A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line

BACKGROUND: The rationale of using bovine papillomavirus-1 (BPV-1) derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR) gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7. RESULTS: The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles. CONCLUSION: Bovine papillomavirus type-1 (BPV-1)-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1–5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles

Mechanism of Genomic Instability in Cells Infected with the High-Risk Human Papillomaviruses

In HPV–related cancers, the “high-risk” human papillomaviruses (HPVs) are frequently found integrated into the cellular genome. The integrated subgenomic HPV fragments express viral oncoproteins and carry an origin of DNA replication that is capable of initiating bidirectional DNA re-replication in the presence of HPV replication proteins E1 and E2, which ultimately leads to rearrangements within the locus of the integrated viral DNA. The current study indicates that the E1- and E2-dependent DNA replication from the integrated HPV origin follows the “onion skin”–type replication mode and generates a heterogeneous population of replication intermediates. These include linear, branched, open circular, and supercoiled plasmids, as identified by two-dimensional neutral-neutral gel-electrophoresis. We used immunofluorescence analysis to show that the DNA repair/recombination centers are assembled at the sites of the integrated HPV replication. These centers recruit viral and cellular replication proteins, the MRE complex, Ku70/80, ATM, Chk2, and, to some extent, ATRIP and Chk1 (S317). In addition, the synthesis of histone γH2AX, which is a hallmark of DNA double strand breaks, is induced, and Chk2 is activated by phosphorylation in the HPV–replicating cells. These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH. We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV. We conclude that the HPV replication origin within the host chromosome is one of the key factors that triggers the development of HPV–associated cancers. It could be used as a starting point for the “onion skin”–type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection

HPV-18 genoomi jagunemine ja säilumine rakkudes

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Pärast papilloomiviiruste (HPV) ja emakakaelavähi vahelise seose avastamist 1980ndate aastate lõpust on HPV alane uurimustöö olnud suunatud nende viiruste poolt indutseeritavate vähkkasvajate tekke molekulaarsete mehhanismide väljaselgitamisele. Preventatiivsete vaktsiinide väljatöötamise järgselt on võimalik HPV nakkust vältida. Samas on suur hulk inimesi, kes praeguseks on HPV-ga juba nakatunud ning neil on reaalne risk saada vähkkasvaja oma hilisematel eluetappidel. Kahjuks pole HPV levikut suudetud siiani efektiivselt veel piirata. Seetõttu on jätkuvalt aktuaalne viirusvastaste ravimite väljatöötamine, mis võimaldaks viirusnakkust efektiivselt elimineerida. Sealjuures on põhirõhk leida ühendeid, mis suudaksid peatada viiruse DNA replikatsiooni või takistada viiruse genoomi säilumist jagunevates rakkudes. Käesolevas doktoritöös anname ülevaate meie tööst - erinevate HPV subtüüpide võimest replitseeruda inimese osteosarkoomi rakuliinis U2OS, kirjeldame HPV-18 transkriptsioonikaarti ning näitame, et HPV replikatsioon ning transkriptsioon U2OS rakkudes on otseses vastavuses varasema informatsiooniga HPV genoomi replikatsiooni ning transkriptsiooni kohta. See annab alust järelduseks, et U2OS rakuliin on relevantne süsteem HPV genoomi funktsionaalseteks uuringuteks. Minu doktoritöö viimases osas esitame tulemused HPV-18 DNA stabiilse säilumise uurimisest, kus me kirjeldame HPV-18 DNA säilumiseks vajalike viiruslike elemente ning valideerime neid HPV-18 genoomi kontekstis. Kokkuvõtvalt, meie töö tulemusena kirjeldame me rakuliini, mille abil on võimalik luua süsteem viirusvastaste replikatsiooni ja HPV-18 DNA säilumist inhibeerivate ühendite skriinimiseks.After the discovery of the link between papillomaviruses (HPV) and cervical cancer in the 1980’s, papillomavirus research has been directed towards the studies of the molecular mechanisms of induction of cervical cancer by the papillomaviruses. The preventive vaccine against HPV makes it is possible to avoid HPV infection, however a large number of people are still infected and HPV infection has not been effectively controlled yet. Thereby it is still important to continue to pursue HPV antivirals that could either block the viral DNA replication or stable maintenance of the viral genome in infected cells. In this thesis we describe immortalized human osteosarcoma cell line U2OS that is capable of supporting replication a number of different HPV subtypes. We describe further the transcriptional map of HPV-18 in U2OS cells and verify the relevancy of this system as the information obtained from these studies correlates with the previous findings of HPV-18 in primary keratinocytes. In the last part of this thesis we go in depth of the stable maintenance and segregation of HPV-18 DNA as we describe the cis-elements crucial for this function. We further validate this information in the context of the U2OS cells. In conclusion we describe a cell line suitable for developing screening system for antivirals against HPV and we go in depth of describing crucial elements of HPV-18 DNA segregation

MANF regulates neuronal survival and UPR through its ER-located receptor IRE1a

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located pro-tein with cytoprotective effects in neurons and pancreatic b cells in vitro and in models of neurodegeneration and diabetes in vivo. However, the exact mode of MANF action has remained elusive. Here, we show that MANF directly interacts with the ER transmembrane unfolded protein response (UPR) sensor IRE1a, and we identify the binding interface between MANF and IRE1a. The expression of wild-type MANF, but not its IRE1a binding-deficient mutant, attenuates UPR signaling by decreasing IRE1a oligomerization; phosphor-ylation; splicing of Xbp1, Atf6, and Txnip levels; and protecting neurons from ER stress-induced death. MANF-IRE1a interaction and not MANF-BiP interaction is crucial for MANF pro-survival activity in neurons in vitro and is required to protect dopamine neurons in an animal model of Parkinson's disease. Our data show IRE1a as an intracellular receptor for MANF and regulator of neuronal survival.Peer reviewe

Increased circulating concentrations of mesencephalic astrocyte-derived neurotrophic factor in children with type 1 diabetes

Mesencephalic astrocyte-derived neurotrophic factor (MANF) was recently shown to be essential for the survival and proliferation of pancreatic beta-cells in mice, where deletion of MANF resulted in diabetes. The current study aimed at determining whether the concentration of circulating MANF is associated with the clinical manifestation of human type 1 diabetes (T1D). MANF expression in T1D or MANF levels in serum have not been previously studied. We developed an enzyme-linked immunosorbent assay (ELISA) for MANF and measured serum MANF concentrations from 186 newly diagnosed children and adolescents and 20 adults with longer-term T1D alongside with age-matched controls. In healthy controls the mean serum MANF concentration was 7.0 ng/ml. High MANF concentrations were found in children 1-9 years of age close to the diagnosis of T1D. The increased MANF concentrations were not associated with diabetes-predictive autoantibodies and autoantibodies against MANF were extremely rare. Patients with conspicuously high MANF serum concentrations had lower C-peptide levels compared to patients with moderate MANF concentrations. Our data indicate that increased MANF concentrations in serum are associated with the clinical manifestation of T1D in children, but the exact mechanism behind the increase remains elusive.Peer reviewe

Koroonaviiruse omikrontüve vastane antikehade kaitse ja T-rakuline immuunvastus pärast BNT162b2-vaktsiini tõhustusdoosi saamist

Eesti Arst 2023; 102(2):11