1,721,022 research outputs found
The role of NK and ILC1 cells in the control of murine cytomegalovirus during ontogeny
No full textLjudski citomegalovirus (HCMV) je jedan od najrasprostranjenijih virusa diljem svijeta. Njegova raširenost predstavlja veliki problem u zdravstvenom sektoru zbog toga što može nakon infekcije ostaviti ozbiljne posljedice, pogotovo ukoliko dođe do prijenosa virusa s trudnice na plod, čime uzrokuje prirođenu infekciju HCMV-om koja može imati i letalan ishod. Imunosni sustav u prenatalnom i ranom postnatalnom razdoblju nije razvijen kao u odraslih te je nedostatak zrelosti razlog tome što infekcije u tom periodu mogu uzrokovati ozbiljne posljedice. Mehanizmi kako nerazvijeni imunosni sustav, odnosno prirođena imunost, štiti plod i novorođenčad od infekcija različitim patogenima su slabo istraženi. Za potrebe istraživanja HCMV-a koristi se mišjicitomegalovirus (MCMV) s kojim dijeli mnoge sličnosti u temeljnoj biologiji i patogenezi, te zbog toga infekcija miševa MCMV-om predstavlja najčešće korišteni model infekcije citomegalovirusom. U ovom istraživanju koristili smo transgenične miševe, pristup uklanjanja imunoloških stanica monoklonskim protutijelima, te rekombinantni MCMV kako bi istražili ulogu stanica NK i ILC1 u nadzoru infekcije MCMV-om u novookoćenih miševa. Transgenične miševe smo genotipizirali PCR-om, prisustvo stanica NK i ILC1 odredili smo metodom protočne citometrije, dok smo testom virusnih čistina odredili količinu virusa u ispitivanim organima. Rezultati ovog rada pokazali su kako stanice urođene imunosti, stanice NK i ILC1, imaju važnu ulogu u nadzoru infekcije MCMV-om u jetri novookoćenih miševa. Daljnja istraživanja će pružiti bolje razumijevanje neonatalnog imunosnog sustava.Human cytomegalovirus (HCMV) is one of the most widespread viruses worldwide. It is a major problem in healthcare because the infecion can leaveserious consequences, especially if the virus is transmitted from the pregnant woman to the fetus, i.e., if it causes congenital HCMV infection that can havea fatal outcome. The immune system in the prenatal and early postnatal period is immature as compared to adults, and the immaturity is the reason why infections in that period can result in serious consequences. The mechanisms by which an underdeveloped immune system, i.e. innate immunity, protects the fetus and newborns from infections by various pathogens have been poorly investigated. To study HCMV, murine cytomegalovirus (MCMV) is often used, which shares many biological similarities and pathogenesis; therefore, infection of mice with MCMV is the most commonly used model of cytomegalovirus infection. In this study, we used transgenic mice, an immune cell depletion approach with monoclonal antibodies, and recombinant MCMV to investigate the role of NK cells and ILC1 cells in the control of MCMV infection in newborn mice. The transgenic mice were genotyped by PCR, the presence of NK and ILC1 cells was determinedby flow cytometry method, while the levels of virus in the examined organs was determined by the plaque assay. The results of this work showed that innate immune cells, NK cells and ILC1, play an important role in controlling MCMV infection in the liver of newborn mice. Further research will provide a better understanding of the neonatal immune system
The role of NK and ILC1 cells in the control of murine cytomegalovirus during ontogeny
No full textLjudski citomegalovirus (HCMV) je jedan od najrasprostranjenijih virusa diljem svijeta. Njegova raširenost predstavlja veliki problem u zdravstvenom sektoru zbog toga što može nakon infekcije ostaviti ozbiljne posljedice, pogotovo ukoliko dođe do prijenosa virusa s trudnice na plod, čime uzrokuje prirođenu infekciju HCMV-om koja može imati i letalan ishod. Imunosni sustav u prenatalnom i ranom postnatalnom razdoblju nije razvijen kao u odraslih te je nedostatak zrelosti razlog tome što infekcije u tom periodu mogu uzrokovati ozbiljne posljedice. Mehanizmi kako nerazvijeni imunosni sustav, odnosno prirođena imunost, štiti plod i novorođenčad od infekcija različitim patogenima su slabo istraženi. Za potrebe istraživanja HCMV-a koristi se mišjicitomegalovirus (MCMV) s kojim dijeli mnoge sličnosti u temeljnoj biologiji i patogenezi, te zbog toga infekcija miševa MCMV-om predstavlja najčešće korišteni model infekcije citomegalovirusom. U ovom istraživanju koristili smo transgenične miševe, pristup uklanjanja imunoloških stanica monoklonskim protutijelima, te rekombinantni MCMV kako bi istražili ulogu stanica NK i ILC1 u nadzoru infekcije MCMV-om u novookoćenih miševa. Transgenične miševe smo genotipizirali PCR-om, prisustvo stanica NK i ILC1 odredili smo metodom protočne citometrije, dok smo testom virusnih čistina odredili količinu virusa u ispitivanim organima. Rezultati ovog rada pokazali su kako stanice urođene imunosti, stanice NK i ILC1, imaju važnu ulogu u nadzoru infekcije MCMV-om u jetri novookoćenih miševa. Daljnja istraživanja će pružiti bolje razumijevanje neonatalnog imunosnog sustava.Human cytomegalovirus (HCMV) is one of the most widespread viruses worldwide. It is a major problem in healthcare because the infecion can leaveserious consequences, especially if the virus is transmitted from the pregnant woman to the fetus, i.e., if it causes congenital HCMV infection that can havea fatal outcome. The immune system in the prenatal and early postnatal period is immature as compared to adults, and the immaturity is the reason why infections in that period can result in serious consequences. The mechanisms by which an underdeveloped immune system, i.e. innate immunity, protects the fetus and newborns from infections by various pathogens have been poorly investigated. To study HCMV, murine cytomegalovirus (MCMV) is often used, which shares many biological similarities and pathogenesis; therefore, infection of mice with MCMV is the most commonly used model of cytomegalovirus infection. In this study, we used transgenic mice, an immune cell depletion approach with monoclonal antibodies, and recombinant MCMV to investigate the role of NK cells and ILC1 cells in the control of MCMV infection in newborn mice. The transgenic mice were genotyped by PCR, the presence of NK and ILC1 cells was determinedby flow cytometry method, while the levels of virus in the examined organs was determined by the plaque assay. The results of this work showed that innate immune cells, NK cells and ILC1, play an important role in controlling MCMV infection in the liver of newborn mice. Further research will provide a better understanding of the neonatal immune system
Infection of neurons with cytomegalovirus
No full textInfekcija HCMV-om, pripadnikom porodice β herpesvirusa, široko je rasprostranjena u ljudskoj populaciji. Kod imunokompetentnih pojedinaca infekcija uzrokuje blaže ili nikakve simptome, dok kod osoba sa oslabljenim ili nezrelim imunološkim sustavom može biti životno ugrožavajuća. Kao najčešći uzročnik kongenitalnih virusnih infekcija, HCMV uzrokuje teška strukturalna i funkcionalna oštećenja CNS-a. Trenutno ne postoji cjepivo za citomegalovirus, a dostupni terapijski postupci pokazuju ograničenu učinkovitost, stoga su istraživanja temeljnih mehanizama nastanka bolesti od izuzetne važnosti.
Skoro sva dostupna istraživanja infekcije neurona CMV-om provedena su u in vitro uvjetima i pokazuju oprečne rezultate o sklonosti CMV-a prema neuronima. Svrha ovog rada bila je provesti in vivo istraživanje na mišjem modelu s reporterskim MCMV virusom kako bi se otkrilo mogu li neuroni biti produktivno inficirani. Rezultati su pokazali da u mozgu miševa dolazi do in vivo konverzije reporterskog virusa 14. dan nakon infekcije. Ovi rezultati ukazuju da inficirani neuroni produciraju virus, ali zbog niskog titra reporterskog virusa u mozgu korištenih miševa nismo saznali koliki postotak neurona ima sposobnost produkcije virusnih čestica. Stoga, ovo istraživanje služi kao preliminarno za daljnja in vivo istraživanja citomegalovirusne infekcije neurona.Infection with HCMV, a member of the β herpesvirus family, is widely distributed within the human population. In immunocompetent individuals the infection causes mild or none symptoms. On the other hand, it can be a life-threatening infection in people with weakened or immature immune system. As the most common cause of congenital viral infections, HCMV leads to severe structural and functional abnormalities of the CNS. At the moment there is no vaccine to prevent cytomegalovirus infection. Also, the available therapeutic procedures show limited efficacy. Therefore, the research on basic mechanisms of CMV infection are of great importance.
Almost all the available research on CMV infection of neurons have been conducted in vitro and suggest conflicting results on CMV preference for neurons. The purpose of this paper was to conduct an in vivo research using mouse model with reporter MCMV virus to determine whether neurons can be productively infected. The results have demonstrated that in vivo conversion of the reporter virus occurs in the brain of mice 14 days after the infection. These results indicate that infected neurons can produce the virus. However, due to the low titer of reporter virus in the brain of mice, we haven't found out the exact percentage of neurons that has the ability to produce the virus. In conclusion, this paper serves as a preliminary research for necessary further in vivo research on CMV infection of neurons
The role of NK and ILC1 cells in the control of murine cytomegalovirus during ontogeny
No full textLjudski citomegalovirus (HCMV) je jedan od najrasprostranjenijih virusa diljem svijeta. Njegova raširenost predstavlja veliki problem u zdravstvenom sektoru zbog toga što može nakon infekcije ostaviti ozbiljne posljedice, pogotovo ukoliko dođe do prijenosa virusa s trudnice na plod, čime uzrokuje prirođenu infekciju HCMV-om koja može imati i letalan ishod. Imunosni sustav u prenatalnom i ranom postnatalnom razdoblju nije razvijen kao u odraslih te je nedostatak zrelosti razlog tome što infekcije u tom periodu mogu uzrokovati ozbiljne posljedice. Mehanizmi kako nerazvijeni imunosni sustav, odnosno prirođena imunost, štiti plod i novorođenčad od infekcija različitim patogenima su slabo istraženi. Za potrebe istraživanja HCMV-a koristi se mišjicitomegalovirus (MCMV) s kojim dijeli mnoge sličnosti u temeljnoj biologiji i patogenezi, te zbog toga infekcija miševa MCMV-om predstavlja najčešće korišteni model infekcije citomegalovirusom. U ovom istraživanju koristili smo transgenične miševe, pristup uklanjanja imunoloških stanica monoklonskim protutijelima, te rekombinantni MCMV kako bi istražili ulogu stanica NK i ILC1 u nadzoru infekcije MCMV-om u novookoćenih miševa. Transgenične miševe smo genotipizirali PCR-om, prisustvo stanica NK i ILC1 odredili smo metodom protočne citometrije, dok smo testom virusnih čistina odredili količinu virusa u ispitivanim organima. Rezultati ovog rada pokazali su kako stanice urođene imunosti, stanice NK i ILC1, imaju važnu ulogu u nadzoru infekcije MCMV-om u jetri novookoćenih miševa. Daljnja istraživanja će pružiti bolje razumijevanje neonatalnog imunosnog sustava.Human cytomegalovirus (HCMV) is one of the most widespread viruses worldwide. It is a major problem in healthcare because the infecion can leaveserious consequences, especially if the virus is transmitted from the pregnant woman to the fetus, i.e., if it causes congenital HCMV infection that can havea fatal outcome. The immune system in the prenatal and early postnatal period is immature as compared to adults, and the immaturity is the reason why infections in that period can result in serious consequences. The mechanisms by which an underdeveloped immune system, i.e. innate immunity, protects the fetus and newborns from infections by various pathogens have been poorly investigated. To study HCMV, murine cytomegalovirus (MCMV) is often used, which shares many biological similarities and pathogenesis; therefore, infection of mice with MCMV is the most commonly used model of cytomegalovirus infection. In this study, we used transgenic mice, an immune cell depletion approach with monoclonal antibodies, and recombinant MCMV to investigate the role of NK cells and ILC1 cells in the control of MCMV infection in newborn mice. The transgenic mice were genotyped by PCR, the presence of NK and ILC1 cells was determinedby flow cytometry method, while the levels of virus in the examined organs was determined by the plaque assay. The results of this work showed that innate immune cells, NK cells and ILC1, play an important role in controlling MCMV infection in the liver of newborn mice. Further research will provide a better understanding of the neonatal immune system
THE ROLE OF NEURONS AND GLIAL CELLS IN PRODUCTIVE AND LATENT CYTOMEGALOVIRUS INFECTION
No full textCilj istraživanja: Humani citomegalovirus (HCMV) je vodeći uzročnik prirođenih virusnih infekcija koji često uzrokuje trajna oštećenja živčanog sustava u djece. Tipovi stanica i mehanizmi uključeni u uspostavljanje cjeloživotne latencije citomegalovirusa (CMV) u mozgu još uvijek su nepoznati. Glavni cilj ovog istraživanja je razjasniti diseminaciju virusa i stanični tropizam, te imunološke mehanizme koji nadziru infekciju i uspostavljanje latencije CMV-a u mozgu. Materijali i metode: U ovom istraživanju koristili smo mišji model prirođene CMV infekcije, u kojem su novookoćeni miševi inficirani mišjim citomegalovirusom (MCMV). Kako bismo izučavali biologiju infekcije MCMV-om u mozgu, koristili smo rekombinantne reporterske viruse i transgenične miševe koji ispoljavaju rekombinazu Cre ili im nedostaju ključni elementi efektorskih funkcija limfocita T. Tropizam, anatomska lokacija i reaktivacija virusa određeni su imunohistokemijskim metodama. Kako bismo testirali ulogu imunoloških medijatora u sprečavanju infekcije neurona HCMV-om, koristili smo staničnu liniju humanog neuroblastoma SH-SY5Y i organotipsku kulturu tkiva ljudskog fetalnog mozga (hfOBSC). Rezultati: Pokazali smo da je tijekom akutne faze infekcije u novookoćenih miševa hipokampus glavno mjesto produktivne infekcije MCMV-om. Infektivni virus prvo su proizveli astrociti, zatim mikroglije i konačno neuroni, koji su bili glavna mjesta replikacije virusa tijekom kasne faze akutne infekcije. Pokazali smo da su CD4+ limfociti T i citokin IFN-γ potrebni za razrješenje produktivne infekcije u neuronima. Citokin IFN-γ također može spriječiti infekciju stanica SH SY5Y i neurona u hfOBSC HCMV-om. Konačno, pokazali smo da MCMV uspostavlja latenciju u neuronima i da su CD4+ limfociti T ključni za sprječavanje reaktivacije virusa iz neurona. Zaključak: Rezultati ovog istraživanja po prvi put pokazuju da CMV uspostavlja latenciju u neuronima, te da su CD4+ limfociti T i citokin IFN-γ ključni za nadzor produktivne i latentne infekcije u neuronima. Ova studija ima važan translacijski potencijal s obzirom da ukazuje na to da bi poticanje odgovora posredovanog CD4+ limfocitima T moglo spriječiti neurološke posljedice uzrokovane prirođenom CMV infekcijom.Objectives: Human cytomegalovirus (HCMV) is the leading cause of congenital viral infections, frequently accompanied with long-term neurological sequelae in children. The cell types and mechanisms involved in establishing lifelong CMV latency in the brain, from which the virus reactivates intermittently, remain enigmatic. This research aims to elucidate viral dissemination and cell tropism, immune mechanisms controlling the infection, and the establishment of MCMV latency in the brain. Materials and methods: In this research, we used mouse model of congenital CMVinfection, in which newborn mice are infected with MCMV postnatally. To study the biology of MCMV infection in the brain, we utilized recombinant reporter viruses and transgenic mouse lines that express Cre recombinase or lack critical elements of T-cell effector functions. Viral tropism, anatomical location, and reactivation were determined with immunohistochemical methods. We employed human cell line SH-SY5Y and human fetal organotypic brain slices cultures (hfOBSCs) to test the role of immune mechanisms in preventing HCMV infection of neurons. Results: We found that the hippocampus is a major site of productive MCMV infection during the acute phase in newborn mice. Infectious virus was first produced by astrocytes, then microglia, and finally by neurons, which were the major sites of viral replication during the late phase of infection. CD4+ T-cells and IFN-γ were pivotal in resolving a productive infection in neurons. IFN-γ can also suppress HCMV infection of SH-SY5Y cell line and neurons in hfOBSCs. Finally, we show that MCMV establishes latency in neurons and that CD4+ T-cells are crucial to prevent virus reactivation. Conclusion: This research shows for the first time that neuron-restricted cytomegalovirus latency in the central nervous system is regulated by CD4+ T cells and IFN-γ. This study has important translational potential as it demonstrates that boosting CD4+ T-cell-mediated immunity could prevent neurological sequelae following congenital CMV infection
Role of interferon gamma in the control of mouse cytomegalovirus infection in neurons
No full textCytomegalovirus (CMV) is a highly prevalent β-herpesvirus that infects the majority of the population worldwide. CMV infections are usually asymptomatic, however, in immunocompromised individuals and newborns they can lead to severe sequelae and life-threatening conditions. Congenital CMV infection is the most common congenital infection that can affect brain development and cause severe neurological disabilities. CMV can infect a broad range of cells including neurons, which employ pro-survival strategies to avoid destruction from the immune system. Generally, CD4+ and CD8+ T cells play a crucial role in the control of CMV infection in the brain. Since these cells secrete IFNγ, the objective of this thesis is to determine the role of IFNγ in the control of MCMV infection in neurons, using a mouse model of congenital infection. For this purpose, newborn mice lacking IFNγR in neurons were infected with MCMV and organs were harvested at three timepoints of infection. Viral titers in brain were determined by plaque assay and the percentage of infected neurons was quantified by dual immunohistochemical analysis. Analysis of viral titers was also performed for congenitally infected mice lacking IFNγR in astrocytes. The results of these experiments show that IFNγ does not directly control MCMV infection in neurons and astrocytes, both during, and after resolution of productive infection. These findings add to the understanding of immune responses to CMV infection which is pivotal for development of effective therapies and vaccines and raise new questions on which molecules and cells are involved in the control of CMV infection in neurons.Citomegalovirus (CMV) je široko rasprostranjen β-herpesvirus koji inficira većinu populacije širom svijeta. CMV infekcije su obično asimptomatske, međutim u imunokompromitiranih pojedinaca i novorođenčadi mogu dovesti do ozbiljnih posljedica te stanja opasnih po život. Kongenitalna CMV infekcija je najčešća kongenitalna infekcija koja može utjecati na razvoj mozga i uzrokovati ozbiljne neurološke poremećaje. CMV može inficirati širok raspon stanica, uključujući neurone koji koriste strategije preživljavanja kako bi izbjegli uništenje od strane imunološkog sustava. Općenito, CD4+ i CD8+ T stanice imaju presudnu ulogu u kontroli CMV infekcije u mozgu. Budući da te stanice izlučuju IFNγ, cilj ovog rada je utvrditi ulogu IFNγ u kontroli MCMV infekcije u neuronima, koristeći mišji model kongenitalne infekcije. U tu svrhu, novorođeni miševi kojima nedostaje IFNγR u neuronima inficirani su MCMV-om te su im organi prikupljeni u tri vremenske točke infekcije. Virusni titar u mozgu je određen analizom plakova te je kvantificiran postotak inficiranih neurona analizom dvojne imunohistokemije. Analiza virusnih titrova provedena je i za kongenitalno-inficirane miševe kojima nedostaje IFNγR u astrocitima. Rezultati ovih eksperimenata pokazuju da IFNγ nema direktnu kontrolu nad MCMV infekcijom u neuronima i astrocitima tijekom te nakon rezolucije produktivne infekcije. Ovi pronalasci pridodaju razumijevanju imunoloških odgovora na CMV infekciju, što je ključno za razvoj učinkovitih terapija i cjepiva te postavljaju nova pitanja o tome koje molekule i stanice su uključene u kontrolu CMV infekcije u neuronima
Karakterizacija protutijela specifičnih za MCMV protein MCK-2 i gO
No full textCytomegalovirus (CMV) is β-herpesvirus, which establishes lifelong latent infection in host. CMV is transmitted through bodily fluids and represents the most frequent congenital infection in humans. Infection of healthy individuals with human CMV (HCMV) is usually asymptomatic, however, it can cause severe disease in immunocompromised individuals, as well as in infants infected in the prenatal period. CMV has broad tropism, infecting majority of cell types and organs. Viral glycoproteins mediate fusion with host cell membranes and entry of virus into target cells. Glycoprotein complexes gH/gL are determining virus specificity for infection of distinct cell types. Due to the species-specific nature of CMV, mouse CMV (MCMV) has been widely used as a model to study HCMV infection. Two gH/gL complexes have been identified in MCMV, gH/gL/gO and gH/gL/MCK-2. However, the biology and role of these complexes is still incompletely defined, in part due to the lack of antibodies specific for these proteins. Here I characterized new antibodies specific for gO and MCK-2 using flow cytometry, Western blot and enzyme-linked immunosorbent assay. Antibodies were generated by immunizing mice with proteins produced in eukaryotic cells and subsequent fusion of B cells with myeloma cells. Obtained hybridomas were preliminary screened by ELISA and positive antibodies were further tested in Western blot and flow cytometry. In total, seven out of fifty-one tested MCK-2 antibodies were specific for MCK-2 in flow cytometry. Five of them were positive in Western blot as well. Furthermore, five out of twelve tested gO antibodies specific for gO were identified by flow cytometry, and additional four by Western blot. Identified antibodies will be used in future studies of MCMV gH/gL complexes.Citomegalovirus (CMV) je β-herpesvirus koji u domaćinu uspostavlja doživotnu latentnu infekciju. CMV se prenosi tjelesnim tekućinama i predstavlja najčešću prirođenu infekciju u ljudi. Infekcija ljudskim CMV-om (eng. Human CMV, HCMV) je kod zdravih ljudi obično asimptomatska, međutim kod osoba sa oslabljenim imunološkim sustavom te djece zaražene u prenatalnom razdoblju može izazvati kliničku manifestaciju bolesti. CMV inficira većinu staničnih tipova i posljedično većinu organa. Fuzija virusne i stanične membrane te ulazak virusa u stanicu su posredovani virusnim glikoproteinima. Specifičnost virusa za ulazak u različite tipove stanica je određena glikoproteinskom kompleksima gH/gL. Budući da je HCMV specifičan za vrstu, mišji CMV (eng. mouse CMV, MCMV) se koristi kao model za istraživanje infekcije ljudskim CMV-om. Dva glikoproteinska kompleksa gH/gL su karakterizirana u mišjem CMV-u: gH/gL/gO i gH/gL/MCK-2. Biološke funkcije i uloge ovih kompleksa su nepotpuno razjašnjene, djelomično zbog nedostatka specifičnih protutijela. U ovom radu sam karakterizirala protutijela specifična za proteine gO i MCK-2 koristeći protočnu citometriju, Western blot i enzimski vezani imunosorbentni test (ELISA). Protutijela su dobivena imunizacijom miševa proteinima proizvedenim u eukariotskim stanicama nakon čega je uslijedila fuzija B limfocita sa stanicama mijeloma. Dobiveni hibridomi preliminarno su analizirani uz pomoć ELISA-e te su pozitivna protutijela dalje analizirana u Western blotu i protočnoj citometriji. Sedam od ukupno pedeset dva testirana MCK-2 protutijela je bilo specifično za MCK-2 u protočnoj citometriji, a njih pet je također bilo pozitivno u Western blotu. Nadalje, pet od ukupno dvanaest testiranih gO protutijela bilo je specifično za gO u protočnoj citometriji, a dodatnih četiri u Western blotu. Identificirana protutijela će biti korištena u daljnim istraživanjima gH/gL kompleksa MCMV-a
NK cells in pathogenesis of congenital cytomegalovirus infection
No full textCilj istraživanja: Prirođena infekcija citomegalovirusom (CMV) najčešća je virusna
prirođena infekcija koja može uzrokovati dugotrajne neurološke posljedice. Uloga
stanica NK u nadzoru virusa tijekom prirođene infekcije CMV-om, kao i utjecaj infekcije
na imunitet posredovan stanicama NK uglavnom su slabo istraženi. Glavni cilj ovog
istraživanja je odrediti ulogu stanica NK u nadzoru citomegalovirusa i promjene u
raznovrsnim podskupinama i funkciji stanica NK inducirane citomegalovirusnom
infekcijom u modelu prirođene infekcije.
Materijali i metode: U ovom istraživanju korišten je model prirođene infekcije, u kojem
su novookoćeni miševi inficirani MCMV-om s ciljem karakterizacije funkcionalnih,
fenotipskih i transkripcijskih promjena u stanicama NK. U istraživanju smo koristili
protočnu citometriju, transkriptomske analize te druge metode molekularne biologije.
Mehanizme odgovorne za promjene u stanicama NK nakon infekcije MCMV-om odredili
smo koristeći različite genetski modificirane miševe kojima nedostaju proupalni
medijatori ili njihovi receptori na stanicama NK. Alternativno smo inaktivaciju navedenih
tipova molekula proveli upotrebom blokirajućih protutijela.
Rezultati: Pokazali smo da infekcija novookoćenih miševa MCMV-om značajno
kompromitira fenotip, sazrijevanje i funkciju stanica NK. Upalni odgovor uzrokovao je
deregulaciju izražaja brojnih transkripcijskih faktora, što je rezultiralo dugotrajno
oslabljenom funkcijom stanica NK. Uz navedeno, utvrdili smo da perinatalna infekcija
narušava proces nastajanja stanica NK u koštanoj srži, što objašnjava navedene
dugotrajne negativne efekte infekcije na njihovu djelotvornost.
Zaključak: Ovo istraživanje pokazuje da perinatalna infekcija MCMV-om uzrokuje
dugotrajnu završnu diferencijaciju stanica NK, njihovu fenotipsku i transkripcijsku
disregulaciju, te narušava funkcionalni status stanica NK.Objectives: Congenital cytomegalovirus (CMV) infection is the most common viral
congenital infection that can cause long-term neurological sequelae. The role of NK
cells in the control of MCMV during congenital infection and the impact of infection on
NK cells in the context of immunity are not investigated. The main goal of this research
is to determine the role of NK cells in the control of MCMV during perinatal infection and
the changes in NK cell compartment and their function induced by CMV infection in the
congenital infection model.
Materials and methods: In this research, we used the congenital model of MCMV
infection, in which newborn mice are infected with MCMV, to assess the functional
phenotypic and transcriptional changes in NK cells. To address the mechanism
responsible for the changes in NK cells induced by MCMV infection, we used transgenic
mice, which lack major proinflammatory mediators or receptors on NK cells, or we used
blocking antibodies for their inactivation.
Results: We found that MCMV infection of newborns severely compromised NK cell
phenotype, maturation and functionality. Inflammatory responses to infection
dysregulated the expression of major transcription factors governing NK cell fate, such
as Eomes, resulting in impaired NK cell function. Moreover, the bone marrow's capacity
to efficiently generate new NK cells was reduced, explaining the prolonged negative
effects of perinatal infection on NK cells.
Conclusion: Results of this research show that perinatal MCMV infection induces
maturation, phenotypic change and transcriptional dysregulation of NK cells. We also
showed that perinatal MCMV infection causes functional changes in NK cells
Infection of astrocytes with mouse cytomegalovirus
No full textCitomegalovirus (CMV) je široko rasprostranjen virus u ljudi čija je karakteristika uspostava cjeloživotne latencije. Kod zdravih osoba infekcija je uobičajeno asimptomatska, ali u osoba s oslabljenim i nezrelim imunosnim sustavom može uzrokovati po život opasne infekcije. Kongenitalna infekcija CMV-om je najčešća transplacentarno prenosiva prirođena infekcija koja može utjecati na razvoj mozga i uzrokovati trajne neurološke poremećaje. Dosad je poznato da in vitro CMV može inficirati većinu staničnih vrsta mozga do određene razine, međutim, nije jasno vrijedi li to in vivo i je li ta infekcija produktivna. Glavni cilj ovog diplomskog rada bio je na mišjem modelu kongenitalne infekcije utvrditi inficira li CMV astrocite in vivo i podržavaju li astrociti uspješnu replikaciju virusa. U svrhu istraživanja korišteni su miševi i mišji citomegalovirus (MCMV) divljeg tipa, ali i inducibilni Cre/loxP reporterski sustav koji se sastoji od rekombinantnog MCMV-flox virusa te specifičnog transgeničnog soja miševa, u kojem je Cre rekombinaza aktivna samo u astrocitima. Nakon infekcije novorođenih miševa i prikupljanja organa u određenim vremenskim točkama, provedena je analiza infekcije astrocita imunohistokemijskom metodom, te virusna titracija mozgova pomoću metode određivanja virusnih čistina (plakova). Dobiveni rezultati su pokazali da MCMV uspješno inficira astrocite in vivo. Štoviše, na vrhuncu infekcije u mozgu, trećina inficiranih stanica su bili upravo astrociti. Osim toga, dokazano je da je infekcija astrocita in vivo produktivna te da astrociti doprinose širenju CMV infekcije u mišjem mozgu. Ova saznanja doprinose boljem razumijevanju neuropatogeneze kongenitalne CMV infekcije i otvaraju nova pitanja o utjecaju infekcije na funkciju astrocita i o ulozi ostalih staničnih vrsta u mozgu tijekom infekcije.Cytomegalovirus (CMV) is a widespread virus, characterized by the establishment of lifelong latency. In healthy individuals the infection is typically asymptomatic, however, in immunocompromised persons and in congenitally infected children CMV infection can lead to a life-threatening condition. Congenital CMV infection is the most common intrauterine infection that can affect brain development and cause permanent neurological disabilities. So far, it was clear that CMV can infect most brain cell types in vitro to a certain level, but whether this is also true in vivo and whether the infection is productive remains unknown. Thus, the main objective of this thesis was to determine whether CMV infects astrocytes in vivo and whether astrocytes support successful viral replication, using a mouse model of congenital infection. In this research we have used wild-type mice and mouse cytomegalovirus (MCMV), but also a conditional Cre/loxP reporter system, consisting of recombinant MCMV-flox virus and transgenic mouse strain in which Cre recombinase is active only in astrocytes. After MCMV infection of newborn mice and organ harvesting at certain time points, analysis of astrocyte infection was performed using immunohistochemical staining, as well as determination of brain virus titers by plaque assay. The results obtained from this study confirmed that MCMV successfully infects astrocytes in vivo, moreover, at the peak of infection in the brain, a third of the infected cells were astrocytes. In addition, it has been shown that astrocytes support productive CMV infection in vivo and that they contribute to virus spread throughout the brain. These findings contribute to a better understanding of the neuropathogenesis of congenital CMV infection and are raising new questions about the impact of infection on astrocyte function and the role of other brain cell types during infection
THE ROLE OF NEURONS AND GLIAL CELLS IN PRODUCTIVE AND LATENT CYTOMEGALOVIRUS INFECTION
No full textCilj istraživanja: Humani citomegalovirus (HCMV) je vodeći uzročnik prirođenih virusnih infekcija koji često uzrokuje trajna oštećenja živčanog sustava u djece. Tipovi stanica i mehanizmi uključeni u uspostavljanje cjeloživotne latencije citomegalovirusa (CMV) u mozgu još uvijek su nepoznati. Glavni cilj ovog istraživanja je razjasniti diseminaciju virusa i stanični tropizam, te imunološke mehanizme koji nadziru infekciju i uspostavljanje latencije CMV-a u mozgu. Materijali i metode: U ovom istraživanju koristili smo mišji model prirođene CMV infekcije, u kojem su novookoćeni miševi inficirani mišjim citomegalovirusom (MCMV). Kako bismo izučavali biologiju infekcije MCMV-om u mozgu, koristili smo rekombinantne reporterske viruse i transgenične miševe koji ispoljavaju rekombinazu Cre ili im nedostaju ključni elementi efektorskih funkcija limfocita T. Tropizam, anatomska lokacija i reaktivacija virusa određeni su imunohistokemijskim metodama. Kako bismo testirali ulogu imunoloških medijatora u sprečavanju infekcije neurona HCMV-om, koristili smo staničnu liniju humanog neuroblastoma SH-SY5Y i organotipsku kulturu tkiva ljudskog fetalnog mozga (hfOBSC). Rezultati: Pokazali smo da je tijekom akutne faze infekcije u novookoćenih miševa hipokampus glavno mjesto produktivne infekcije MCMV-om. Infektivni virus prvo su proizveli astrociti, zatim mikroglije i konačno neuroni, koji su bili glavna mjesta replikacije virusa tijekom kasne faze akutne infekcije. Pokazali smo da su CD4+ limfociti T i citokin IFN-γ potrebni za razrješenje produktivne infekcije u neuronima. Citokin IFN-γ također može spriječiti infekciju stanica SH SY5Y i neurona u hfOBSC HCMV-om. Konačno, pokazali smo da MCMV uspostavlja latenciju u neuronima i da su CD4+ limfociti T ključni za sprječavanje reaktivacije virusa iz neurona. Zaključak: Rezultati ovog istraživanja po prvi put pokazuju da CMV uspostavlja latenciju u neuronima, te da su CD4+ limfociti T i citokin IFN-γ ključni za nadzor produktivne i latentne infekcije u neuronima. Ova studija ima važan translacijski potencijal s obzirom da ukazuje na to da bi poticanje odgovora posredovanog CD4+ limfocitima T moglo spriječiti neurološke posljedice uzrokovane prirođenom CMV infekcijom.Objectives: Human cytomegalovirus (HCMV) is the leading cause of congenital viral infections, frequently accompanied with long-term neurological sequelae in children. The cell types and mechanisms involved in establishing lifelong CMV latency in the brain, from which the virus reactivates intermittently, remain enigmatic. This research aims to elucidate viral dissemination and cell tropism, immune mechanisms controlling the infection, and the establishment of MCMV latency in the brain. Materials and methods: In this research, we used mouse model of congenital CMVinfection, in which newborn mice are infected with MCMV postnatally. To study the biology of MCMV infection in the brain, we utilized recombinant reporter viruses and transgenic mouse lines that express Cre recombinase or lack critical elements of T-cell effector functions. Viral tropism, anatomical location, and reactivation were determined with immunohistochemical methods. We employed human cell line SH-SY5Y and human fetal organotypic brain slices cultures (hfOBSCs) to test the role of immune mechanisms in preventing HCMV infection of neurons. Results: We found that the hippocampus is a major site of productive MCMV infection during the acute phase in newborn mice. Infectious virus was first produced by astrocytes, then microglia, and finally by neurons, which were the major sites of viral replication during the late phase of infection. CD4+ T-cells and IFN-γ were pivotal in resolving a productive infection in neurons. IFN-γ can also suppress HCMV infection of SH-SY5Y cell line and neurons in hfOBSCs. Finally, we show that MCMV establishes latency in neurons and that CD4+ T-cells are crucial to prevent virus reactivation. Conclusion: This research shows for the first time that neuron-restricted cytomegalovirus latency in the central nervous system is regulated by CD4+ T cells and IFN-γ. This study has important translational potential as it demonstrates that boosting CD4+ T-cell-mediated immunity could prevent neurological sequelae following congenital CMV infection
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