velikost textu

Patogenní působení ptačích leukózových virů u kuřat

Upozornění: Informace získané z popisných dat či souborů uložených v Repozitáři závěrečných prací nemohou být použity k výdělečným účelům nebo vydávány za studijní, vědeckou nebo jinou tvůrčí činnost jiné osoby než autora.
Název:
Patogenní působení ptačích leukózových virů u kuřat
Název v angličtině:
Pathogenic Effect of Avian Leukosis Viruses in Chicken
Typ:
Disertační práce
Autor:
Ing. Dana Průková, Ph.D.
Školitel:
MVDr. Josef Geryk, CSc.
Oponenti:
RNDr. Šárka Němečková, DrSc.
prof. RNDr. Libor Grubhoffer, CSc.
Id práce:
112464
Fakulta:
Přírodovědecká fakulta (PřF)
Pracoviště:
Katedra genetiky a mikrobiologie (31-140)
Program studia:
Genetika (P1504)
Obor studia:
Genetika (XGEN)
Přidělovaný titul:
Ph.D.
Datum obhajoby:
15. 9. 2008
Výsledek obhajoby:
Prospěl/a
Jazyk práce:
Čeština
Abstrakt:
7. SOUHRN Ptačí leukosové viry (ALV) jsou nejběžnějšími přirozeně se vyskytujícími retroviry u domácí drůbeže, vyvolávají neoplasie, mají nepříznivé vlivy na imunitní systém a způso- bují syndrom wasting disease. Jednotlivé virové kmeny se liší v účinnosti s jakou vyvolávají wasting disease. Jako nejúčinnější způsob se jeví intraembryonální inokulace viru, po vylíhnutí vnímavost kuřat rychle klesá. Nejnápadnější příznaky onemocnění byly indukovány viry podskupiny C (ALV-C), ale i jednotlivé kmeny této podskupiny se významně liší ve schopnosti induko- vat wasting disease. Téměř srovnatelnou patogenitu jako ALV-C vykazoval virus podsku- piny D (ALV-D), zatímco viry podskupin B (ALV-B) jsou jen mírně patogenní a viry pod- skupiny A (ALV-A) nevyvovolávají wasting disease. Minimální dávka ALV-C spolehlivě vyvolávající wasting disease po intraembryonální inokulaci je 102 infekčních partikulí viru. Imunohistochemická analýza ukázala, že wasting disease je doprovázen poškoze- ním lymfoidních orgánů, přičemž ALV-C vykazuje největší patogenitu. Typickým přízna- kem je deplece lymfocytů, v thymu, burse a ve slezině, ale poškozeny jsou i ostatní buňky imunitního systému. Zatímco počet dendritických buněk v burse se zvyšuje, jejich zastou- pení v thymu a slezině klesá. Ve slezině ubývá dendritických buněk hlavně v elipsoidu, který sám podléha redukci. Zvýšený počet makrofágů v thymu a slezině je v souladu se všeobecně pozorovanou aktivací monocytomakrofágového systému. Ve slezině bylo také prokázáno menší zastupení CD4+ T buněk, avšak počet CD8+ T buněk byl zvýšený. Pouze u kuřat infikovaných viry podskupiny C a D klesal poměr CD4+/CD8+ lym- focytů v periferní krvi pod hodnotu 1, viry podskupiny A a B nevyvolávaly signifikantní pokles. V souladu s morfologickými změnami v lymfoidních orgánech u kuřat infikova- ných ALV-C byla i jejich špatná odpovídavost na antigen Brucella abortus a neschopnost jejich slezinných buněk reagovat na konkanavalin A. Stejně tak absence morfologických změn v lymfoidních orgánech koreluje s dobrou protilátkovou odpovědí na bakteriální antigen a reaktivitou slezinných buněk na lektin u kuřat infikovaných ALV-A. Z analýzy patogení aktivity chimérických virů mezi ALV-A a ALV-C vyplynulo, že determinanty akutní ALV patogenity lze lokalizovat do genu env. Zbývající oblasti viro- vého genomu mají jen mírný modulační efekt, nápadný zejména u C oblasti. Všechny pa- rametry wasting disease (snížení tělesné hmotnosti, involuce thymu, sleziny a bursy) stej- 83 ně jako pokles poměru CD4+/CD8+ T lymfocytů v periferní krvi segregovaly s podskupi- novou specifitou C chimérických virů. Nefroblastomy se objevují po infekci embryí a mladých kuřat virem MAV2(N), zástupcem podskupiny B ALV. Jak potvrzují histologické nálezy, tato skupina klonálních nádorů je velmi heterogenní. Mezi nejvíce nápadné histologické změny ledvinné tkáně patří výskyt neobvyklých struktur, které jsme nazvali jako hnízda pseudonefrogeneze. Pozorovaná přítomnost cystických dilatovaných kanálků, nižší četnost vyvinutých glome- rulů a proximálních kanálků souvisí se špatnou fyziologickou funkcí ledviny. Analýza integračních míst viru MAV2(N) odhalila, že obecným místem integrace je také transkripční faktor FoxP1 (byl nalezen v 5% ledvinových nádorů). Kuřecí foxP1 obsahuje několik alternativních promotorů a sestřihových míst, proto mohou vznikat různé izoformy tohoto genu. Hladina foxP1 mRNA v nefroblastomech byla rovnocenná, nebyla pozorovaná zvýšená exprese genu ovlivněná retrovirou integrací. Rozdíly byly patrné při proteinové expresi, nejen v rozsahu zastoupení FoxP1 v tkáni, ale také v jeho lokalizaci - v jádře nebo cytoplasmě. Tato skutečnost, spolu s možnými funkcemi jednotlivých izofo- rem foxP1 by mohla přispívat k nádorové přeměně buněk. 84
Abstract v angličtině:
comparativestudy of pďhomorphologicalchangesin lymphoid tissuesinducedby ALVs of different subgroup specificity, has not been performed.Using the chicken model we aimed at clarification of additional features,especially pathomorphological and immunological chara- cteristics of the wasting disease induced by different strains of ALV{. In order to achieve a propercompďison, also additionalALV subgroup(A'B'D) were included. on the otlrer hand, several retroviruses have been found to induce kidney fumoÍs . MAv2 (N) is an avian nonacuteoncogenic Íetrovirusof subgroupB ALv. In chickens infec- ted in ovo or early afrer hatching it induces, with high efÍrciency'multip|e clonal embryonic- type tumors of kidney - nephnoblastomas (Watts and Smith, 1980).The model of MAV2 (N)- induced chicken nephroblastomais based on the assumed abiliý of MAV2(N) retrovirus to that is, by a deregulationof expressionof geneshit transform cells by insertional mutagenesis, by the proviral integration.This avian model ofnephroblastoma representsa valuable tool for identi$ing genes involved in the maligrant transformationof cells. It is assumedthat macro- scopic nepfuoblastomasarise by clonal expansion of blastema cells in which MAV2 (N) provirus has deregulatedspecific genes controling differenciation and proliferation @ajer et ď.' 2003). Summary ALVs have a large potential to induce a broad specÚum of neoplastic and non-neo- plastic disorders,particulary when congenitalytansmitted or inÚaembryonally inoculated, sinoethis modeof infectionprovidesoptimalconditionsfor induďion of persistentviraemia after hatching.Actively rep|icatingvirus reintegratesinto the genomeof targetc|ls, which is for integration thevicinity of a cellularprotooncogene, a prerequisite its accidental into leading frnally to oncogeneactivationand tumour conversionof the cell. AlV-induced tu- mours could thereforebe detectedafter a long latent period. In the meantime,however,the infectedorganismis continuďly suppliedby a large quantityofviral antigens,which may differentphysiological disturbeitherdirectlyor indirectlyby so far unknownmechanisms functions. The outcomeofthis disturbanceďe acutenon-neoplasticdiseasessuch as u,asting disease detectedsoon after hatching' Fřst of all, the role of age and the administeredvirus dose on AlV-C-induced wasting diseasein chickens was examined.For effrcientwasting diseaseinduďon, the embryonicstageof the host plays a critica|role.Thelowestvirus dose administered into chicken embryos and exertingthe pathologicalactivity intraembryonally approachesthe va|ue of l - l0 infectious units. As a quantitativeparameteÍ the wasting of diseasewe chosethe body mass and lymfoid oÍgan mass of chickenstwo weeks afterhatch- ing, when the diseasewas fully developed.Anefficient induction of wasting disease in chickensby AIV, particularyby subgroup requires>102infectiousunits inoculatedin mid C, embryogenesis. The subsequenteffort ofpresent studywas to define the potentialofseveral ALV-C virus strainsto inducewastingdiseasewithin the first two weeks afterhatching,and to com- paÍe to otherALVs of subgroups B, and D. it A, ln our case,a high virus dose was used for inoculationand all testedALVs replicated well in vivo. Hence,they should readily producewastingdisease.Surprisingly,the opposite is true since ALV-A (RAV-I) and ALV-B (RAV-2), reachingthe highesttitresin vivo, exhi- pathogeniciý,respectively, virusesof subgroups and D, which rep|i- bit no or moderate and C cate less efficiently,are highly pathogenic. Apparently,viral genepÍoductsparticipatein the acutepathogenicicity ALVs and,of them,the Env proteinappeaÍs be the main candida. of to knowledgehas been accumulated far on the role ofthe Env proteinin re- te. An abundant so tooviruspathogenicity (reviewedin Svobodaet al. 2003). When comparingthe acutepathogenicity ALV virusesof A, B, C and D subgroup of speciÍiciýwe haveencountered notab|e differences amongindividualviral strains. The most distinctive changeswere producedby viruses of subgroupC, among them only RAV-49 appeared|esseficient in wasting diseaseinduction.A moderatepathogeniciý not accompa- nied by wastingwas scored in ALV-B (RAV-2)- infectedchickens,in which involutiononly pathogenicity RAV-2 was surprising,as of the thymusandbuna were found.The moderate of other viral strains employingthe same receptoÍ associatedwith cytotoxicity' MAv-2(o) of subgroup (Hirotaet al.' 1980;Smith and Ivanyi' 1980)and RÁV.50 of subgroup (pÍesent B D study),were provento be higbly pathogenicin vivo. The dataon RAV-2 and RAV-49 docu- ment that within both subgroups,B and C, significant differencesin pathogenicityexist betweenindividual viral strainsand indicatethat both,wasting and the lymphoid tissue alte- rationmay be inducedindependently. The most conspicuous histopathological were foundin chickensinfectedwith changes subgroup and D, and less distinctivelywith subgroup viruses.Alterationsrevealedin the C B thymustissueof theseanimals,accompanied somecasesevenwitÍtcollapsedlobular archi- in tectureand slight fibrosis, resembledprecociouspuberty(Seemayeret al., 1984)or thymic epithelial injury found in both children and adult patientssuffering acquired immunodefi- ciency syndrome(Ye et al., 2004',HazraandMackal, 2005). Detection ofp27gagprotein indicated replication ofthe virus in thymicepitheliďcells. pÍoteins Expressedretrovira| which p|ays may injure functionsof this cellular compartÍnent, an importantrole in promotionand maturation thymocytesthroughthe action of secreted of humoral substances through the direct contact with thymocltes (Lobach and Ha1,nes, or 1987). An observedincreaseof TCR2* T cells in the thymusmight represent consequence a of such functionalthymic disturbance, detailedmechanismof which remains,however, the unclear.An essentialfeaturewas depletionof lymphocytesin the thymus.bursa and spleen. While the numberof dendriticcells in the bursawas increased, theiÍÍepresentation the thy- in mus and spleenwas Íeduced. the spleen,however,the reductionof dendriticcells concer- In ned only an elipsoid compartment, which in itself was also markedly reduced. The most re- markablechangesin the spleenwere encountered again in chickensinfectedwith subgroupC and D viruses.Namely ALV-C virusesproducedan impairmentof PElS(peri-ellipsoid lym- phocýe sheaths),consideÍed a functional equivalentof the mammalianmarginal zone for (Jeurissen a|.' |992) and PALS regions(peri.arteriolat et lymphocýe sheaths),which is chara- cterizedby a markedreduction B- andT cells with increased of CD8' and depletedCD4* lym- phocytes. Thesechangesresembled both damageofthe marginalzone and shifts in CD8'and CD4+TJymphocyterepresentation the spleen of HlV-infected patients (Wilkins et a1., in 2003).An increasednumerof macrophages the thymusand spleenconesponded in with the observedgeneralactivationof the monocyte-macrophage system.However,intact ellipsoids in RAV-I- and RAV-2- infected chickens would indicate some selectivity of this cellular compartment responding differentviral antigens. in to Morphologicalalteration lymphoid organsof diseased of chickensindicateda heavily impaired immune system.Ratio CD4'/CD8- T lymfocytes in peripheralblood decreased undervalue I in chickensinfectedwith ALV-C and ALV-D, while virusesof subgroup and A B do not inducedsignificantdecÍease this ratio. We have no data aboutc}.totoxic of activity increasedCD8* ly,rnfocytes, it is possiblethatthesecells don't lack such activity.Zinker- but (1994)thought,that cytotoxic cD8* T cells could be responsiblefor nagel and HengartneÍ destruction infectedcells of immunesystem,in late stagesof HIV, and by this way they of could be ableto inducedimmunodeficiency. In accordwith alteredbursal morphologywas a finding of severeimpairmentof hu- moral immunityin AlV-C-infected chickensthat failed in mountingan antibodyresponse to Brucella abortus anÍigen. severealteration lynphoid tissuesagreedwe|l with the in- The of efficiencyofsplenic lymphocýes isolatedfrom ALV-C-infected chickensin responding pro- perly to concanavalin On the otherhand,two weeks afterhatchingAlV-A-infected chic- A. kens have apparentlypreservedintact humoralimmunity,and sp|eniclymphocýes of ALV- A-infectedchickensresponded stimulation concanavalinA,which is in accordance to by with oflymphoid organsin thesechickens. ofmorphologicalchanges absence The affrnityof ALVs for lymphoidtissuesresultingin severemorphological alteration ( of the thymus,bursaand spleenfollowed by subversion immuneregulatory of functions appears be an unifuingaspectofALV acutepathogenicity. to However,in tissuesections ( from infectedanimalsirrespective the viral strain,only someB lymphocýes stainedposi. of tively for the viral Gag protein,indicatingactiveviral replication. otherlymphoid Instead, suchas macrophages, cellularcompartrnents follicular dendriticcells and thymic epithelium, splenicellipsoid cells were largely infected. the pathogenesis ALV-induced immune In of damagethe directtoxic effect ofviruses, namelythoseofsubgroup B and D, has been invo- ked. However,the direct toxicity may not be a principal causeof lymphoid tissuedepletion; more likely lymphocytesare attackedin an indirectway, as it has been shown in another re- (Alimontiet al.,2003). troviralinfection The striking differencesin acute pathogenicityof individual strains of ALVs offer systemfor analysisof viral genomeregionsplaying a significantrole a uniqueexperimental in establishingwasting disease.Based on analysis of pathogenicactivity chimeric viruses betweenALV-A and ALV C' geneený, respectivelyits surfaceunit, is the primaIy doter- minantof pathogenic resultingfrom ALV infection.The otherregionsof viral geno- response me have only slight moduleeffect,especiallyC (constant region).All observedparameters of wastingdisease(reductionof body mass,involutionofthymus, bursa and spleen,also ratio CD4./CD8. T |ymphocytes peripherď blood) was exhibitedin chickensinfectedwith chi- in meric virusesof subgroup specifity. C Despite a large body of our knowledgeaboutadverseeffectsof retroviralinfections, the mechanismresponsiblefor wastingdiseasein infectedhostshas not yet been fully clari- fied. In this respect,comparativestudies of the wasting disease and immunosuppression employing different experimentalmodels appearvďuable, becausethey could bring new insightsinto the pathogenesis acuteretrovirus-induced of diseases. Contraryto pleiotropiceffectsofwasting disease, nephroblastomas ÍepÍesentsretro. a virus pathogenic within one cell transformedin consequence manifestation ofretroviral mutagenesis give rise to macroscopic insertional and tumorof kidney. Morphological picture of nephroblastomas confirms,that this group of clonal tumors The most prominentalterationsthat was observedwere imperfectly is very heterogenous. tubules-with with-outglomeruli,smalleror larger aggregations unorgani- differentiated or of zed and apparentlyundifferentiated cells and unusualsphericalformationsnot known from normalnephrogenesis reminiscent originsof the normaltubuleformation. call them but of We nestsof pseudonephrogenesis. more differentiated In with glomeruli, nephronssupplemented the cystic dilationsofnephrontubularsegments appeared Based on qualitative frequently. and quantitative representations ofthese structures, sampleswere divided into four major classes, infectedtissuewith a prevalenceof normal renal 0, L II, and III. Class 0 samplesrepresent structuresand sporadic cystic dilations of tubules.The nests of abnormalnephrogenesis, whichwereconsidered mostevident the symptoms malignant of *ere transformation. missing present all otherclasses. in class0, but were constantly in ClassesI and II includedtumors with moreor less differentiated nephrons, respectively, variousnumbenofc)stic dilations of tubules, a growingproportion and cells.Samples ofunorganized belonging classIII contai- to nedonly thenests pseudonephrogenesis unorganized of and cells. TheÍe was a correlationbetween tumormorphologyclass and the fumor size. For. the ty nephroblastomas were distributedinto classes I to III, as describedabove, and ordered accordingto their mass. In general,class III members,the least differentiated nephroblasto- mas,clearly displayedgreater size, althougha ratherhigh size variationwithin eachclasswas registered. suggestthat the size variation is mainly causedby differentgrowth ratesof We each individual tumor clone and not by a differenttime of a targetcell infectionbecausethe poo|oftarget cells for transformďion(nephrogenicblastemacells) fadesaway rapidly within the first few days after hatching. It has beenassumedthatthe malignantrenal cell arisesas late as severalspeciťtcregu- latorypathwaysin it havebeendistorted MAv2 integration. by Since thesepathways con- aÍe stituted cascades by genes, provirusdoesnot haveto hit one offunctionally connected the particulargenein orderto deregulate pathway. the That is probablyuhl the effortsto identi! integrations only a limited success(Westawayet al., the crucial nephroblastoma-specific had 1986; That is alsoprobably Joliot et al.,1992). why nephroblastomas displayhistopathologi- geneshave a different impact on the phenoýpe of cal variations,since distinct deregulated transformed deregulation some specific genesmight contribute ma- cells. Nevertheless, of to more efficiently than activatioďinhibitionof others.and such genes lignant transformation shouldconstitute set of 'commonintegration a sites'of the MAV2 provirusin nephroblasto- ma. sinc retoviral integration is in principle site-unspecifio@rown' 1997)' the existence of a common site of integration found in a limited number of independenttumor clones must be a result of the selection process:only cells in which the MAV2 provirus has hit the proper ge- ne or combination of genesgive rise to a tumor. Analysis of integration sites have reveďed the tanscriptional factotfoxPl to be one of the common sites of MAV2 provřal integrationin chicken nephrob|astoma.Theintegration events are clustered around the second coding exon. Therefore N-tuncated protein could ari- se. on the one hand, full-length FoxPl is consider to be a fumor suppressorgene in epitheliď malignancies (Banham et al., 2001), on the other hand N-truncated small isoforms seemsto contributeto oncogeniotransformation(Brown et al., 2008). ChickenfoxPl included severď altemative promotoÍsand altemďive splicing sites, for thď reason various foxPl isoform could arise. A level offoxPl mRNA in nepfuoblasto- mas was equivalen! no increased gene expression influnced by reÚoviral integrďion was observed.Difrerences were noticed in the protein expressioq nuclear or oytoplasmic location ofFoxPl was detected.It is possiblethat the expressionofaltematively spliced Foxpl pro- teins may explain the presence of cytoplasmic staining thď was observed in some tumors (Fox et al., 2004). The virally ďteredýxP/ might inteďere (in a dominant-negďive fashion) witlr a normal funďion of the gene and supportmďipant transformďion.
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