Preeklampsia riski ennustustesti ja -mudeli väljatöötamine

Eesti Arst 2021; 100(3):173 &nbsp

Molekulaarse kompleksdiagnostika olulisus suguteede infektsioonide diagnoosimisel

Taust. Molekulaarne kompleksdiagnostika võimaldab kliinilisest materjalist määrata palju erinevaid haigust tekitavaid mikroorganisme korraga. Eesmärk. Selgitada senist suguteede infektsioonide diagnoosimise praktikat, kompleksuuringu mõju selle kvaliteedile. Metoodika. Quattromed HTI laborisse ühe kuu jooksul saabunud suguteedest võetud proove (n = 4985) uuriti sõltumata arsti tellimusest Luminexi xMAP-paneeliga suguteede infektsioonide selliste võimalike tekitajate suhtes nagu N. gonorrhoeae, C. trachomatis, C. trachomatis LGV, T. vaginalis, M. genitalium, M. hominis, U. urealyticum, U. parvum. Tulemused. Enamasti tellis arst uuringu ühe (31%), kahe (22%) või kolme (20%) obligaatse või oportunistliku patogeeni suhtes. Enim telliti C. trachomatis’e uuringut (91% proovidest). Uuritud proovidest leiti N. gonorrhoeae 0,2%-l, C. trachomatis 3,5%-l, T. vaginalis 0,5%-l, M. genitalium 1,2%-l, M. hominis 7,9%-l, U. urealyticum 7,7%-l ja U. parvum 32%-l juhtudest. Kasutatud meetodiga tuvastasime uuritud materjalis ka teisi STLI tekitajaid, mille suhtes arst ei olnud uuringut tellinud (sagedamini mitmeid STLI-d tekitavaid mikroorganisme, nt N. gonorrhoeae ja M. genitalium). Analüüsides proove vaid arsti tellimuse järgi, oleks suguhaigusi põhjustavatest patogeenidest jäänud avastamata 40% N. gonorrhoeae, 0,6% C. trachomatis’e, 88% T. vaginalis’e ja 48% M. genitalium’i esinemisest. Järeldused. Kompleksdiagnostika kasutamine võimaldaks mitmete sugulisel teel levivate mikroorganismide (eelkoige N. gonorrhoeae ja T. vaginalis’e) paremat avastamist ning võiks seeläbi parandada suguhaiguste ravi ja diagnoosimise kvaliteeti.Eesti Arst 2014; 93(8):450–45

Novel Early Pregnancy Multimarker Screening Test for Preeclampsia Risk Prediction

Preeclampsia (PE) is a common pregnancy-linked disease, causing preterm births, complicated deliveries, and health consequences for mothers and offspring. We have previously developed 6PLEX, a multiplex assay that measures PE-related maternal serum biomarkers ADAM12, sENG, leptin, PlGF, sFlt-1, and PTX3 in a single test tube. This study investigated the potential of 6PLEX to develop novel PE prediction models for early pregnancy. We analyzed 132 serum samples drawn at 70–275 gestational days (g days) from 53 pregnant women (PE, n = 22; controls, n = 31). PE prediction models were developed using a machine learning strategy based on the stepwise selection of the most significant models and incorporating parameters with optimal resampling. Alternative models included also placental FLT1 rs4769613 T/C genotypes, a high-confidence risk factor for PE. The best performing PE prediction model using samples collected at 70–98 g days comprised of PTX3, sFlt-1, and ADAM12, the subject's parity and gestational age at sampling (AUC 0.94 [95%CI 0.84–0.99]). All cases, that developed PE several months later (onset 257.4 ± 15.2 g days), were correctly identified. The model's specificity was 80% [95%CI 65–100] and the overall accuracy was 88% [95%CI 73–95]. Incorporating additionally the placental FLT1 rs4769613 T/C genotype data increased the prediction accuracy to 93.5% [AUC = 0.97 (95%CI 0.89–1.00)]. However, 6PLEX measurements of samples collected at 100–182 g days were insufficiently informative to develop reliable PE prediction models for mid-pregnancy (accuracy <75%). In summary, the developed model opens new horizons for first-trimester PE screening, combining the easily standardizable 6PLEX assay with routinely collected antenatal care data and resulting in high sensitivity and specificity

Preeklampsia riski ennustustesti ja -mudeli väljatöötamine

Väitekirja elektrooniline versioon ei sisalda publikatsiooneSõltuvalt maailmajaost ja riigis kättesaadavast tervishoiuteenuse kvaliteedist mõjutab preeklampsia (PE) 3–5% kõigist rasedustest. Selle peamisteks kliinilisteks sümptomiteks on raseduse teisel poolel arenev hüpertensioon ja proteinuuria. PE võib põhjustada emal organpuudulikkust ning ka ema ja/või loote surma. PE-d iseloomustav uteroplatsentaarne puudulikkus põhjustab raskendatud ainevahetust ema ja loote vahel, laguproduktide eemaldamist ja üldist regulatiivset pärsingut. Kliiniliselt eristatakse varajast, enne 34. rasedusnädalat avalduvat ja hilist, alates 34. rasedusnädalast avalduvat PE-d. Tänapäeval peetakse oluliseks varajast PE ennustust aspiriinil põhineva profülaktika alustamiseks ning hilisemas raseduse faasis haiguse kinnitamist või välistamist. Varajane ennustus põhineb ema baasnäitajatel (eelnev raseduste arv, vanus, varasem PE, rass), ultraheli uuringul ja vereseerumist määratavatel PlGF või PAPP-A tasemetest. Kombineeritud riskihinnang võimaldab tuvastada 90% varajastest PE juhtudest, hilise PE korral aga ainult 40%. PE riskihinnang sFlt-1/PlGF kaudu on raseduse teisel poolel efektiivne ainult varajase PE korral. Hilise PE korral on see lähenemine spetsiifiline ainult 75% juhtudel. Käesoleva doktoritöö eesmärk oli luua uuenduslik multimarker-immuunuuring ja kombineerida selle abil saadud mõõtmiste andmestikust PE ennustusmudelid: 1. Töötada välja uudne Luminex® xMAP-il põhinev immuunuuring 6PLEX PE seoseliste seerumi biomarkerite määramiseks: ADAM12, sENG, leptiin, PlGF, sFlt-1 ja PTX3. 2. PE ennustus oli III trimestril kogutud proovidest kõige parem, kui ennustusmudelisse kaasati viis biomarkerit (sFlt-1, PlGF, ADAM12, sENG, leptiin) koos ema lisa-faktoritega. Nimetatud mudeli PE ennustustäpsus oli 96,5%. 3. I trimestri PE ennustusmudeli väljatöötamisel kasutati masinõppel põhinevat algoritmi, mis tagas parima PE ennustusmudelina 88,2% täpsuse (kaasates ADAM12, PTX3, sFlt-1 ja emapoolsed faktorid). Platsenta FLT1 rs4769613 genotüübi kaasamisel paranes I trimestri PE ennustusmudeli täpsus 93,5%-ni. Doktoritöö tulemusena töötasin välja kõrge tundlikkuse ja spetsiifilisusega innovaatilise multimarker-immuunuuringu 6PLEX, mis võimaldab ema vereproovi alusel kõrge täpsusega hinnata PE tekkeriski raseduse esimesel ja teisel poolel. Sellise uuringu eeliseks on kuluefektiivsus ja aja kokkuhoid, kuna huvipakkuvad biomarkerid määratakse samaaegselt ühest proovimaterjalist.Preeclampsia (PE) affects in total close 3-5% pregnancies worldwide, depending on the access and level of local healthcare. PE is defined as mother`s new hypertension after 20 weeks of gestation with most common additional symptom being proteinuria. It can also cause maternal organ dysfunction (hepatic, renal, haematological) and in worst case mother`s/baby`s death. One of the core causes of PE is uteroplacental pathology, leaded by inadequate spiral artery modification and poor villous development. PE is divided based on its symptomatic presence to be early- (before 34th gestational week) or late-onset (from 34th gestational week). Modern practice for PE management holds two concepts - early phase prediction for prophylaxis with aspirin or late pregnancy PE rule-out in-case of PE suspicion. First, general today to assess PE prediction based on FMF model (screening on gestational weeks 11-14) and assessing maternal characteristics and measuring preferably UtA-PI, PAPP-A or PlGF reaches to correct prognosis in 90% of cases with early-onset PE but moderate 40% with late-onset. PE rule-out method, in case of developed symptoms, by assessing sFlt-1/PLGF ratio only works effectively for early-onset situation. Use in case of late-onset increases false-positive PE predictions sharply. The aim of this thesis was to explore the dynamics and value of proposed serum biomarkers of PE in predicting the risk for the disease, and to combine informative biomarkers and clinical data to develop novel PE prediction models applicable either in early or late pregnancy: 1. The novel Luminex® based 6PLEX assay detects six PE related biomarkers ADAM12, PlGF, sENG, sFlt1, PTX3 and leptin in multiplex format with high precision and low intra- and interassay variation. 2. 6PLEX assay based multicomponent modelling (sFlt-1, PlGF, ADAM12, sENG, leptin) enables superior PE prediction in III trimester with 96.5% accuracy. 3. Applying the 6PLEX assay on I trimester, specifically between 10-14 gestational weeks, allows 88.2% precise PE prediction without any false negative cases and 80% specificity (ADAM12, PTX3, sFlt-1). Incorporation of fetal FLT1 rs4769613 data even improves the prediction performance to 93.5%, raising the precificity metrics. This thesis presents innovative experimental research developing and applying multiplex immunoassay measurements of PE related biomarkers in either early or the late pregnancy. Modelling the PE prediction using this novel assay enable timely identification of high-risk pregnancies, application of preventive measures and targeted monitoring of patients. Benefit of such approach is cost-effectiveness and time-savings as multiple biomarkers are detected at the same time in single reaction.https://www.ester.ee/record=b556115

Control of the rescue and replication of Semliki Forest virus recombinants by the insertion of miRNA target sequences.

Due to their broad cell- and tissue-tropism, alphavirus-based replication-competent vectors are of particular interest for anti-cancer therapy. These properties may, however, be potentially hazardous unless the virus infection is controlled. While the RNA genome of alphaviruses precludes the standard control techniques, host miRNAs can be used to down-regulate viral replication. In this study, target sites from ubiquitous miRNAs and those of miRNAs under-represented in cervical cancer cells were inserted into replication-competent DNA/RNA layered vectors of Semliki Forest virus. It was found that in order to achieve the most efficient suppression of recombinant virus rescue, the introduced target sequences must be fully complementary to those of the corresponding miRNAs. Target sites of ubiquitous miRNAs, introduced into the 3' untranslated region of the viral vector, profoundly reduced the rescue of recombinant viruses. Insertion of the same miRNA targets into coding region of the viral vector was approximately 300-fold less effective. Viruses carrying these miRNAs were genetically unstable and rapidly lost the target sequences. This process was delayed, but not completely prevented, by miRNA inhibitors. Target sites of miRNA under-represented in cervical cancer cells had much smaller but still significant effects on recombinant virus rescue in cervical cancer-derived HeLa cells. Over-expression of miR-214, one of these miRNAs, reduced replication of the targeted virus. Though the majority of rescued viruses maintained the introduced miRNA target sequences, genomes with deletions of these sequences were also detected. Thus, the low-level repression of rescue and replication of targeted virus in HeLa cells was still sufficient to cause genetic instability

Illustration of used miR target cassette design.

<p>Identical miRNA target sites are shown using the same colour code (grey, white or striped); the 8-nucleotide spacers between miRNA target sites are shown in black.</p

Northern blot analysis of SFV RNAs in transfected cells.

<p>BHK-21 cells were transfected with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1) in the presence of 300 picomol of each miRNA inhibitors (designated with “+”) or 900 pmol of irrelevant control oligonucleotide (designated with “−”). Total RNA was isolated from cells at 12 h p.t. or 36 h p.t.. RNA (5 µg) was loaded on a 1.4% denaturing agarose gel, resolved by electrophoresis and visualised by northern blotting with a DIG-labelled RNA probe complementary to the 3′ UTR of SFV4 or to β-actin mRNA. Arrows left of the panel designate viral genomic RNA (A), SG mRNA made from the native SG promoter (B), additional SG RNA synthesised from the duplicated SG promoter in SFV4-2SG-Gluc (C) and β-actin mRNA, used as loading control (β-act). The panel is composed of pictures obtained by two different exposures of the same filter, which was necessary due to the huge difference in viral RNAs levels at 12 and 36 h p.t.. After the shorter exposition (36 h p.t.), mRNA of β-actin is hardly detectable. Longer exposure (12 h p.t.) detects mRNA of β-actin in mock-samples at 36 h. p.t., but it is masked by the strong signal from viral RNAs in samples from cells transfected with DNA/RNA layered SFV replication-competent vectors. The experiment was repeated twice with similar results.</p

Infectivity of DNA/RNA layered SFV replication-competent vectors carrying miR-cl2 targets.

<p>(A) General design of the vectors; the same symbols as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075802#pone-0075802-g003" target="_blank">Fig. 3A</a> are used to designate different sequences. (B, C) Infectivity of recombinant vectors carrying different miR-cl2 targets was measured by ICA in BHK-21 (B) and HeLa (C) cells. The vertical axes represent infectivity normalised to that of pCMV-SFV4-2SG-Gluc (designated as Control), which was taken as 100%. The inserted miR-cl2 cassettes are indicated under the drawing. Columns on the graph represent an average of three independent experiments; error bars represent standard deviation. * designates a statistically significant difference (p<0.05), as determined by a Mann – Whitney U test.</p

Effects of miRNA inhibitors on the rescue, multiplication and Gluc expression of recombinant vectors.

<p>BHK-21 cells were electroporated with 1 µg pCMV-SFV4-2SG-Gluc (Control) or pCMV-SFV4-2SG-Gluc-miR-cl1P (miR-cl1P) in the presence of 2-0-Met-RNA oligonucleotides complementary to <i>Let-7</i>, <i>miR-17</i> and <i>miR-19</i> (300 pmol of each; indicated with black symbols and “+”) or in the presence of 900 pmol irrelevant control oligonucleotide (indicated with open symbols and “−”). Titres of rescued viruses and Gluc activity in growth media were monitored up to 48 h p.t. (horizontal axes). (A) Titres of rescued recombinant viruses. Vertical axes represent the virus titre (pfu/ml) in the growth medium. (B) Expression of Gluc by recombinant viruses. Vertical axes represent the Gluc activity (in relative luciferase activity units) in the growth medium. Representative data from one from three reproducible experiments is shown in each panel.</p