Title Imunološki nadzor prirođene citomegalovirusne infekcije mozga u novookoćenih miševa - zaštitna uloga stanične imunosti i protuvirusnih protutijela : doktorski rad Author Đurđica Cekinović Grbeša Mentor Ester Pernjak-Pugel (mentor)Committee member Stipan Jonjić (predsjednik povjerenstva)Committee member Vlatka Mejaški-Bošnjak (član povjerenstva)Committee member Igor Prpić (član povjerenstva)Committee member Ester Pernjak-Pugel (član povjerenstva)Granter University of Rijeka Defense date and country 2011-01-01, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 616 - Pathology. Clinical medicine Abstract Cilj istraživanja: analizirati koje stanice imunog sustava imaju ulogu u kontroli infekcije u mozgu novookoćenih miševa inficiranih mišjim citomegalovirusom (MCMV). Ispitati ulogu protutijela specifičnih za virus u sprječavanju ulaska virusa u središnji živčani sustav (SŽS), razvoja encefalitisa i posljedičnih neuroloških poremećaja u inficiranih novookoćenih miševa. Materijal i metode: novookoćene miševe intraperitonealno smo inficirali divljim tipom MCMV-a 6 do 12 sati po okotu u dozi od 5 x 102 infektivnih čestica virusa (engl. plaque forming units, PFU). Kinetiku MCMV infekcije (razmnožavanja virusa) u mozgu i parametre razvoja encefalitisa (infiltrati mononuklearnih stanica, zadebljanja stijenki krvnih žila, postojanje glijalnih čvorića, upalne promjene moždanih ovojnica) smo pratili tijekom tri tjedna po infekciji. Imune stanice smo izolirali iz mozga inficiranih miševa te protočnim citometrom pomoću specifičnih protutijela analizirali njihov fenotip i aktivacijski status. Neutralizirajuća protutijela specifična za virus injicirali smo inficiranim miševima petog ili devetog dana nakon infekcije te analizirali razinu virusne infekcije u mozgu, broj patohistoloških lezija nastalih tijekom razvoja encefalitisa i parametre neurološkog razvoja mozga (debljina vanjskog zrnatog sloja kore i veličina malog mozga te morfologija Purkinjeovih stanica). Rezultati: u novookoćenih miševa inficiranih MCMV-om sedmog dana po infekciji razvija se encefalitis karakteriziran infiltracijom mononuklearnih stanica, aktivacijom mikroglije i promjenom morfologije stijenki krvnih žila. Kao odgovor na nazočnost virusa u SŽS ulaze stanice imunog sustava. Prve stanice koje nalazimo u mozgu inficiranih životinja su prirodne ubilačke stanice (stanice NK), koje možemo izolirati iz mozga krajem prvog tjedna po infekciji. U drugom tjednu nakon infekcije u mozgu inficiranih novookoćenih miševa nalazimo aktivirane V mikroglija stanice, monocite i limfocite T. Najveći udio imunih stanica u inficiranom mozgu zauzimaju CD4+ i CD8+ limfociti T. CD8+ limfociti T brojčano su dominantne imune stanice u inficiranom mozgu te posreduju u rezoluciji aktivnog umnožavanja virusa u SŽS-u. Značajan udio CD8+ limfocita T jesu limfociti specifični za proteine MCMV-a i ove stanice ostvaruju svoj antivirusni učinak lučenjem interferona gamma (IFN-γ). CD8+ limfociti T izolirani iz mozga inficiranih novookoćenih miševa imaju zaštitni učinak na razvoj MCMV infekcije što smo pokazali prijenosom ovih stanica u odrasle imunodeficijentne miševe inficirane MCMV-om. U ovih životinja je titar virusa u organima bio značajno niži u odnosu na inficirane kontrolne miševe koji nisu primili izolirane stanice. Protutijela specifična za virus također su se pokazala zaštitnima pri infekciji mozga u novookoćenih miševa inficiranih MCMV-om. U životinja kojima smo prenijeli protuvirusna protutijela petog dana nakon infekcije umnožavanje virusa u mozgu bilo je značajno manje u odnosu na kontrolne životinje koje nisu primile protutijela. Također, životinje kojima smo prenijeli protuvirusna protutijela razvile su blaži oblik encefalitisa. Posljedično, u ovih životinja poremećaji razvoja malog mozga opisani u novookoćenih miševa inficiranih MCMV-om bili su manje izraženi u odnosu na inficirane novookoćene miševe koji nisu primili imunoterapiju. Zaključak: u ovom radu opisane su komponente imunog odgovora koje sudjeluju u nadzoru MCMV infekcije u mozgu novookoćenih miševa. Po prvi puta je dokazana uloga CD8+ limfocita T u nadzoru virusa u SŽS-u u novookoćenih miševa, kao i protektivni kapacitet ovih stanica, izoliranih iz mozga inficiranih novookoćenih miševa, da kontroliraju MCMV infekciju u odraslih imunodeficijentnih miševa. Nadalje, pokazana je zaštitna uloga protutijela specifičnih za virus u sprječavanju razmnožavanja virusa u mozgu. Posljedično, pokazali smo da životinje tretirane VI protuvirusnim protutijelima razvijaju blaži oblik encefalitisa što rezultira poboljšanim razvojem malog mozga.
Abstract (english) Objectives: to analyze the components of the immune response that play a role in the control of the infection in brain of newborn mice infected with murine cytomegalovirus (MCMV). To analyze the role of virus-specific antibodies in the prevention of MCMV infection in the central nervous system (CNS), development of encephalits and neurological disorders that accompany MCMV infection in brain of newborn mice. Material and methods: newborn mice were intraperitoneally infected with 5 x 102 PFU of MCMV (wild type, Smith strain) 6 to 12 hours after birth. Kinetics of virus replication and development of encephalitis was followed through three weeks period following infection. Immune cells were isolated from infected brains and analyzed by flow cytometry. Virus-specific antibodies were inoculated into infected newborn mice at day 5 or day 9 post infection in order tho analyze the effect of antibodies onto virus entry into the CNS, virus replication in the brain and development of encephalitis. Additionaly, we analyzed the role of antibody treatment on the cerebellum development of infected newborn mice (cerebellum external granular layer thickness and the morphology of Purkinje neurons). Results: in newborn mice infected with MCMV the virus can be detected in the CNS at day 7 post infection which is accompanied by development of encephalits. Inflammatory response is characterized with the infiltration of mononuclear cells, microglia activation and perivascular cuffing. Immune cells infiltrate into infecetd brain. First cells which can be detected in brain parenchyma are NK cells, already at day 7 post infection. Infiltration of NK cell is followed by activation of microglia cells, infiltration of monocytes and T lymphocytes. CD4+ and CD8+ T lymphocytes predominate in the brain of infected newborn mice, but CD8+ T cells highly VIII outnumber CD4+ T cells. Significant amount of CD8+ T lymphocytes is specific for several MCMV encoded proteins. These immune cells mediate their antiviral activity via production of IFN-γ. Activation of CD8+ T lymphocytes results in resolution of active virus replication in infected brain which makes these cells the key components of the immune response in control of MCMV infection in developing brain. CD8+ T lymphocytes isolated from brains of infected newborn mice showed protective capacity onto MCMV replication in various organs of infected adult immunodeficient mice. This result confirms that CD8+ T lymphocytes in newborn mice are fully competent immune cells to control MCMV infection. Virus-specific antibodies transferred into infected newborn mice showed to be protective in preventing virus replication in the brain and consequent development of MCMV-induced encephalitis. Infected newborn mice that received virus-specific antibodies on day 5 or 9 post infection had decreased virus titers in the brain, reduced inflammatory response and improved postnatal development of the cerebellum as compared to infected newborn mice that received control, non-specific antibodies. Conclusion: here we characterized the components of the immune response that play a role in control of MCMV-induced encephalitis in brains of infected newborn mice. We showed that CD8+ T lymphocytes have a predominant role in resolution of active virus replication in the newborn CNS and, when isolated from brains of infected newborn mice and passively transferred into adult immunocompromised recipients, are fully capable to control MCMV infection. Also, we showed that passive immunization of mice with virus-specific antibodies has a protective effect onto development of MCMV-induced encephalitis in newborn mice. Virus-specific antibodies reduce virus replication in newborn brain which results in decreased inflammatory response and improved cerebellum development. IX
Keywords
Citomegalovirus
Encefalitis
Imunološki nadzor
Novookoćeni miševi
Prirođena infekcija
Protutijela.
Keywords (english)
Antibodies
Congenital infection
Cytomegalovirus
Encephalitis
Immune control
Newborn mice.
Language croatian URN:NBN urn:nbn:hr:188:594385 Promotion 2011 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Catalog URL https://libraries.uniri.hr/cgi-bin/ucat/unilib.cgi?form=D1121030072 Type of resource Text Extent 83 str; 30 cm File origin Born digital Access conditions Closed access Terms of use Created on 2017-01-19 18:13:59
