tumor char-acteristics(n=241)are given in Supplementary Material,and these are comparable with previously published cohorts with similar inclusion criteria(26).These clinical standards may differ somewhat from current guidelines as shown by the fact that only104(43%)patients received additional adjuvant endocrine treatment,although their tumors had positive steroid hormone receptor status.All patients had received adjuvant anthracycline-based chemotherapy according to clinical stand-ards at the time of their primary therapy.Tumor size(HR, 1.77;P=0.0225),grade(HR, 2.07; P=0.004),and adjuvant endocrine therapy(HR,0.49; P=0.0025)were significantly associated with TDM in this cohort(Table1).Adjuvant endocrine therapy,grade,and progesterone receptor(PgR)were significantly associated with DFS(data not shown).Tumor size,grade,endocrine therapy, and PgR status were significantly associated with OS(data not shown).For all clinical endpoints,risk of recurrence was lower for patients who received adjuvant endocrine therapy. Univariate analysis of methylation markers.Because we have observed DNA methylation of PITX2as a strong marker in previous studies,we first analyzed the performance of this marker.The methylation score measured with the amplificate designed for the promoter of transcripts A and B of PITX2 (PITX2P2),located upstream of the first transcriptional start, showed that PITX2P2hypermethylation was associated with a high risk of distant recurrence in this patient cohort[HR,1.66; 95%confidence interval(95%CI), 1.21-2.28;P=0.002;C index=0.624;see Table1and Fig.1].PITX2P2hyper-methylation was also associated with poor DFS(HR,1.47;95% CI,1.11-1.96;P=0.0084)and OS(HR,2.07;95%C,1.40-3.06;P=0.0003).However,methylation of the amplificate designed for the promoter regulating transcript C of PITX2 (P I TX2P1)was not associated with distant recurrence in this patient cohort(HR,1.23;95%CI,0.89-1.68;P=0.21). Among the other62tested amplificates,15amplificates, derived from14genes,were associated with TDM(false discovery rate<5%;Table2).Methylation of BMP4,FOXL2, LMX1A,C20orf55,BARX1,PLAU(both amplificates),FGF4, NR5A1,BRCA1,TLX3,NR2E1,LHX4,ZNF1A1,and CCND2 and BMP4,FOXL2,LMX1A,FGF4,and BRCA1showed C indices between0.58and0.63comparable with that of PITX2P2.For DFS,BMP4,FOXL2,and C20orf55and for OS all15amplificates described above were significantly associated with the respective clinical endpoint(false discovery rate<5%; 7DNA Methylation Markers and Anthracyclines Clin Cancer Res2009;15(1)January1,2009317data not shown).For all markers,with the exception of C20orf55,lower methylation scores were associated with better clinical outcome.Multivariate analysis of methylation markers and clinical variables.PITX2P2methylation was a significant marker in the multivariate analysis including age at surgery,pathologic T stage and grade,PgR status,and adjuvant endocrine therapy (P=0.05;Table1).To show clinical usefulness of a biomarker, it is necessary that the marker increases the predictive performance of established clinical variables.For the multivar-iate model including PITX2P2,the bootstrap-corrected C index, which is an unbiased estimate of the effect size for regression models,is larger than that for the model including the clinical variables only(0.647compared with0.61,respectively;Table 3).Therefore,PITX2P2provides additional information for prediction of TDM independent of established clinical variables.Most of the15amplificates identified in univariate analysis were also a significant marker in multivariate analysis when combined with age,pathologic T stage,PgR status,and adjuvant endocrine therapy.The bootstrap-corrected C indices of the multivariate models(range,0.620-0.665)were larger than those of the multivariate model using only clinical variables(0.610;Table2).Marker panel selection and performance prediction.Having established the role of several DNA methylation markers(PITX2 and15other markers)for outcome prediction,we assessed if combining these markers into a panel resulted in improved performance compared with that of the individual markers alone.Overfitting becomes a critical issue if feature selection, model building,and performance estimation are done within one data set(27).Therefore,the bootstrapped-corrected C index is used in the feature selection step.To select relevant markers that could improve the performance of our already well-established marker PITX2,we evaluated the gain in estimated effect size(C index)by stepwise adding the best markers from univariate analysis to PITX2P2.Figure2A illustrates the results of the feature selection step.With PITX2P2as‘‘anchor,’’BMP4, C20orf55,and FGF4were selected to define a four-marker panel for prediction of TDM in the currentcohort.Fig.1.Prediction of TDM survivalprobabilities based on PITX2P2DNAmethylation.Kaplan-MeierTDM curves ofpatients categorized in subgroups basedon PI TX2P2DNAmethylation scores(P=0.00014).The methylation score wasgrouped into quartiles.Log-rank test wasused to test for significant separationbetween the groups.C index was0.624.Patients at risk for the different quartiles areindicated in12-mo intervals.Cancer Therapy:Clinical Clin Cancer Res2009;15(1)January1,2009318To estimate the performance regarding outcome prediction of the four-marker panel,feature selection was nested within a cross-validation procedure.This corrects for overfitting due to feature selection,and the resulting cross-validated C index is an unbiased estimate of the performance of the marker panel in a future data set.The C index of the cross-validated prediction score is0.668compared with0.624for PITX2P2alone (Table3).We replicated the estimation procedure100times, thereby generating a range of C indices for our four-marker panel.As a result,a median C index of0.654(range,0.622-0.676)was obtained for the prediction score,and in98of100 replications,the estimated performance of our four-marker panel was better than that of PITX2P2alone(Fig.2B).Based on these results,we conclude that a four-marker panel can indeed improve outcome prediction over a single marker.To further illustrate the performance of our outcome predictor,we report the results of the first cross-validation replicate in more detail.The four markers selected for our marker panel represent a stable selection:C20orf55was selected in all cross-validation runs,FGF4in16of20runs, and BMP4in14of20runs.Only two other markers were selected in any run:APC and BRCA1(Fig.2C).The C index of the cross-validated prediction score for a multivariate model including the cross-validated prediction score as well as age at surgery,pathologic T stage,PgR,and adjuvant endocrine therapy was0.695(P<0.01,LRT;Table3).The Wald P value for the cross-validated prediction score in the multivariate model was<0.01.Kaplan-Meier estimates illustrate the difference(P<0.01,log-rank test;Fig.2D)between the performance of the identified marker panel(Fig.3,left)and PITX2P2alone(Fig.1).For all patients,the5-year survival prediction of the quartile with the highest cross-validated prediction scores for the four-marker panel is<50%,whereas that of the quartile with the lowest cross-validated prediction scores is>85%.Similar to TDM,significant prediction scores were derived for DFS(P=0.01)and OS(P<0.01).Since today all patients with steroid hormone receptor-positive breast cancer will receive adjuvant endocrine therapy,we performed a subgroup analysis for those patients in our cohort who had received adjuvant endocrine treatment. Although the size of cohort was limited to104patients,a highly statistically significant separation of survival groups was observed when stratifying the patients according to the prediction score(Fig.3,right).DiscussionAccording to current guidelines,adjuvant chemotherapy needs to be considered for the majority of patients with primary breast cancer,with the exception of a small subgroup with very favorable outcome prediction based on clinical factors(1).In node-positive breast cancer,anthracycline-based adjuvant chemotherapy has become the standard of care during the1990s(28).Moreover,several studies showed that adding a taxane(29),or,more recently,administering dose-dense chemotherapy regimes(including anthracyclines;ref.30), further improves outcome in node-positive breast cancer. However,not all patients will benefit from such more aggressive therapies and therefore only face their more severe side effects,because these patients either have an excellent prognosis or have tumors that do not respond to certain chemotherapies.Thus,prognostic and predictive markers are urgently needed to tailor the best possible therapy regimen for the individualpatients.DNA Methylation Markers and Anthracyclines Clin Cancer Res2009;15(1)January1,2009319In previous studies,we found that DNA methylation of PITX2reliably predicts the risk of distant recurrence in node-negative breast cancer (13,15,17).These results prompted us to analyze PITX2methylation in node-positive breast cancer and to measure other DNA methylation markers,with the goal to develop a marker panel for improved outcome prediction.For this purpose,we have analyzed a cohort of 241patients with lymph node-positive,ER-positive,and HER-2-negative breast cancer,who had all received adjuvant anthracycline-based chemotherapy.We focused on patients with these tumor characteristics,because they constitute a clinically relevant subgroup for which standardized treatment guidelines are available (1,26).Among the node-positive breast cancer patients,our subpopulation is considered to have a relatively favorable outcome because of their positive steroid hormone receptor status and the absence of HER-2amplification.Therefore,we hypothesized that it might be possible to further stratify this group of patients so that a good prognosis group could be defined that would derive sufficient benefit from conventional anthracycline-based therapy and would not require more aggressive therapy such as,for example,dose-dense regimens or addition of taxanes.In the current study,we were able to show that PITX2DNA methylation also predicts outcome in node-positive breast cancer.Because clinical factors such as grade,tumor stage,or age have been described previously as outcome predictors in primary breast cancer,we first calculated a base model.The predictive accuracy of the multivariate Cox model with established clinical variables,quantified using the bootstrap-corrected C index as an unbiased measure of the predictive strength of a model,was 0.604.Adding PITX2to the base model resulted in a significantly improved outcome prediction (C index =0.651).This confirms the utility of PITX2as a marker for outcome prediction in primary breast cancerandFig.2.Marker panel feature selection and marker panel validation.A,marker panel selection.Result of the feature selection step using bootstrap-corrected C index to select markers for the panel to predictTDM.Marker models built using PITX2P2and the first n amplificates (X axis ,markers ranked according to univariate performance)areevaluated using the bootstrap-corrected C index (Yaxis ).Closed circles,selected markers.These are,from left to right ,amplificates for PITX2P2,BMP4,C20orf55,and FGF4.B ,histogram of cross-validated C index from 100repetitions.C index for model with PITX2P2alone was 0.624(thick vertical black line );98of 100repetitions for the four-marker panel are larger.C,stability of marker selection.Frequency of the indicated markers in cross-validation runs.In each of the 20cross-validation runs,featureselection is repeated on 95%of the samples.C20orf55was selected in all cross-validation runs,FGF4in 16of 20runs,and BMP4in 14of 20runs.Only two other markers were selected by this procedure:APC and BRCA1,which were both selected in only 3of 20runs.D,histogram of the null distribution of the log-rank test statistic of our data set.Histogram shows the distribution of log-rank test statistic of 100data sets,of which survival time and censoring indicator were randomly permutated.This permutated data set was used to derive P values of the LRT (Cox model)for the cross-validated prediction score.Thick vertical black line,log-rank statistic for the cross-validated prediction score of the Kaplan-MeierTDM curves shown in Fig.3(left panel ).Cancer Therapy:ClinicalClin Cancer Res 2009;15(1)January 1,2009320。

流行病学常用指标一．实习目的：1．掌握流行病学研究中常用的测量疾病和死亡频率指标的计算方法。

2．掌握这些指标的用途和意义。

3．理解率标化的目的，熟悉率标化的具体方法。

4．理解“人时”单位的含义，了解其适用条件，熟悉其具体计算方法。

二．学时：3学时三．实习内容：1．1992年我国某市肺癌流行病学调查资料。

2．某小学麻疹流行资料分析。

3．1952~1953年某市推行白喉预防接种资料分析。

4．两城市1990年死亡率比较。

【课题一】某市有10万人口，其中男性45,000人，女性55,000人，1992年死亡总数为1,000人（男600，女400）。

当年，肺癌发病50例（男40，女10），其中45例死亡（男36，女9）。

作业要求：计算下列各指标。

1．1992年该市粗死亡率2．1992年该市肺癌死亡率3．1992年该市性别死亡率4．1992年该市肺癌病死率5．1992年该市肺癌死亡构成比6．1992年该市肺癌性别死亡率比（男/女）【课题二】某小学共有学生300名，某年1~5月份发生了麻疹流行，共发生病例20名。

经调查发现，300名学生中有190名以往接种过麻疹疫苗，另有10名曾患过麻疹，其中2例发生在前一年的12月份。

本次麻疹流行资料见图1。

图1 学生麻疹发病时间分布示意图作业要求：完成下表计算。

表1 计算结果记录1月2月3月4月5月1~5月罹患率（%）期间患病率（%）【课题三】 某市人口30多万，全市共分10个区，1952~1953两年内该市推行白喉预防接种。

接种对象是14岁以下托儿所、幼儿园和小学校儿童。

第七区为该市白喉预防重点区。

该区1952~1954年期间人口基本没有变动，共有14岁以下接种对象5000人。

接种从1952年1月开始进行，连续不断地延至1953年9月，共接种3000人。

在此接种期间及此后9个月至1954年6月为止，曾对接种者及接种对象未接种者中发生的白喉病例进行登记，结果如下：表2 某市第七区1952.1~1954.6白喉预防接种情况及白喉发病情况 日 期 接种人数 接种者中发病人数未接种者中发病人数*1952 1~3月 250 0 10 4~6月 400 2 5 7~9月 600 0 8 10~12月 700 1 12 1953 1~3月 550 0 4 4~6月 350 2 3 7~9月 150 1 6 10~12月 0 2 1954 1~3月 3 4 4~6月0 3 合 计3000957*若接种前发病，不再考虑接种依据该资料，某大夫计算接种者和未接种者白喉年发病率结果如下：0002.15.230009=÷=接种者年发病率 0004.115.2200057=÷=未接种者年发病率作业要求：回答下列问题：1．该大夫的计算结果是否正确？为什么？2．完成下表并重新计算发病率。

预防医学、卫生学营养卫生、食品卫生1. 中华职业医学.2. 实用医学多因素统计方法.3. 美国妇女自我保健经典——我们的身体我们自己.4. 食物营养成分表（北京地区）.5. 现代流行病学方法与应用.6. 医学实验设计与统计分析w.7. 美国最新临床医学问答——营养学.8. 科学补钙.9. 现代职业医学_王莹1996.10. 中国人怎么吃.11. 女性的身体个人必备手册.12. 食品营养和食品卫生.13. 性的报告 21世纪版性知识手册.14. 英汉-汉英美容医学词汇.15. 医学多变量统计与统计软件.16. 3岁宝宝技能训练与对策问答.17. 夜间育儿：怎样让婴幼儿入睡.18. 教养难带宝宝百科：养育0-5岁高需求孩子的必备知识.19. 自我分析情态的更新.20. 临床流行病学：临床科研设计、衡量与评价.21. 医学美学.22. 住院医师规范化培训手册.23. 伤害流行病学.24. 病历书写错误400例.25. 医患沟通理论与实践.26. 戒烟者的自述“吸烟与我”真人真事征稿选.27. 放松自己大步走！：雅各布松（Jacobson）肌肉放松法.28. 医学营养学_黄承钰2003.29. 现代性医学（第二版）.30. 实用卫生统计学学习指导.31. 超级育儿通.32. 新生儿营养学+.33. 病历书写与示范.34. 病历书写基本规范实用手册+.35. 中国营养科学全书下.36. 食物与食物相克.37. 健康育婴魔法书.38. 医院健康教育处方.39. 医院内感染的预防与控制（第四版）.40. 卫生保健学学习指导.41. 医疗机构医务人员三基训练指南医学影像科.42. 百年医学科技进展.43. 中国儿童骨龄评分法.44. 中国食物成分表2004 第2册.45. 中国营养科学全书上.46. 医院管理制度.47. 职业健康安全专业基础.48. 新生儿回家了.49. 化学品职业危害分类控制技术.50. 医学创新由来.51. 北京大学医学教材营养与食品卫生学.52. 母乳哺养完全指南.53. 不再疲惫战胜疲劳和不良感觉的十种方法.54. 如何喂养挑食孩子.55. 宝宝安睡魔法书.56. 常用食物相克1000问.57. 现场流行病学案例与分析.58. 医学数据挖掘.59. 健康在我心中.60. 医院感染学.61. 中国医院院长手册(第二版).62. 职业卫生也职业医学.63. 实用医院财务管理.64. _医学统计学习题全解指南.65. 求医不如求己.66. 实用流行病学学习指导.67. 医疗文书书写规范与病案管理.68. 中国医学人文教育历史、现状与前景.69. 不生病的智慧.70. SPSS医学统计速学速用.71. 伤害流行病学现场研究方法.72. 医学史十五讲.73. 中国医学人文评论：第1卷.74. 协和医事.75. 护士站必备丛书住院病人健康教育指南.76. 医学统计实战练习.77. 实用医院形象策划.78. 职业卫生与职业医学（第六版）.79. 婴幼儿的科学喂养：构筑一生健康的基石.80. 环境卫生学（第六版）.81. 营养与食品卫生学（第六版）.82. 营养与食品卫生学学习指导及习题集.83. 流行病学(第6版).84. 用人单位职业病防治实用指南.85. 企业职业卫生管理培训教材.86. 研究生规划教材统计分析在医学课题中的应用.87. 卫生统计学学习指导与习题集.88. 医院规范化管理操作范本.89. 医院规范化管理操作范本.90. 儿童少年卫生学学习指导与习题集.91. 预防医学学习指导与习题集(第2版).92. 卫生统计学(第6版).93. 2008执业药师过关必做3000题(中药学类).94. 从头到脚说健康.95. 病历书写规范（修订版）.96. 研究生教学用书医学统计学第二版.97. 2001年世界卫生报告精神卫生：新的了解、新的希望.98. 2008年世界卫生报告初级卫生保健.99. 医学统计学与 SPSS 软件应用.100. 中国医学人文评论：第2卷.101. 卫生学学习指导与习题集.102. 病案信息技术精选模拟习题集.103. 性高潮的科学.104. 8年制医学文献信息检索.105. 现代医院消毒学(第2版).106. 2009全国卫生专业技术资格考试指导预防医学.107. 不生病的智慧4 易经内病外治法.108. 患者安全医疗救治的核心作用：国际版.109. 医疗机构医务人员三基训练指南：精神科2008.110. 2009药师(药剂士)职称考试强化训练习题集.111. 医院管理流程图解.112. 病理学与病理学技术精选模拟习题集.113. 2009国家医师资格考试医学综合笔试应试指南公共卫生执业医师（上册）.114. 2009最新修订版：国家医师资格考试医学综合笔试应试指南公共卫生执业医师下. 115. 国家医师资格考试模拟试题解析临床执业助理医师.116. 不生病的智慧2 健康饮食精华·吃好不吃药.117. 不生病的智慧3 易经养生说明书.118. 新编医院管理教程（第二版）.119. 国家医师资格考试医学综合笔试应试指南临床执业助理医师 2009最新修订版. 120. 职业卫生与职业医学案例版供预防医学类专业使用.121. 医院科室管理学.122. 英文病历书写基础.123. 21世纪外科医师锻造之路124. 不生病的智慧5：佛道武药中的养生保命真法125. 专科医院经营管理实录126. 医院安全目标管理实践127. 食物与食物相克128. 简明食物营养成分表129. 中国食物成分表130. 养生师技能培训教材中医养生简论中国医学1. 目录--中国医学理论2.北京中医学院首届研究生论文汇编.3.世界易医传统生命科学大会论文集.4.手法医学与传统疗法研究新进展 '九七北海国际手法医学与传统疗法学术会议论文集.5.伤科论文汇编第三辑.6.医学科学论文汇编七.7.山东省中药学会第四次学术经验交流会（上册）论文选编.8.中医的科学原理.9.中西医结合思路与方法.10.第四届国际中西医结合肾臓病学术会议论文汇编.11.中医火神派探讨.12.全国中医结合学会骨伤科专业委员会第十二次学术年会浙江省中西医结合学会骨伤科专业.13.四诊研究论文汇编.14.阴阳五行数学及其在中医学上的应用.15.全息诊疗学.16.论文汇编南京中医药大学附属医院建院五十周年.17.医学中西结合录.18.中医与传统文化.19.社区中医诊疗实用教程上.20.社区中医诊疗实用教程下.21.目录--.-.习题、试题及题解22.中医急诊医学.23.国家执业中医师资格考试复习应试强化训练.24.原发性肾小球肾炎的中医特色疗法（英汉对照）.25.走进中医对生命和疾病的全新探索.26.二零零六中医执业医师资格考试复习应试全书（上册）.27.中医执业医师资格考试全真模拟题及真题汇析.28.二零零七年全国临床中医全科医学专业中级技术资格考试大纲与指南.29.中医各家学说习题集.30.中药学(师)考试过关必做三千题.31.医师资格考试中西医结合执业医师资格考试习题集.32.医师资格考试医师资格考试中西医结合执业医师资格考试应试指南.33.中西医结合医学主治医师资格考试全真模拟.34.目录--.-.中医学丛书、文集、连续出版物35.中国历代中医格言大观.36.中国特色医疗新技术+.37.中华传世医方下民间秘方＝.38.中华传世医方上名医验方＝.39.明清名医全书大成喻嘉言医学全书.40.明清名医全书大成孙一奎医学全书.41.明清名医全书大成李中梓医学全书.42.明清名医全书大成冯兆张医学全书.43.明清名医全书大成王肯堂医学全书.44.明清名医全书大成沈金鳌医学全书.45.明清名医全书大成叶天士医学全书.46.明清名医全书大成薛立斋医学全书.47.明清名医全书大成万密斋医学全书.48.明清名医全书大成缪希雍医学全书.49.明清名医全书大成汪昂医学全书.50.明清名医全书大成龚廷贤医学全书.51.明清名医全书大成王孟英医学全书.52.明清名医全书大成武之望医学全书.53.明清名医全书大成陈士铎医学全书.54.轩岐救正论.55.中医药学高级丛书—医古文.56.中华经典医书第二集.57.中华经典医书第三集.58.中华经典医书第五集.59.中华经典医书第七集.60.中华经典医书第四集.61.秦伯未医学名著全书.62.中华经典医书第六集.63.中华经典医书第一集.64.中华名医高新诊疗通鉴+.65.刘柏龄治疗脊柱病经验撷要.66.科学研究论文汇编失眠症用“水针疗法”疗效观察.67.柯新桥中医医学论文集.68.名师与高徒第二届著名中医药学家学术传承高层论坛选粹.69.名师与高徒：第三届著名中医药学家学术传承高层论坛选粹.70.徐大椿医书全集（下册）.71..医古文-学习重点、复习要点、考试难点_.72..医古文习题集_.73..医古文自学辅导_.74.目录--.中医预防、卫生学75.目录--.养生76.气功谚语手册.77.金华养生秘旨与分析心理学.78.中医补气血集成.79.中医预防医学.80.人体使用手册(二零零六年版).81.人体使用手册.82.太医养生宝典_中医世家漫谈三分治七分养.83.中华养生智慧.85.黄帝内经·养生智慧.86.和谐中华丛书曲黎敏养生十二说.87.黄帝内经·养生智慧二.88.中医养生保健百科.89.中国龙文化养生之道.90.人体通补手册.91.中医养生学.92.目录--.气功93.气功秘旨.94.气功精选三辑佛家、道家气功精选.95.中国自在气功.96.新灵子术.97.生命的修炼易道气功养生.98.静坐修道与长生不老.99.灵子术秘传.100.中国硬气功功法荟萃.101.双人瑜伽教程.102.武林秘籍珍本八宝硬所功阐秘.103.目录--.中医基础理论104.经络功法——古代秘传长寿术.105.金匮要略易解.106.伤寒论新义.107.伤寒论集注.108.图表注释伤寒论新义.109.内经五运六气学中医时间气象医学.110.金匮玉函经二注二十二卷.111.伤寒论今释.112.易医妙用.113.中国医学大成（八）重刊金匮玉函经二注.114.珍本医籍丛刊身经通考.115.中医药学高级丛书—伤寒论.116.中医药学高级丛书—金匮要略.117.中医运气学解秘医易宝典.118.中医药学高级丛书—内经.119.阴阳五行运气八卦及其在中医学中的应用.120.黄帝内经导读.121.聿修堂医书选素问识素问绍识灵枢识难经疏证. 122.经筋疗法.123.中医望诊相法.124.中医基础理论体系现代研究：基础与临床.125.中医药学高级丛书—中医基础理论.126.中医基础理论图表解（第二版）.127.实用整脊医术.129.中医基础理论.130.思考中医对自然与生命的时间解读伤寒论导论.131.中国针灸穴位通鉴（上卷）.132.中国针灸穴位通鉴（下卷）.133.中国针灸经络通鉴.134.五运六气临床应用大观：天人合一诊疗法.135.当中医遇上西医历史与省思.136.人体腧穴全真解剖图谱.137.活解伤寒论.138.类经（附：类经图翼类经附翼）.139.中医经络探秘经络整体调整对皮肤医疗·美容·抗衰老的突出效果（上册）. 140.中医经方临床入门.141.新世纪全国高等中医药院校规划教材配套教学用书中医学基础习题集.142.高等中医药院校教学参考丛书伤寒论（第二版）.143.伤寒论方证新识.144.全身经穴应用解剖图谱日汉对照_严振国二零零六.145.循经推运医学（下册）+.146.循经推运医学（上册）+.147.经络图解(第三版).148.针灸经络穴位图解.149.伤寒论案例版.150.医道求真《黄帝内经》学术体系研究.151.中医学基础修订版.152.开启中医之门：运气学导论.153.圆运动的古中医学.154.中医名家师讲稿丛书印会河中医学基础讲稿.155.伤寒发微.156.郝万山伤寒论讲稿.157.任应秋中医各家学说讲稿.158.高等中医药院校教学参考丛书金匮要略（第二版）.159.子午流注与灵龟八法.160.《黄帝内经》告诉了我们什么？关于生态医学思想的溯源及其现代意义研究. 161.黄帝内经太素.162.实用人体腧穴解剖图谱.163.全注全译黄帝内经上.164.全注全译黄帝内经下.165.何平叔解读《黄帝内经》古中医学的人体疾病观.166.胡希恕病位类方解.167.中国中医药学主题词表（上卷）.168.中国中医药学主题词表（下卷）.169.现代中医药文库中医时间医学全书.170.伤寒论阴阳图说P.171.黄帝内经使用手册阳气启动人体大药.172.黄帝内经使用手册阳气启动人体大药.173.古中医学堂丛书-圆运动的古中医学（续）. 174.跟《黄帝内经》学养生.175.目录--.中医临床学176.目录--.中医诊断学177.中医证候诊断治疗学.178.痰饮浅说.179.中医痛证诊疗大全.180.掌纹与疾病.181.中医舌苔图谱.182.现代掌纹诊病.183.中医手掌诊疗学.184.现代中西医诊疗丛书中西医临床疼痛学.185.中医药学高级丛书—中医诊断学.186.中医诊断与鉴别诊断学.187.金氏脉学.188.中医症状鉴别诊断学第二版.189.症因脉治.190.手掌诊病基础图解.191.脉法精粹.192.现代掌纹诊病图谱.193.周楣声脉学.194.百病望诊与图解.195.人体全息诊疗学.196.中医证候鉴别诊断学.197.中医证候鉴别诊断学（第二版）.198.常见症状中医鉴别诊疗学.199.中医诊断学.200.医宗金鉴四诊心法要诀白话解.201.耳穴诊断学.202.实用中医诊断学.203.掌纹诊病实例分析图谱.204.最新实用诊脉法.205.五天学会望手诊病.206.观手知健康：经络全息手诊.207.国际剑锋手健康法学术论文集.208.中医望诊彩色图谱.209.实用中医瘀血病证治.210.中医探解基因密码干支医学新探.211.我的脉学探索八五%的精准脉诊是如何炼成的. 212.中医诊断学新世纪第二版.213.中医证候学（上册）.214.中医证候学（中册）.215.中医证候学（下册）.216.朱文锋中医辨证学讲课实录.217.中医望诊彩色图谱.218.目录--.中医治疗学219.保健针灸与按摩.220.活血化瘀疗法临床实践增订本.221.中医熏洗疗法大全.222.推拿手法技巧图解P.223.实用中医内科大膏药手册.224.熏洗疗法.225.点穴疗法.226.实用按摩推拿大全P.227.中国民间推拿术.228.中国推拿治疗学P.229.百病中医熏洗熨擦疗法.230.火罐疗法.231.足药浴疗法.232.实用推拿手法彩色图谱.233.经穴点压疗法.234.新兴膏药应用指南.235.现代疑难病中医治疗精粹.236.推拿手法彩色图谱.237.中医病机治法学.238.捏脊.239.新手法治疗颈臂腰腿痛.240.全息经络刮痧法.241.捏脊疗法.242.实用穴位埋线疗法.243.药浴学.244.穴敷疗法聚方镜.245.中国手法诊治大全.246.人体X形平衡法.247.中医治法与方剂（第四版）.248.拍打疗法.249.拔罐疗法.250.熏洗疗法.251.疑难顽怪病论治.252.中国脊柱推拿手法全书.253.中医症状治疗学.254.实用美容美体熏浴术.255.中医理论研究与临床实践大系中医诊断治疗学.256.中医汽雾透皮治疗新法.257.应激反应与中医证候中医治疗及中西医结合中英文本. 258.捏脊.259.祖传点穴解穴疗伤秘诀.260.(便携式)推拿学学习记忆手册.261.捶背捏脊敲出身体免疫力.262.疑难病症中医治疗研究.263.实用图示捏脊疗法.264.中医辨证论治学.265.《孙子兵法》与疑难疾病的治疗.266.目录--.针灸学、针灸疗法267.重订铜人腧穴针灸图经.268.中国灸疗学.269.针灸补泻手法.270.大成水针刀疗法.271.灸疗治百病.272.太乙神针.273.中国针灸復健医学+.274.针灸玉龙经神应结合注.275.中国针灸荟萃第二分册现存针灸医籍+.276.中国针灸年纪（一九九一年卷）+.277.神经疾病针灸疗法.278.针灸有效病症.279.中国当代针灸名家医案.280.针灸临证指南.281.医学手相微经络平衡整体诊疗论.282.针灸经络生物物理学——中国第一大发明的科学验证（增订版）. 283.针灸资生经.284.中国灸法治疗学.285.针灸聚英.286.董氏奇穴针灸学.287.五行磁吸针磁疗针灸学.288.中风病与醒脑开窍针刺法.289.中国针灸证治通鉴+.290.一二五种常见病穴位疗法秘验.291.实用灸法手册.292.灸法养生.293.中国平衡针灸.294.常见病的一针疗法——单穴疗法验案汇萃.295.浮针疗法.296.平衡针法临床精要.297.肩周炎一针治疗法.298.郑氏针灸全集.299.点压特定穴治疗小儿脑瘫.300.腹针疗法.301.针灸临床辨证论治.302.针灸推拿全书.303.针灸大成.304.皮下针疗法治百病.305.针刀疗法.306.小针刀疗法.307.实用保健灸法.308.水针疗法.309.切脉针灸治癌.310.中国水针治疗学.311.特种灸法临床精要.312.针灸秘验.313.太乙神针灸临证录.314.穴位温灸疗百病.315.实用水针注射技巧.316.实用腕踝针疗法.317.手到病除中国神奇手疗大全.318.针刀医学原理_朱汉章.319.水针疗法与穴位埋藏.320.刀中刀疗法骨伤疼痛的新疗法.321.灸具灸法.322.实验针灸学.323.针灸学.324.灸疗法.325.管氏针灸经验集.326.针灸治疗临床配穴处方手册.327.金针王乐亭经验集.328.中医药学高级丛书—针灸治疗学.329.浮针疗法速治软组织伤痛.330.艾灸疗法.331.人体药库学.332.临床灸学汉英对照.333.中国针灸史图鉴（上下卷）.334.实用针刀术.335.针灸治痛（第二版）.336.中国针灸刺灸法通鉴_黄龙祥二零零三.337.水针疗法治百病.338.放血疗法控制论在中医和放血疗法中的应用. 339.实用动物针灸手册.340.中国刺血疗法大全_王峥二零零五.341.实用灸疗.342.实用水针疗法.343.耳穴贴压疗法治百病.344.艾灸疗法治百病.345.中国特种针法丛书皮肤针.346.针灸学推拿(按摩)学.347.针灸穴位图解.348.藏医火灸疗法（藏汉对照）.349.兽医针灸学.350.一针疗法《灵枢》诠用.351.三棱针.352.经穴减肥.353.针灸治验录.354.火柴棒医生手记.355.针法灸法图解（修订版）.356.现代中风针灸康复术.357.谢锡亮灸法.358.自然疗法丛书灸疗治百病.359.勉学堂针灸集成.360.习题集针灸推拿学分册（供中医药类专业用）.361.针灸基本功.362.针刀临床治疗学.363.针灸六绝：神经病针灸现代疗法（第二版）.364.针灸学_石学敏二零零七.365.针灸学（第二版）.366.针灸聚英.367.实用时间针法秘要周易与针灸学.368.针灸时间医学概论.369.中国民间刺血术.370.金针秘传.371.温灸疗法.372.图解艾灸疗法.373.实验针灸学供针灸推拿学专业五年制、七年制、研究生使用. 374.简明针灸学.375.实用舌针学.376.银质针导热治疗软组织痛.377.古法针刺举隅_张士杰一九九五.378.实验针灸学习题集.379.浮针疗法（第二版）.380.艾灸美容技法(图解).381.特色疗法丛书刺络放血疗法.382.当代名老中医经典-新针灸学.383.中医独特疗法·针刺拔罐发泡疗法(第二版).384.实测经络针灸学概要.385.实用火针疗法.386.小动物针灸技法手册.387.火针临床应用.388.循证针灸学.389.虚者灸之.390.筋长一寸寿延十年香港名医朱增祥拉筋复位法.391.对症穴位按摩大图典.392.热敏灸实用读本.393.目录--.其他疗法394.常用腧穴临床发挥.395.内丹养生功法指要.396.气功经脉入门附：全息诊疗与经络诊疗.397.治癌秘方——我治癌三拾四年医案.398.清·黄元吉养生静功心法注释.399.手足纹诊病及图解医学肤纹学.400.生活百味山药治百病.401.神仙济世良方.402.埋线疗法治百病.403.中国药膳大辞典（修订版）.404.中国药膳辨证治疗学.405.中医饮食调补学.406.中国空劲气功术（初级教材）.407.西方现代临床按摩系列基础临床按摩疗法：解剖学与治疗学的结合. 408.疾病与饮食丛书-肝·胆·胰腺病食谱.409.护肝食疗必读.410.腰腿痛——患者最想知道什么.411.中风后遗症靳三针特效治疗.412.乙肝病药膳食疗.413.图解小儿按摩除百病.414.中医药膳学.415.绿色健康通道实用足浴自我治病大全.416.自然疗法丛书足浴治百病.417.刃针微创治疗术.418.家庭足浴全书.419.足浴足疗治百病.420.痛证等常见病症的病因与手法治疗：关节肌肉的失衡与矫正.421.西方现代临床按摩系列按摩药理学.422.西方现代临床按摩系列按摩师临床指南（第三版）.423.儿科推拿学.424.西方现代临床按摩系列运动保健按摩疗法_李国惠二零零八译b. 425.临床筋伤推拿学.426.从头到脚自我按摩法.427.└─目录--.医案、医话（临床经验）428.内蒙古名老中医临床经验选粹.429.中医临证录.430.长江医话.431.增评柳选四家医案.432.回春錄新诠.433.海外医话.434.中医临证薪传录.435.医林五十年.436.向日老中医临证实录.437.名医别录（辑校本）.438.中医百家药论荟萃.439.二续名医类案（上集）.440.医学微言.441.中国名老中医药专家学术经验集三.442.赵绍琴临证验案精选.443.黄元御医书十一种下四圣心源四圣悬枢素灵微蕴长沙药解玉楸药解. 444.三十年临证探研录.445.古今名医临证金鉴咳喘肺胀卷（上卷）.446.古今名医临证金鉴外感热病卷下卷.447.古今名医临证金鉴·消渴卷.448.古今名医临证金鉴·中风卷.449.古今名医临证金鉴·男科卷.450.古今名医临证金鉴·儿科卷（上卷）.451.古今名医临证金鉴·五官科卷.452.古今名医临证金鉴·胸痹心痛卷.453.古今名医临证金鉴·奇症卷.454.古今名医临证金鉴·心悸怔忡卷.455.古今名医临证金鉴·痹证卷（下卷）.456.明清十八家名医医案.457.医方集解.458.古今名医临证金鉴·头痛眩晕卷.459.古今名医临证金鉴·妇科卷（上卷）.460.古今名医临证金鉴·淋证癃闭卷.461.古今名医临证金鉴水肿关格卷下卷.462.古今名医临证金鉴·腹泻痢疾卷.463.古今名医临证金鉴·胃痛痞满卷（下卷）.464.古今名医临证金鉴·癫狂痫卷.465.古今名医临证金鉴·黄疸胁痛臌胀卷（上卷）.466.古今名医临证金鉴·外科卷.467.古今名医临证金鉴·血证卷.468.痹症治验.469.中医临床经验选编.470.麻瑞亭治验集.471.伤寒六经求真.472.军马常发病教材（三年制试用本）（上册）.473.军马常发病教材（三年制试用本）（下册）.474.闻过喜医辑.475.清宫医案研究：横排简体字本第一册.476.曹沧洲医案.477.清宫医案研究：横排简体字本第二册.478.清宫医案研究：横排简体字本第三册.479.清宫医案研究：横排简体字本第四册.480.张岫云医案百例附：张岫云老大夫学术思想.481.现代著名老中医名著重刊丛书施今墨临床经验集.482.清代名医医话精华（精装）.483.施今墨医案解读.484.杏林霜华北京铁路总医院名老中医经验集.485.中国百年百名中医临床家丛书叶桔泉.486.广州近代老中医医案医话选编.487.上海名老中医金匮典藏.488.郑钦安医书阐释.489.上海市名中医学术经验集.490.沪上中医名家名科.491.中医名家学术经验集（二）古法针刺灵方治验.492.陈可冀学术思想及医案实录.493.当代名医临证治验实录.494.名医师承讲记.495.中医临证思辨录全国优秀中医临床人才研修项目策论精选. 496.经典传承临证录全国优秀中医临床人才研修项目医案精选. 497.薪火传承集第三批全国老中医药专家学术经验传承精选. 498.冷庐医话.499.中华名老中医学验传承宝库.500.周仲瑛医论选.501.津沽中医名家学术要略第一辑.502.二续名医类案（下集）.503.目录--.中医内科504.中医内伤火病学.505.眩晕、中风证治.506.温病纵横.507.痧胀玉衡.508.实用中医心血管病学.509.温病浅谈.510.今日中医内科（下卷）.511.今日中医内科（中卷）.512.中医药学高级丛书—温病学.513.杂病辨治.514.心脑血管疾病中医诊治.515.中医理论研究与临床实践大系中医内科五脏病学.516.今日中医内科（上卷）.517.实用中医心病学.518.实用中医消化病学.519.中医药学高级丛书—中医内科学.520.现代中医心病学.521.中医治疗强直性脊柱炎.522.赵绍琴内科学.523.现代中医肝脏病学.524.中医内科学习题集.525.现代名中医哮喘诊治绝技.526.中医内科学（供中医类专业用）.527.现代名中医冠心病治疗绝技.528.现代名中医高血压中风治疗绝技.529.中医内科证候辨析与应用.530.中医治疗强直性脊柱炎（第二版）.531.现代名中医糖尿病治疗绝技.532.中医睡眠医学.533.糖尿病的中医特色疗法（英汉对照）.534.医宗金鉴杂病心法要诀白话解.535.中医内科急症学精要.536.心血管科专病中医临床诊治（第二版）.537.4.呼吸科专病中医临床诊治(第二版).538.4.中医题库丛书中医内科学考试题解.539.全国临床中医学中西医结合医学中药学专业技术资格考试大纲与指南中医内科学. 540.中医肝病案例选.541.颜德馨中医心脑病诊治精粹.542.中医内科学习题集.543.高等中医药院校教学参考丛书中医内科学（第二版）.544.肝病中医治疗合理用药与常用中药肝损伤.545.高等中医药院校教学参考丛书温病学（第二版）.546.中医讲稿系列刘景源温病学讲稿.547.现代中医心血管病学.548.中医肾脏病学.549.痰瘀相关学说与疑难病治疗.550.实用中医内科学（第二版）.551.中医五脏相关学说研究：从五行到五脏相关.552.实用中医心血管病诊疗学.553.邵长荣实用中医肺病学.554..中医肾脏病学_.555.目录--.中医外科556.中国大肠肛门病学.557.错骨缝的诊断与治疗.558.中医美容学.559.今日中医外科.560.中国肛肠病学+.561.中医临床大系中医外科治疗大成.562.外科全生集四卷.563.外科十三方考.564.中医药学高级丛书—中医外科学.565.丁氏痔科学.566.中医外科学（供中医药类专业用）.567.中国古代医学骨科源流略.。

流行病学常用指标一．实习目的：1．掌握流行病学研究中常用的测量疾病和死亡频率指标的计算方法。

2．掌握这些指标的用途和意义。

3．理解率标化的目的，熟悉率标化的具体方法。

4．理解“人时”单位的含义，了解其适用条件，熟悉其具体计算方法。

二．学时：3学时三．实习内容：1．1992年我国某市肺癌流行病学调查资料。

2．某小学麻疹流行资料分析。

3．1952~1953年某市推行白喉预防接种资料分析。

4．两城市1990年死亡率比较。

【课题一】某市有10万人口，其中男性45,000人，女性55,000人，1992年死亡总数为1,000人（男600，女400）。

当年，肺癌发病50例（男40，女10），其中45例死亡（男36，女9）。

作业要求：计算下列各指标。

1．1992年该市粗死亡率2．1992年该市肺癌死亡率3．1992年该市性别死亡率4．1992年该市肺癌病死率5．1992年该市肺癌死亡构成比6．1992年该市肺癌性别死亡率比（男/女）【课题二】某小学共有学生300名，某年1~5月份发生了麻疹流行，共发生病例20名。

经调查发现，300名学生中有190名以往接种过麻疹疫苗，另有10名曾患过麻疹，其中2例发生在前一年的12月份。

本次麻疹流行资料见图1。

图1 学生麻疹发病时间分布示意图作业要求：完成下表计算。

表1 计算结果记录1月2月3月4月5月1~5月罹患率（%）期间患病率（%）【课题三】 某市人口30多万，全市共分10个区，1952~1953两年内该市推行白喉预防接种。

接种对象是14岁以下托儿所、幼儿园和小学校儿童。

第七区为该市白喉预防重点区。

该区1952~1954年期间人口基本没有变动，共有14岁以下接种对象5000人。

接种从1952年1月开始进行，连续不断地延至1953年9月，共接种3000人。

在此接种期间及此后9个月至1954年6月为止，曾对接种者及接种对象未接种者中发生的白喉病例进行登记，结果如下：表2 某市第七区1952.1~1954.6白喉预防接种情况及白喉发病情况 日 期 接种人数 接种者中发病人数未接种者中发病人数*1952 1~3月 250 0 10 4~6月 400 2 5 7~9月 600 0 8 10~12月 700 1 12 1953 1~3月 550 0 4 4~6月 350 2 3 7~9月 150 1 6 10~12月 0 2 1954 1~3月 3 4 4~6月0 3 合 计3000957*若接种前发病，不再考虑接种依据该资料，某大夫计算接种者和未接种者白喉年发病率结果如下：0002.15.230009=÷=接种者年发病率 0004.115.2200057=÷=未接种者年发病率作业要求：回答下列问题：1．该大夫的计算结果是否正确？为什么？2．完成下表并重新计算发病率。

胶南市1984～2004年肾综合征出血热人间疫情分析薛志坤;管勤世;张德磊【期刊名称】《预防医学论坛》【年(卷),期】2005(11)5【摘要】[目的]了解胶南市HFRS发病及死亡状况,为HFRS的防治提供科学依据。

[方法]对1984～2004年HFRS疫情资料进行分析。

[结果]1984～2004年共报告HFRS病人2633例,死亡171例。

1984～1990年发病率最高,年均发病率为29.49/10万。

10～4月高发,发病数占全年的84.9%;发病率男性为20.24/10万,女性为9.66/10万;发病以中青年为主,15～45岁发病率为15.42/10万;农民发病2053例,占发病总数的77.98%。

[结论]胶南市是HFRS高发区,发病以青壮年农民为主,且有周期性规律。

【总页数】3页(P614-616)【关键词】HFRS;疫情【作者】薛志坤;管勤世;张德磊【作者单位】山东省胶南市疾病控制中心【正文语种】中文【中图分类】R512.8【相关文献】1.周宁县1984-2002年肾综合征出血热疫情分析 [J], 肖树生;刘景荣2.集安市2003-2007年肾综合征出血热宿主动物监测及人间疫情分析 [J], 夏永发3.空军某场站1984～1994年鼠情与肾综合征出血热疫情分析 [J], 贺志平;乔忠田;李群喜;何建军4.承德市1984年-2006年肾综合征出血热疫情分析 [J], 赵国良;魏淑惠;吕静;季文波;杜明涛5.遵义县1996至2000年肾综合征出血热宿主动物监测及人间疫情分析 [J], 王定明;王昭孝;童亦兵;刘铭;胡丽娟;蔡星和;黄艳萍;张仁俊;唐朝忠;黄仁托;李昭毅;王建民;张安均;罗小波;杨明辉;乐午生;任忠秋因版权原因，仅展示原文概要，查看原文内容请购买。