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We evaluated rotational thromboelastometry tracings in 44 critically ill coronavirus disease 2019 patients, to determine whether there is a viscoelastic fingerprint and to test the hypothesis that the diagnosis and prediction of venous thromboembolism would be enhanced by the addition of rotational thromboelastometry testing.Rotational thromboelastometry values reflected an increase in clot strength for the EXTEM, INTEM, and FIBTEM assays beyond the reference range. No hyperfibrinolysis was noted. Fibrinolysis shutdown was present but did not correlate with thrombosis; 32% (14/44) of patients experienced a thrombotic episode. For every 1 mm increase of FIBTEM maximum clot formation, the odds of developing thrombosis increased 20% (95% confidence interval, 0-40%, P = .043), whereas for every 1,000 ng/mL increase in D-dimer, the odds of thrombosis increased by 70% (95% confidence interval, 20%-150%, P = .004), after adjustment for age and sex (AUC 0.96, 95% confidence interval, 0.90-1.00). There was a slight but significant improvement in model performance after adding FIBTEM maximum clot formation and EXTEM clot formation time to D-dimer in a multivariable model (P = .04).D-dimer concentrations were more predictive of thrombosis in our patient population than any other parameter. Rotational thromboelastometry confirmed the hypercoagulable state of coronavirus disease 2019 intensive care unit patients. FIBTEM maximum clot formation and EXTEM clot formation time increased the predictability for thrombosis compared with only using D-dimer. Rotational thromboelastometry analysis is most useful in augmenting the information provided by the D-dimer concentration for venous thromboembolism risk assessment when the D-dimer concentration is between 1,625 and 6,900 ng/dL, but the enhancement is modest. Fibrinolysis shutdown did not correlate with thrombosis.

Aim. To analyze the topographic patterns of valvular and atherosclerotic calcification growth.Methods. Dysfunctional aortic valves (n = 18) and atherosclerotic plaques (n = 20) were fixed in formalin, postfixed in 1% osmium tetroxide, consecutively stained by 2% osmium tetroxide and 2% uranyl acetate, and embedded into epoxy resin (Epon) with the further grinding and polishing ofthe samples. Upon the counterstaining by lead citrate and sputter coating with carbon, samples were visualized by backscattered scanning electron microscopy. Elemental analysis was conducted via energy-dispersive X-ray spectroscopy. Measurement of Ca/P ratio within the mineral deposits was carried out employing a pool table principle (i.e., in the center of the deposit, in the near and far circumferences (clockwise), and in control regions around the mineral deposit). Topographic patterns of calcifications were modeled using the correlation analysis. Results. Significant correlation was revealed between the Ca/P ratio in the deposit center and in the near and far circumferences of deposit in both in valvular (r = 0,35-0,78 - near circumference; r = 0,63-0,69 - far circumference) and atherosclerotic mineral deposits (r = 0,37-0,56 - near circumference; r = 0,48-0,63 - far circumference), suggesting the hierarchical growth of cardiovascular calcification around the initial nucleation sites.Conclusion. Valvular and atherosclerotic calcifications development is concentric.

Background The benefit of adjuvant therapy in synovial sarcoma (SS) treatment is under debate. Long-term follow-up data are missing. Methods SS patients treated in the consecutive trials CWS-81, CWS-86, CWS-91, CWS-96, CWS-2002-P, and the SoTiSaR-registry till 2013 were analyzed. Results Median age of 185 patients was 13.9 years (0.1–56)—with median follow-up of 7.4 years for 163 survivors. Most tumors (76%) were located in extremities. Size was 10 cm in 13 (7%) (13 missing). In 84 (45%) tumors, first excision was complete (R0 corresponding to IRS-I-group) and in 101 (55%) marginal (R1 corresponding to IRS-II-group). In a subsequent surgical intervention during chemotherapy, R0-status was accomplished in 23 additional IRS-II-group patients with secondary surgery. Radiotherapy was administered to 135 (73%), thereof 62 with R0-status and 67 R1-status (6 missing information). Adjuvant chemotherapy was administered to all but six patients. 5-year event-free (EFS) and overall survival (OS) was 82.9% ± 5.7 (95%CI) and 92.5% ± 3.9. Local and metastatic relapse-free survival was 91.3% ± 4.3 and 92.3% ± 4.1 at 5 years, respectively. In the multivariate analysis, tumor size and no chemotherapy were independently associated with EFS. Size and site were associated with OS. In a detailed analysis of local and metastatic events, tumor size was associated with an independent risk for developing metastases. No independent factor for suffering local recurrence could be identified. Discussion Omission of chemotherapy in a non-stratified way seems not justified. Size governs survival due to high linear association with risk of suffering metastatic recurrence in a granular classification.

A 2-stage comprehensive psychological and physiological examination of freshmen of a higher naval educational institution was carried out. At the first stage, characteristic profiles of cadets were determined. At the second stage, the identified characteristic profiles were correlated with the traditional criteria for military-professional adaptation. It was found that the most often examined young men (according to the Leonhard Schmiszek technique) have such character accentuations as hypertimicity (81.8%) and demonstrability (53.1%), less often stuck (39.3%), pedanticity (19.6%), emotion (18.2%), exaltation (15.4%), and even less often cyclothymic (8.4%), excitability (7.7%), anxiety (2.8%) and dysthymic (2,1%). Cadets assigned to the passive-aggressive stereotype group manage worse in the main disciplines than cadets from other groups. Cadets with passive-aggressive stereotype have lower academic performance as compared to other groups. They have lower expert scores in discipline, neuropsychic stability, communication skills, credibility in a team and level of general culture. In addition, in cadets of this group, the mobility of the main nervous processes at the level of the motor analyzer is somewhat lower to the comparison group. The study showed that the characterological features in the form of mixing or opposing accentuations of characters revealed in cadets play in favor of their personal development, health and successful study only in about 2040% of cases. In other cases, these character features do not contribute to this and can develop into personal disorders incompatible with military service during the training process. Based on the results of the study, psychological and pedagogical tasks were formulated to improve the system of selecting of cadets for naval educational institutions and to improve their professional education.

The expression of genes involved in DNA repair (DDB1, ERCC4, ERCC5), leukocyte adhesion (VCAM1, ICAM1, SELE, SELP), endothelial mechanotransduction (KLF4), endothelial differentiation (PECAM1, CDH5, CD34, NOS3), endothelial-to-mesenchymal transition (SNAI1, SNAI2, TWIST1, GATA4, ZEB1, CDH2), and also coding scavenger-receptors (LOX1, SCARF1, CD36, LDLR, VLDR), antioxidant system (PXDN, CAT, SOD1) and transcription factor (HEY2) has been studied using the quantitative PCR in primary human coronary (HCAEC) and internal thoracic (HITAEC) arteries endothelial cells exposed to alkylating mutagen mitomycin C (MMC). The study was carried out two time points after 6 h of incubation with MMC and after 6 h of the genotoxic load followed by 24 h of incubation in pure culture medium. After the exposure to MMC, a decreased expression of almost all studied genes was noted in the exposed HCAEC and HITAEC; the only exception was found in the case of SNAI2, which demonstrated a 4-fold increase in its expression compared to the unexposed control. Elimination of MMC from the both cell cultures was accompanied by increased expression of the VCAM1, ICAM1, SELE, SNAI2, KLF4 genes and decreased expression of the PECAM1, CDH5, CD34, ZEB1, CAT, PXDN genes. In addition, HITAEC cells were characterized by decreased expression of the SOD1, SCARF1, CD36 genes and an increased expression of the SNAI1 and TWIST1 genes; in HCAEC, increased expression of the LDLR and VLDLR genes was noted. Thus, the genotoxic stress, induced by the alkylating mutagen MMC, is associated with the endothelial dysfunction, manifested by the altered gene expression profile of the endothelial cell cultivated under conditions of the genotoxic load.

Hypercalcemia and hyperphosphatemia associate with an elevated risk of cardiovascular events, yet the pathophysiological basis of this association is unclear. Disturbed mineral homeostasis and the associated hypercalcemia and hyperphosphatemia may result in the formation of circulating calciprotein particles (CPPs) that aggregate the excessive calcium and phosphate ions. If not counteracted, the initially formed harmless amorphous spherical complexes (primary CPPs) may mature into damaging crystalline complexes (secondary CPPs). Secondary CPPs are internalized by vascular cells, causing a massive influx of calcium ions into the cytosol, leading to a proinflammatory response, cellular dysfunction, and cell death. Although the pathophysiological effects induced by CPPs in vascular cells receive increasing attention, a complete picture of how these particles contribute to the development of atherosclerosis and vascular calcification remains elusive. We here discuss existing knowledge on CPP formation and function in atherosclerosis and vascular calcification, techniques for investigating CPPs, and models currently applied to assess CPP-induced cardiovascular pathogenesis. Lastly, we evaluate the potential diagnostic value of serum CPP measurements and the therapeutic potential of anti-CPP therapies currently under development.

The purpose of the present study is to determine the prevalence of genital mycoplasmas (M. genitalium, M. hominis, U. parvum, U. urealyticum) in pregnant women by molecular biological methods. Material/Methods: A prospective epidemiological study of 107 pregnant women hospitalized in the Clinic of Obstetrics and Gynecology, University Hospital-Pleven, Bulgaria, was conducted. Vaginal secretion samples were taken from all 107 pregnant women. A Polymerase chain reaction (PCR) assay was used to detect the genomic DNA of the bacteria in pregnant women. Results: The highest is the relative share of women in the age group from 20 to 35 years - 66 (64.68%), followed by women under 20 years - 27 (25.23%) and women over 35 years - 14 (13.08%). Detection of bacterial DNA was found in 85 (79.44%) of the cases, with present Ureaplasma spp. Colonization in 42 women (39.25%). Although no statistical dependence was found on open bacteria and age groups (p-value = 0.4688), it is noteworthy that the prevalence of Mycoplasma spp. and Ureaplasma spp. as a whole in the age group from 20 to 35 years, which has the highest birth rate, is more than twice higher than the group of up to 20 years and more than five times higher compared to the group over 35 years. Conclusions: Studies on the incidence of Mycoplasma spp. and Ureaplasma spp. in pregnant women is important for controlling the pregnancy, predicting the risk of developing maternal-fetal infection and discussing the options for timely treatment.

Background . Atherosclerosis is frequently accompanied by an extensive calcification, yet the mechanisms behind its initiation and progression remain obscure. In particular, there is unclear whether large mineral deposits grow concentrically or by merging of microcalcifications. Aim of the study was to investigate calcium phosphate maturation during atherosclerotic calcification employing an elemental analysis approach. Material and methods . We collected 20 calcified atherosclerotic plaques excised during carotid endarterectomy. After being fixed in formalin and postfixed in osmium tetroxide, plaques were dehydrated and stained in uranyl acetate with the subsequent embedding into epoxy resin, grinding, polishing, and lead citrate counterstaining. Upon the sputter coating with carbon, we visualised the plaque microanatomy by means of backscattered scanning electron microscopy. Elemental analysis was carried out using energy-dispersive X-ray spectroscopy in the backscattered mode at high vacuum and 20 kV voltage. The analysis was performed at 11 radial points within the calcium deposit and 4 control points at ascending distance from the deposit. Results . Calcium to phosphate ratio differed between the calcium deposits in distinct plaques and also within the same plaque. We found a statistically significant correlation between calcium to phosphate ratio in the center and periphery of the calcium deposit. Areas with distinct electron density had different calcium to phosphate ratio; however, there was no clear correlation between these parameters. Conclusion . Correlation of calcium to phosphate ratio in the center and periphery of the calcium deposit suggests that its maturation develops from the center to the periphery rather than by merging of neighboring calcium deposits.

Azole resistance is a concern for the management of diseases caused by Aspergillus fumigatus in humans. Azole fungicide use in the environment has been identified as a possible cause for development of resistance, which increases the complexity and number of stakeholders involved in this emerging problem. A workshop was held in Amsterdam early 2019 in which stakeholders, including medical and agricultural researchers, representatives from the government, public health, fungicide producers and end-users, reviewed the current evidence supporting environmental selection for resistance and to discuss which research and measures are needed to retain the effectiveness of the azole class for environmental and medical applications. This paper provides an overview of the latest insights and understanding of azole resistance development in the clinical setting and the wider environment. A One Health problem approach was undertaken to list and prioritize which research will be needed to provide missing evidence and to enable preventive interventions.

An association between high serum calcium/phosphate and cardiovascular events or death is well-established. However, a mechanistic explanation of this correlation is lacking. Here, we examined the role of calciprotein particles (CPPs), nanoscale bodies forming in the human blood upon its supersaturation with calcium and phosphate, in cardiovascular disease. The serum of patients with coronary artery disease or cerebrovascular disease displayed an increased propensity to form CPPs in combination with elevated ionised calcium as well as reduced albumin levels, altogether indicative of reduced Ca2+-binding capacity. Intravenous administration of CPPs to normolipidemic and normotensive Wistar rats provoked intimal hyperplasia and adventitial/perivascular inflammation in both balloon-injured and intact aortas in the absence of other cardiovascular risk factors. Upon the addition to primary human arterial endothelial cells, CPPs induced lysosome-dependent cell death, promoted the release of pro-inflammatory cytokines, stimulated leukocyte adhesion, and triggered endothelial-to-mesenchymal transition. We concluded that CPPs, which are formed in the blood as a result of altered mineral homeostasis, cause endothelial dysfunction and vascular inflammation, thereby contributing to the development of cardiovascular disease.

We analyzed literature data and our results of treatment of 37 patients with cancer of the middle and lower rectal ampulla (Т2-4аN0-2М0) who underwent low anterior resection of the rectum with colorectal anastomosis. The purpose of the study was to reveal characteristics of late colorectal anastomotic leakage. The results demonstrated differences in the clinical course of early and late colorectal anastomotic leakage, the site of anastomotic defect, anastomosis characteristics and the need for repeat surgery. There were some pathogenetic aspects distinguishing early and late anastomotic leakage, such as technical problems in early leakage and association with preoperative radiotherapy in late anastomotic leakage. Conclusions: timely diagnostics of microleakage of colorectal anastomosis will allow changing the further tactics of the patient management and avoiding the manifestation of late anastomotic leakage.

BACKGROUND: The aim of this study was pure tone audiometry (PTA) evaluation in normal individuals exposed to hypobaric hypoxia, taking into account the influence of air rarefaction on sound transmission via a standard earphone.METHODS: The study was conducted in a hypobaric chamber using a standard audiometer and a TDH-39P earphone whose performance at altitudes was analyzed in a previous research. Eight male volunteers underwent PTA testing at ground level and at 15,000 ft under normoxia (via an oxygen mask) and after 20 min of hypoxia. Auditory threshold at 500, 1000, 2000, and 4000 Hz was recorded from the right ear while monitoring arterial oxygen saturation (Sao2). The PTA data obtained at high altitude were corrected according to a specific recalibration table.RESULTS: During hypoxia, a significant threshold shift was observed only at 4000 Hz, with respect to ground level recording, for the sole not-corrected data. At the same frequency a significant threshold shift was also observed between the ground level recording and normoxia at 15,000 ft, confirming the presence of a hypobaric effect not related to hypoxia. After the recalibration procedure, this hearing impairment was not significant. No correlation with Sao2 levels was observed.DISCUSSION: The mild and not significant presence of high altitude-induced PTA derangements in healthy normal individuals was documented, although a stimulus recalibration was needed for a correct interpretation of our data.Lucertini M, Lancia S, Sanjust F, Guadagno AG, Lucertini L, Sisto R. Lack of significant audiometric changes under hypobaric hypoxia at 15,000 ft. Aerosp Med Hum Perform. 2020; 91(1):32–36.

Frailty is increasingly recognized as a better predictor of adverse postoperative events than chronological age. The objective of this review was to systematically evaluate the effect of frailty on postoperative morbidity and mortality. Studies were included if patients underwent non-cardiac surgery and if frailty was measured by a validated instrument using physical, cognitive and functional domains. A systematic search was performed using EMBASE, MEDLINE, Web of Science, CENTRAL and PubMed from 1990 - 2017. Methodological quality was assessed using an assessment tool for prognosis studies. Outcomes were 30-day mortality and complications, one-year mortality, postoperative delirium and discharge location. Meta-analyses using random effect models were performed and presented as pooled risk ratios with confidence intervals and prediction intervals. We included 56 studies involving 1.106.653 patients. Eleven frailty assessment tools were used. Frailty increases risk of 30-day mortality (31 studies, 673.387 patients, risk ratio 3.71 [95% CI 2.89-4.77] (PI 1.38-9.97; I2=95%) and 30-day complications (37 studies, 627.991 patients, RR 2.39 [95% CI 2.02-2.83). Risk of 1-year mortality was threefold higher (six studies, 341.769 patients, RR 3.40 [95% CI 2.42-4.77]). Four studies (N=438) reported on postoperative delirium. Meta-analysis showed a significant increased risk (RR 2.13 [95% CI 1.23-3.67). Finally, frail patients had a higher risk of institutionalization (10 studies, RR 2.30 [95% CI 1.81- 2.92]). Frailty is strongly associated with risk of postoperative complications, delirium, institutionalization and mortality. Preoperative assessment of frailty can be used as a tool for patients and doctors to decide who benefits from surgery and who doesn’t.

Extrachromosomal circularization of DNA is an important genomic feature in cancer. The structure, composition and genome-wide frequency of extrachromosomal circular DNA, however, have not yet been extensively profiled. Here, we combined genomic and transcriptomic approaches to describe the landscape of extrachromosomal circular DNA in neuroblastoma, a tumor arising in childhood from primitive cells of the sympathetic nervous system. Our analysis identifies and characterizes a wide catalog of somatically acquired and undescribed extrachromosomal circular DNAs. Moreover, we find that extrachromosomal circular DNAs are an unanticipated major source of somatic rearrangements, contributing to oncogenic remodeling through chimeric circularization and reintegration of circular DNA into the linear genome. Cancer-causing lesions can emerge out of circle-derived rearrangements and are associated with adverse clinical outcome. It is highly probable that circle-derived rearrangements represent an ongoing mutagenic process. Thus, extrachromosomal circular DNAs represent a multi-hit mutagenic process, with important functional and clinical implications for the origins of genomic remodeling in cancer.

Calcific aortic valve disease (CAVD), previously thought to represent a passive degeneration of the valvular extracellular matrix (VECM), is now regarded as an intricate multistage disorder with sequential yet intertangled and interacting underlying processes. Endothelial dysfunction and injury, initiated by disturbed blood flow and metabolic disorders, lead to the deposition of low-density lipoprotein cholesterol in the VECM further provoking macrophage infiltration, oxidative stress, and release of pro-inflammatory cytokines. Such changes in the valvular homeostasis induce differentiation of normally quiescent valvular interstitial cells (VICs) into synthetically active myofibroblasts producing excessive quantities of the VECM and proteins responsible for its remodeling. As a result of constantly ongoing degradation and re-deposition, VECM becomes disorganised and rigid, additionally potentiating myofibroblastic differentiation of VICs and worsening adaptation of the valve to the blood flow. Moreover, disrupted and excessively vascularised VECM is susceptible to the dystrophic calcification caused by calcium and phosphate precipitating on damaged collagen fibers and concurrently accompanied by osteogenic differentiation of VICs. Being combined, passive calcification and biomineralisation synergistically induce ossification of the aortic valve ultimately resulting in its mechanical incompetence requiring surgical replacement. Unfortunately, multiple attempts have failed to find an efficient conservative treatment of CAVD; however, therapeutic regimens and clinical settings have also been far from the optimal. In this review, we focused on interactions and transitions between aforementioned mechanisms demarcating ascending stages of CAVD, suggesting a predisposing condition (bicuspid aortic valve) and drug combination (lipid-lowering drugs combined with angiotensin II antagonists and cytokine inhibitors) for the further testing in both preclinical and clinical trials.

Tissue-engineered vascular graft for the reconstruction of small arteries is still an unmet clinical need, despite the fact that a number of promising prototypes have entered preclinical development. Here we test Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)Poly(ε-caprolactone) 4-mm-diameter vascular grafts equipped with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and stromal cell-derived factor 1α (SDF-1α) and surface coated with heparin and iloprost (PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo, n = 8) in a sheep carotid artery interposition model, using biostable vascular prostheses of expanded poly(tetrafluoroethylene) (ePTFE, n = 5) as a control. Primary patency of PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo grafts was 62.5% (5/8) at 24 h postimplantation and 50% (4/8) at 18 months postimplantation, while all (5/5) ePTFE conduits were occluded within the 24 h after the surgery. At 18 months postimplantation, PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo grafts were completely resorbed and replaced by the vascular tissue. Regenerated arteries displayed a hierarchical three-layer structure similar to the native blood vessels, being fully endothelialised, highly vascularised and populated by vascular smooth muscle cells and macrophages. The most (4/5, 80%) of the regenerated arteries were free of calcifications but suffered from the aneurysmatic dilation. Therefore, biodegradable PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo grafts showed better short- and long-term results than bio-stable ePTFE analogues, although these scaffolds must be reinforced for the efficient prevention of aneurysms.

Genetic causes of the global decline in male fertility are among the hot spots of scientific research in reproductive genetics. The most common way to evaluate male fertility in clinical trials is to determine semen quality. Lower semen quality is very often accompanied by subfertility or infertility, occurs in many diseases and can be caused by many factors, including genetic ones. The following forms of lowered semen quality (pathozoospermia) are known: azoospermia, oligozoospermia, asthenozoospermia, teratozoospermia, and some combined forms. To systematize information about the genetic basis of impaired spermatogenesis, we created a catalog of human genes associated with lowered semen quality (HGAPat) and analyzed their functional characteristics. The catalog comprises data on 126 human genes. Each entry of the catalog describes an association between an allelic variant of the gene and a particular form of lowered semen quality, extracted from the experimental study. Most genes included into the catalog are located on autosomes and are associated with such pathologies as non-obstructive azoospermia, oligozoospermia or asthenozoospermia. Slightly less than half of the included genes (43%) are expressed in the testes in a tissue-specific manner. Functional annotation of genes from the catalog showed that spermatogenic failure can be associated with mutations in genes that control biological processes essential for spermiogenesis (regulating DNA metabolism, cell division, formation of cellular structures, which provide cell movement) as well as with mutations in genes that control cellular responses to unfavorable conditions (stress factors, including oxidative stress and exposure to toxins).

For many years, the risk-based therapy stratification of children with neuroblastoma has relied on clinical and molecular covariates. In recent years, genome analysis has revealed further alterations defining risk, tumor biology, and therapeutic targets. The implementation of a robust and scalable method for analyzing traditional and new molecular markers in routine diagnostics is an urgent clinical need. Here, we investigated targeted panel sequencing as a diagnostic approach to analyze all relevant genomic neuroblastoma risk markers in one assay. Our “neuroblastoma hybrid capture sequencing panel” (NB-HCSP) assay employs a technology for the high-coverage sequencing (&gt, 1000×) of 55 selected genes and neuroblastoma-relevant genomic regions, which allows for the detection of single nucleotide changes, structural rearrangements, and copy number alterations. We validated our assay by analyzing 15 neuroblastoma cell lines and a cohort of 20 neuroblastomas, for which reference routine diagnostic data and genome sequencing data were available. We observed a high concordance for risk markers identified by the NB-HSCP assay, clinical routine diagnostics, and genome sequencing. Subsequently, we demonstrated clinical applicability of the NB-HCSP assay by analyzing routine clinical samples. We conclude that the NB-HCSP assay may be implemented into routine diagnostics as a single assay that covers all essential covariates for initial neuroblastoma classification, extended risk stratification, and targeted therapy selection.

Currently, transcatheter aortic valve implantation (TAVI) represents the most efficient treatment option for patients with aortic stenosis, yet its clinical outcomes largely depend on the accuracy of valve positioning that is frequently complicated when routine imaging modalities are applied. Therefore, existing limitations of perioperative imaging underscore the need for the development of novel visual assistance systems enabling accurate procedures. In this paper, we propose an original multi-task learning-based algorithm for tracking the location of anatomical landmarks and labeling critical keypoints on both aortic valve and delivery system during TAVI. In order to optimize the speed and precision of labeling, we designed nine neural networks and then tested them to predict 11 keypoints of interest. These models were based on a variety of neural network architectures, namely MobileNet V2, ResNet V2, Inception V3, Inception ResNet V2 and EfficientNet B5. During training and validation, ResNet V2 and MobileNet V2 architectures showed the best prediction accuracy/time ratio, predicting keypoint labels and coordinates with 97/96% accuracy and 4.7/5.6% mean absolute error, respectively. Our study provides evidence that neural networks with these architectures are capable to perform real-time predictions of aortic valve and delivery system location, thereby contributing to the proper valve positioning during TAVI.

The expression of DNA repair (DDB1, ERCC4, ERCC5), leukocyte adhesion (VCAM1, ICAM1, SELE, SELP), endothelial mechanotransduction (KLF4), endothelial differentiation (PECAM1, CDH5, CD34, NOS3), endothelial-to-mesenchymal transition (SNAI1, SNAI2, TWIST1, GATA4, ZEB1, CDH2), scavenger receptors (LOX1, SCARF1, CD36, LDLR, VLDR), antioxidant system (PXDN, CAT, SOD1) and transcription factor (HEY2) genes in primary human coronary (HCAEC) and internal thoracic (HITAEC) arteries endothelial cells exposed to alkylating mutagen mitomycin C (MMC) was studied at two time points - after 6 h of incubation with MMC and after 6 h of the genotoxic load followed by 24 h of incubation in pure culture medium using the quantitative PCR. Immediately after MMC exposure, in the exposed HCAEC and HITAEC a decreased expression of almost all studied genes was noted excepted SNAI, which demonstrated a 4-told increase in its expression compared to the unexposed control. Elimination of MMC from the cultures, an increased expression of the VCAM1, ICAM1, SELE, SNAI2, KLF4 genes and a decreased the mRNA level of the PECAM1, CDH5, CD34, ZEB1, CAT, PXDN genes were observed in both cell lines. In addition, HITAEC cells were characterized by a decreased expression of the SOD1, SCARF1, CD36 genes and an increased expression of the SNAI1 and TWIST1 genes; in HCAEC, an increased mRNA level of the LDLR and VLDLR genes was noted. Thus, MMC-induced genotoxic stress is associated with the endothelial dysfunction.Provedena otsenka ékspressii genov reparatsii DNK (DDB1, ERCC4, ERCC5), adgezii leĭkotsitov (VCAM1, ICAM1, SELE, SELP), éndotelial'noĭ mekhanotransduktsii (KLF4), éndotelial'noĭ differentsirovki (PECAM1, CDH5, CD34, NOS3), éndotelial'no-mezenkhimal'nogo perekhoda (SNAI1, SNAI2, TWIST1, GATA4, ZEB1, CDH2), skavendzher-retseptorov (LOX1, SCARF1, CD36, LDLR, VLDR), antioksidantnoĭ zashchity (PXDN, CAT, SOD1) i transkriptsionnogo faktora HEY2 v kul'turakh pervichnykh éndotelial'nykh kletok koronarnoĭ (HCAEC) i vnutrenneĭ grudnoĭ (HITAEC) arteriĭ cheloveka, éksponirovannykh alkiliruiushchim mutagenom mitomitsinom S (MMS). Uroven' ékspressii opredeliali s pomoshch'iu metoda kolichestvennoĭ PTsR v dvukh vremennykh tochkakh: cherez 6 ch kul'tivirovaniia kletok v prisutstvii MMS i cherez 6 ch ékspozitsii mutagenom s posleduiushchimi 24 ch kul'tivirovaniia v chistoĭ kul'tural'noĭ srede. Neposredstvenno posle vozdeĭstviia mutagena v éksponirovannykh kul'turakh HCAEC i HITAEC bylo otmecheno snizhenie ékspressii prakticheski vsekh izuchennykh genov, za iskliucheniem SNAI2 (ego ékspressiia byla povyshena v 4 raza) po sravneniiu s neéksponirovannym kontrolem. Posle éliminirovaniia iz kul'tur MMS v obeikh kletochnykh liniiakh bylo otmecheno uvelichenie ékspressii genov VCAM1, ICAM1, SELE, SNAI2, KLF4 i snizhenie ékspressii genov PECAM1, CDH5, CD34, ZEB1, CAT, PXDN. Krome togo, kletki HITAEC takzhe kharakterizovalis' snizheniem ékspressii genov SOD1, SCARF1, CD36 i povysheniem ékspressii genov SNAI1 i TWIST1; v kletkakh HCAEC bylo otmecheno uvelichenie ékspressii genov LDLR i VLDLR. Takim obrazom, genotoksicheskiĭ stress, vyzvannyĭ mutagenom alkiliruiushchego mekhanizma deĭstviia, assotsiirovan s razvitiem éndotelial'noĭ disfunktsii, chto proiavliaetsia v izmenenii profilia gennoĭ ékspressii éndotelial'nykh kletok, kul'tiviruemykh v usloviiakh genotoksicheskoĭ nagruzki.

Simple Summary Efficient trafficking and survival of CAR T cells within the hostile tumor microenvironment are important prerequisites for potent solid tumor attack that have not yet been achieved. We deployed monospecific murine instead of polyclonal human T cells for CAR T cell generation to evaluate second generation L1CAM- and HER2-specific CARs with different spacer length and either the CD28 or 4-1BB co-stimulatory domain in mouse models of neuroblastoma and ovarian carcinoma. This mouse-in-mouse approach ensured CAR T cell trafficking unhindered by species-specific discrepancies and demonstrated superior solid tumor attack by CAR T cells harboring the CD28 compared to 4-1BB co-stimulatory domain. Our approach has the potential to improve prediction and selection of promising clinical CAR candidates against solid tumors in the future. Abstract Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.

Amplification of MYCN is the driving oncogenic change in a subset of high-risk neuroblastomas. The MYCN protein and the Aurora-A kinase form a complex during the S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in the S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription–replication conflicts and activates ataxia telangiectasia and Rad3-related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR kinases induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription–replication conflicts is an effective therapy for MYCN-driven neuroblastoma. Eilers and colleagues report that Aurora-A suppresses transcription–replication conflicts in MYCN-driven neuroblastoma, a vulnerability that can be targeted with a combination of Aurora-A and ATR kinase inhibitors.

Tissue-engineered vascular grafts are widely tested as a promising substitute for both arterial bypass and replacement surgery. We previously demonstrated that incorporation of VEGF into electrospun tubular scaffolds from poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(ε-caprolactone) enhances formation of an endothelial cell monolayer. However, an overdose of VEGF can induce tumor-like vasculature, thereby, other bioactive factors are needed to support VEGF-driven endothelialization and successful recruitment of smooth muscle cells. Utilizing emulsion electrospinning, we fabricated one-layer vascular grafts with either VEGF, bFGF, or SDF-1α, and two-layer vascular grafts with VEGF incorporated into the inner layer and bFGF and SDF-1α incorporated into the outer layer with the following structural evaluation, tensile testing, and in vivo testing using a rat abdominal aorta replacement model. The latter graft prototype showed higher primary patency rate. We found that the two-layer structure improved surface topography and mechanical properties of the grafts. Further, the combination of bFGF, SDF-1α, and VEGF improved endothelialization compared with VEGF alone, while bFGF induced a rapid formation of a smooth muscle cell layer. Taken together, these findings show that the two-layer structure and incorporation of bFGF and SDF-1α into the vascular grafts in combination with VEGF provide a higher primary patency and therefore improved in vivo performance.

Introduction: Clinical benefits of laparoscopic surgery are well established, but evidence for financial benefits is limited. This study aimed to compare the financial impact of the introduction of laparoscopic colorectal surgery. Methods: This study included patients who underwent colorectal surgery between January 2010 and 2015. We collected a range of financial data and divided the patients into 2 groups. Primary outcome was total cost defined by surgical-related costs. Results: A total of 1,246 patients were included, of which 440 surgeries were performed laparoscopically. The total median cost of laparoscopy was higher compared to open surgery (EUR 4,665 vs. EUR 4,268, p = 0.001). Laparoscopy was associated with higher equipment costs (EUR 857 vs. EUR 232, p < 0.001), longer operating time (3.2 vs. 2.5 hours, p < 0.001), and more readmissions (10.9 vs. 8.5%, p < 0.001). However, after adjusting for heterogeneity, no difference was found in total cost. Surgical-related costs were counterbalanced by lower costs associated with shorter median hospital stay (6 vs. 9 days, p < 0.001), less morbidity (37.3 vs. 55.1%, p < 0.001), and less mortality (1.8 vs. 5.6%, p = 0.013) for laparoscopy. Conclusion: During the introduction of laparoscopy for colorectal surgery, no significant differences were found in total cost between laparoscopic and open colorectal surgery. However, favorable postoperative outcomes were achieved with laparoscopic surgery.

Summary This study aimed to determine the bacterial profile of vaginal samples from pregnant women and the prevalence of identified microorganisms. Materials and methods: A prospective epidemiological study was conducted on 150 pregnant women admitted to the Clinic of Obstetrics and Gynecology at Dr. G. Stranski University Hospital in Pleven. Vaginal secretion samples were collected from all the 150 women. Direct microscopy of native smears prepared by the wet mount method to detect Trichomonas vaginalis and direct microscopy of Gram-stained smears was performed. Aerobic culture studies were conducted to identify pathogenic bacteria and Sabouraud glucose agar (SGA) to detect the presence of Candida spp. Samples were also collected from 107 of the women for PCR tests for mycoplasma detection. Results: The highest relative share in the study was women aged 21 to 35 years 104 (69.33%). Of all the hospitalized and examined pregnant women, 87 (57.99%) had medical problems during the current pregnancy and postpartum complications, and 63 women (42%) gave birth without accompanying complications. In 86 (57.32%) of the women, bacteria were detected by direct microscopy of Gram-stained smears. Of these, 24 (16%) had gram-negative rods, 28 (18.66%) had gram-positive cocci. In 34 (22.66%), we found gram-negative and gram-variable pleomorphic bacteria. The aerobic culture study revealed microbiological growth in 44 (29.33%) of the women investigated. Conclusions: Studies on the profile of the vaginal microbiota in pregnant women and the spread of pathogenic microorganisms are essential for make prognoses for pregnancy outcome, evaluating the risk of developing a maternal-fetal infection, and discussing options for timely treatment.

Pathognomonic PAX3-FOXO1 fusion oncogene expression is associated with poor outcome in rhabdomyosarcoma. Combining genome-wide CRISPR screening with cell- based functional genetic approaches, we here provide evidence that PAX3-FOXO1 induces replication stress, resulting in a synthetic lethal dependency to ATR-mediated DNA damage-response signaling in rhabdomyosarcoma. Expression of PAX3-FOXO1 in muscle progenitor cells was not only sufficient to induce hypersensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibited increased sensitivity to structurally diverse inhibitors of ATR, a dependency that could be validated genetically. Mechanistically, ATR inhibition led to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increased sensitivity of rhabdomyosarcoma cells to PARP inhibitionin vitro, and combined ATR and PARP inhibition induced regression of primary patient-derived alveolar rhabdomyosarcoma xenograftsin vivo. Moreover, a genome-wide CRISPR activation screen (CRISPRa) identifiedFOSgene family members as inducers of resistance against ATR inhibitors. Mechanistically,FOSgene family members reduced replication stress in rhabdomyosarcoma cells. Lastly, compassionate use of ATR inhibitors in two pediatric patients suffering from relapsed PAX3-FOXO1-expressing alveolar rhabdomyosarcoma showed signs of tolerability, paving the way to clinically exploit this novel synthetic lethal dependency in rhabdomyosarcoma.

Infectious complications are the major cause of morbidity and mortality after solid organ and stem cell transplantation. To better understand host and environmental factors associated with an increased risk of infection as well as the effect of infections on function and survival of transplanted organs, we established the DZIF Transplant Cohort, a multicentre prospective cohort study within the organizational structure of the German Center for Infection Research. At time of transplantation, heart-, kidney-, lung-, liver-, pancreas- and hematopoetic stem cell- transplanted patients are enrolled into the study. Follow-up visits are scheduled at 3, 6, 9, 12 months after transplantation, and annually thereafter; extracurricular visits are conducted in case of infectious complications. Comprehensive standard operating procedures, web-based data collection and monitoring tools as well as a state of the art biobanking concept for blood, purified PBMCs, urine, and faeces samples ensure high quality of data and biosample collection. By collecting detailed information on immunosuppressive medication, infectious complications, type of infectious agent and therapy, as well as by providing corresponding biosamples, the cohort will establish the foundation for a broad spectrum of studies in the field of infectious diseases and transplant medicine. By January 2020, baseline data and biosamples of about 1400 patients have been collected. We plan to recruit 3500 patients by 2023, and continue follow-up visits and the documentation of infectious events at least until 2025. Information about the DZIF Transplant Cohort is available at https://www.dzif.de/en/working-group/transplant-cohort.

Ewing sarcoma (EwS) is an aggressive childhood cancer likely originating from mesenchymal stem cells or osteo-chondrogenic progenitors. It is characterized by fusion oncoproteins involving EWSR1 and variable members of the ETS-family of transcription factors (in 85% FLI1). EWSR1-FLI1 can induce target genes by using GGAA-microsatellites as enhancers.Here, we show that EWSR1-FLI1 hijacks the developmental transcription factor SOX6 - a physiological driver of proliferation of osteo-chondrogenic progenitors - by binding to an intronic GGAA-microsatellite, which promotes EwS growth in vitro and in vivo. Through integration of transcriptome-profiling, published drug-screening data, and functional in vitro and in vivo experiments including 3D and PDX models, we discover that constitutively high SOX6 expression promotes elevated levels of oxidative stress that create a therapeutic vulnerability toward the oxidative stress-inducing drug Elesclomol.Collectively, our results exemplify how aberrant activation of a developmental transcription factor by a dominant oncogene can promote malignancy, but provide opportunities for targeted therapy.

The implantation of bioprosthetic heart valves (BHVs) is increasingly becoming the treatment of choice in patients requiring heart valve replacement surgery. Unlike mechanical heart valves, BHVs are less thrombogenic and exhibit superior hemodynamic properties. However, BHVs are prone to structural valve degeneration (SVD), an unavoidable condition limiting graft durability. Mechanisms underlying SVD are incompletely understood, and early concepts suggesting the purely degenerative nature of this process are now considered oversimplified. Recent studies implicate the host immune response as a major modality of SVD pathogenesis, manifested by a combination of processes phenocopying the long‐term transplant rejection, atherosclerosis, and calcification of native aortic valves. In this review, we summarize and critically analyze relevant studies on (1) SVD triggers and pathogenesis, (2) current approaches to protect BHVs from calcification, (3) obtaining low immunogenic BHV tissue from genetically modified animals, and (4) potential strategies for SVD prevention in the clinical setting.

Atherosclerosis, calcific aortic valve disease (CAVD), and bioprosthetic heart valve degeneration (alternatively termed structural valve deterioration, SVD) represent three diseases affecting distinct components of the circulatory system and their substitutes, yet sharing multiple risk factors and commonly leading to the extraskeletal calcification. Whereas the histopathology of the mentioned disorders is well-described, their ultrastructural pathology is largely obscure due to the lack of appropriate investigation techniques. Employing an original method for sample preparation and the electron microscopy visualisation of calcified cardiovascular tissues, here we revisited the ultrastructural features of lipid retention, macrophage infiltration, intraplaque/intraleaflet haemorrhage, and calcification which are common or unique for the indicated types of cardiovascular disease. Atherosclerotic plaques were notable for the massive accumulation of lipids in the extracellular matrix (ECM), abundant macrophage content, and pronounced neovascularisation associated with blood leakage and calcium deposition. In contrast, CAVD and SVD generally did not require vasculo- or angiogenesis to occur, instead relying on fatigue-induced ECM degradation and the concurrent migration of immune cells. Unlike native tissues, bioprosthetic heart valves contained numerous specialised macrophages and were not capable of the regeneration that underscores ECM integrity as a pivotal factor for SVD prevention. While atherosclerosis, CAVD, and SVD show similar pathogenesis patterns, these disorders demonstrate considerable ultrastructural differences.

OBJECTIVES: To describe the predictive utility of the D-dimer assay among patients with the coronavirus disease 2019 syndrome for unprovoked lower extremity deep venous thrombosis. DESIGN: Prospective observational study with retrospective data analysis. SETTING: Academic medical center surgical ICU. PATIENTS: Seventy-two intubated patients with critical illness from coronavirus disease 2019. INTERVENTIONS: Therapeutic anticoagulation after imaging diagnosis of the first three deep venous thrombosis cases was confirmed; therapeutic anticoagulation as prophylaxis thereafter to all subsequent ICU admissions. MEASUREMENTS AND MAIN RESULTS: Seventy-two patients with severe coronavirus disease 2019 were screened for deep venous thrombosis after ICU admission with 102 duplex ultrasound examinations, with 12 cases (16.7%) of lower extremity deep venous thrombosis identified. There were no differences between groups with respect to age, renal function, or biomarkers except for D-dimer (median, 12,858 ng/mL [interquartile range, 3,176-30,770 ng/mL] for lower extremity deep venous thrombosis vs 2,087 ng/mL [interquartile range, 638-3,735 ng/mL] for no evidence of deep venous thrombosis; p < 0.0001). Clinical screening tools (Wells score and Dutch Primary Care Rule) had no utility. The C-statistic for D-dimer concentration was 0.874 ± 0.065. At the model-predicted cutoff value of 3,000 ng/mL, sensitivity was 100%, specificity was 51.1%, positive predictive value was 21.8%, and negative predictive value was 100%. CONCLUSIONS: Lower extremity deep venous thrombosis is prevalent in coronavirus disease 2019 disease and can be present on ICU admission. Screening has been recommended in the context of the pro-inflammatory, hypercoagulable background milieu. D-dimer concentrations are elevated in nearly all coronavirus disease 2019 patients, and the test appears reliable for screening for lower extremity deep venous thrombosis at or above a concentration of 3,000 ng/mL (more than 13-fold above the normal range). Full anticoagulation is indicated if the diagnosis is confirmed, and therapeutic anticoagulation should be considered for prophylaxis, as all coronavirus disease 2019 patients are at increased risk.

In late December 2019, a cluster of cases of pneumonia of unknown etiology were reported linked to a market in Wuhan, China1. The causative agent was identified as the speciesSevere acute respiratory syndrome-related coronavirusand was named SARS-CoV-2 (ref. 2). By 16 April the virus had spread to 185 different countries, infected over 2,000,000 people and resulted in over 130,000 deaths3. In the Netherlands, the first case of SARS-CoV-2 was notified on 27 February. The outbreak started with several different introductory events from Italy, Austria, Germany and France followed by local amplification in, and later also outside, the south of the Netherlands. The combination of near to real-time whole-genome sequence analysis and epidemiology resulted in reliable assessments of the extent of SARS-CoV-2 transmission in the community, facilitating early decision-making to control local transmission of SARS-CoV-2 in the Netherlands. We demonstrate how these data were generated and analyzed, and how SARS-CoV-2 whole-genome sequencing, in combination with epidemiological data, was used to inform public health decision-making in the Netherlands. Metastava project.

To identify predictors of progression of precerebral atherosclerosis in long-term period after coronary artery bypass surgery.There were 97 procedures of carotid endarterectomy in patients after previous coronary artery bypass grafting for the period from 2006 to 2017. Inclusion criteria were previous CABG, no significant (over 60%) stenosis of internal carotid arteries at discharge after CABG. The control group included 447 patients without progression of precerebral atherosclerosis in long-term period after CABG.Careful monitoring of progression of precerebral atherosclerosis and therapeutic prevention of ischemic stroke are required in patients with mild-to-moderate ICA stenosis after CABG. The most significant predictors of progression of precerebral atherosclerosis after CABG were AF (OR=1.97, 95% CI 1.04-3.73), previous occlusion of stent (OR=7.89, 95% CI=2.3-27.0), chronic brain ischemia grade II or III (OR=22.45, 95% CI=11.9-42.3), chronic kidney disease (OR=15.8, 95% CI=5.04-49.5).It was revealed that the majority of predictors of adverse ischemic cerebral and myocardial events are indirectly associated with atrial fibrillation.Выявить предикторы прогрессирования прецеребрального атеросклероза (ПЦАС) у пациентов в отдаленном периоде после коронарного шунтирования (КШ).В 2006—2017 гг. выполнено 97 каротидных эндартерэктомий пациентам с КШ в анамнезе. Критерии включения в исследование: КШ в анамнезе и отсутствие гемодинамически и клинически значимых (свыше 60%) стенозов внутренних сонных артерий на момент выписки после КШ. В группу контроля вошло 447 пациентов без прогрессирования ПЦАС в отдаленном периоде после КШ.Пациентам, перенесшим КШ и исходно имеющим незначимые стенотические поражения внутренней сонной артерии, необходим особо тщательный мониторинг прогрессирования ПЦАС, а также назначение оптимальной медикаментозной терапии, направленной на профилактику ишемического инсульта. Наиболее высокий риск прогрессирования ПЦАС после операции КШ у пациентов с фибрилляцией предсердий (ОШ 1,97; 95% ДИ 1,04—3,73), наличием окклюзии стента в анамнезе (ОШ 7,89; 95% ДИ 2,3—27,0), хронической ишемией головного мозга II или III степени (ОШ 22,45; 95% ДИ 11,9—42,3), хронической почечной недостаточностью (ОШ 15,8; 95% ДИ 5,04—49,5).Выявлено, что большинство предикторов, вызывающих неблагоприятные ишемические события в головном мозге и миокарде, действуют опосредованно — через развитие фибрилляции предсердий. Доказано, что хроническая обструктивная болезнь легких является протективным фактором в отношении прогрессирования ПЦАС (ОШ 0,2; 95% ДИ 0,04—0,96) и мощным предиктором летального исхода в отдаленном периоде наблюдения после КШ.

The immunosuppressive microenvironment in solid tumors is thought to form a barrier to the entry and efficacy of cell-based therapies such as chimeric antigen receptor (CAR) T cells. Combining CAR T cell therapy with checkpoint inhibitors has been demonstrated to oppose immune escape mechanisms in solid tumors and augment antitumor efficacy. We evaluated PD-1/PD-L1 signaling capacity and the impact of an inhibitor of this checkpoint axis in an in vitro system for cancer cell challenge, the coculture of L1CAM-specific CAR T cells with neuroblastoma cell lines. Fluorescence-activated cell sorting-based analyses and luciferase reporter assays were used to assess PD-1/PD-L1 expression on CAR T and tumor cells as well as CAR T cell ability to kill neuroblastoma cells. Coculturing neuroblastoma cell lines with L1CAM-CAR T cells upregulated PD-L1 expression on neuroblastoma cells, confirming adaptive immune resistance. Exposure to neuroblastoma cells also upregulated the expression of the PD-1/PD-L1 axis in CAR T cells. The checkpoint inhibitor, nivolumab, enhanced L1CAM-CAR T cell-directed killing. However, nivolumab-enhanced L1CAM-CAR T cell killing did not strictly correlate with PD-L1 expression on neuroblastoma cells. In fact, checkpoint inhibitor success relied on strong PD-1/PD-L1 axis expression in the CAR T cells, which in turn depended on costimulatory domains within the CAR construct, and more importantly, on the subset of T cells selected for CAR T cell generation. Thus, T cell subset selection for CAR T cell generation and CAR T cell prescreening for PD-1/PD-L1 expression could help determine when combination therapy with checkpoint inhibitors could improve treatment efficacy.

BackgroundAdoptive cell therapy (ACT) with tumor-reactive T cells has shown consistent clinical efficacy. We evaluated the response to ACT in combination with interferon alpha (IFNa) preconditioning in patients with stage IV metastatic melanoma, most of which were progressive on cytotoxic T-lymphocyte-associated protein 4 and/or programmed cell death protein 1 checkpoint blockade therapy.MethodsThirty-four patients were treated with ex vivo expanded tumor reactive T cells, derived from mixed lymphocyte autologous tumor cultures, or with autologous tumor-infiltrating lymphocytes and evaluated for clinical response. Clinical and immunological parameters associated with response were also evaluated.ResultsBest overall response defined as clinical benefit, comprising either complete response, partial response or stable disease >6 months, was observed in 29% of the patients. Forty-three per cent of the 14 immunotherapy-naïve patients and 20% of the 20 patients progressive on prior immunotherapy benefited from ACT. The overall survival (OS) was 90% versus 28.6% at 1 year and 46.7% versus 0% at 3 years follow-up, of responder and non-responder patients, respectively. Median OS was 36 versus 7 months, respectively. IFNa pretreatment resulted in leukopenia, neutropenia and lymphopenia, which was sustained during the treatment in clinical responders and associated with response. Differences in antigen specificity, but not in phenotype, cytokine profile or CD8+ T cell number of the ACT products correlated with clinical response. Cross-reactivity of the ACT products to one or more allogeneic human leukocyte antigen-matched melanoma cell lines was associated with short OS after treatment while the ACT products of very long-term survivors showed no cross-reactivity but recognized patient-specific neoantigens.ConclusionThis study demonstrates that ACT in combination with a mild IFNa preconditioning regimen can induce clinical benefit even in immunotherapy pretreated patients, although with lower success than in immunotherapy-naïve patients. ACT products comprising neoantigen reactivity may be more effective.

Mitomycin C (MMC) is an alkylating chemotherapy drug that causes DNA crosslinking resulting in transcription arrest and apoptosis. DNA crosslinking is a critical damage to DNA that can be caused not only by MMC and other antitumor drugs, but also by various environmental and anthropogenic endo- and exogenous agents. Mammalian cells exposed to alkylating mutagens are characterized by severe genotoxic stress. Somatic mutations and genotoxic stress may lead to endothelial dysfunction, which is the initial stage of atherosclerosis, a leading cause of morbidity and mortality worldwide. Here we studied DNA damage, protein secretion and gene expression of IL6 and IL8 in primary human coronary artery endothelial cells (HCAEC) and human internal thoracic artery endothelial cells (HITAEC) in vitro exposed to 500 ng/mL MMC. We observed an increase in levels of cytogenetic damage (micronuclei, nucleoplasmic bridges and nuclear buds) in MMC-treated cells compared to control cells. After 6 h incubation with MMC, both HCAEC and HITAEC displayed a decrease in IL8 concentration and the mRNA level of IL6 and IL8 compared to control cells. Removal of MMC from cultures after 6 h followed by 24 h incubation of cells in complete growth media led to a sharp increase in secretion and gene expression of the studied cytokines in both HCAEC and HITAEC. Moreover, HCAEC were more susceptible to mutagenic exposure compared to HITAEC. These findings suggest that the MMC-induced genotoxic stress in endothelial cells derived from different arteries is associated with differential secretion and gene expression of proinflammatory cytokines IL6 and IL8.

Ewing sarcoma (EwS) is an aggressive childhood cancer likely originating from mesenchymal stem cells or osteo-chondrogenic progenitors. It is characterized by fusion oncoproteins involving EWSR1 and variable members of the ETS-family of transcription factors (in 85% FLI1). EWSR1-FLI1 can induce target genes by using GGAA-microsatellites as enhancers. Here, we show that EWSR1-FLI1 hijacks the developmental transcription factor SOX6 – a physiological driver of proliferation of osteo-chondrogenic progenitors – by binding to an intronic GGAA-microsatellite, which promotes EwS growth in vitro and in vivo. Through integration of transcriptome-profiling, published drug-screening data, and functional in vitro and in vivo experiments including 3D and PDX models, we discover that constitutively high SOX6 expression promotes elevated levels of oxidative stress that create a therapeutic vulnerability toward the oxidative stress-inducing drug Elesclomol. Collectively, our results exemplify how aberrant activation of a developmental transcription factor by a dominant oncogene can promote malignancy, but provide opportunities for targeted therapy., Ewing sarcoma is characterized by the fusion of EWSR1 and FLI1. Here, the authors show that EWSR1-FLI1 increases the activity of the developmental transcription factor SOX6, which promotes tumor growth but also increases sensitivity to oxidative stress.

BACKGROUND: Use of single-bed rooms for control of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is under debate; the added value when applying contact precautions has not been shown. We aimed to assess whether an isolation strategy of contact precautions in a multiple-bed room was non-inferior to a strategy of contact precautions in a single-bed room for preventing transmission of ESBL-producing Enterobacteriaceae.METHODS: We did a cluster-randomised, crossover, non-inferiority study on medical and surgical wards of 16 Dutch hospitals. During two consecutive study periods, either contact precautions in a single-bed room or contact precautions in a multiple-bed room were applied as the preferred isolation strategy for patients with ESBL-producing Enterobacteriaceae cultured from a routine clinical sample (index patients). Eligible index patients were aged 18 years or older, had no strict indication for barrier precautions in a single-bed room, had a culture result reported within 7 days of culture and before discharge, and had no wardmate known to be colonised or infected with an ESBL-producing Enterobacteriaceae isolate of the same bacterial species with a similar antibiogram. Hospitals were randomly assigned in a 1:1 ratio by computer to one of two sequences of isolation strategies, stratified by university or non-university hospital. Allocation was masked for laboratory technicians who assessed the outcomes but not for patients, treating doctors, and infection-control practitioners enrolling index patients. The primary outcome was transmission of ESBL-producing Enterobacteriaceae to wardmates, which was defined as rectal carriage of an ESBL-producing Enterobacteriaceae isolate that was clonally related to the index patient's isolate in at least one wardmate. The primary analysis was done in the per-protocol population, which included patients who were adherent to the assigned room type. A 10% non-inferiority margin for the risk difference was used to assess non-inferiority. This study is registered with Nederlands Trialregister, NTR2799.FINDINGS: 16 hospitals were randomised, eight to each sequence of isolation strategies. All hospitals randomised to the sequence single-bed room then multiple-bed room and five of eight hospitals randomised to the sequence multiple-bed room then single-bed room completed both study periods and were analysed. From April 24, 2011, to Feb 27, 2014, 1652 index patients and 12 875 wardmates were assessed for eligibility. Of those, 693 index patients and 9527 wardmates were enrolled and 463 index patients and 7093 wardmates were included in the per-protocol population. Transmission of ESBL-producing Enterobacteriaceae to at least one wardmate was identified for 11 (4%) of 275 index patients during the single-bed room strategy period and for 14 (7%) of 188 index patients during the multiple-bed room strategy period (crude risk difference 3·4%, 90% CI -0·3 to 7·1).INTERPRETATION: For patients with ESBL-producing Enterobacteriaceae cultured from a routine clinical sample, an isolation strategy of contact precautions in a multiple-bed room was non-inferior to a strategy of contact precautions in a single-bed room for preventing transmission of ESBL-producing Enterobacteriaceae. Non-inferiority of the multiple-bed room strategy might change the current single-bed room preference for isolation of patients with ESBL-producing Enterobacteriaceae and, thus, broaden infection-control options for ESBL-producing Enterobacteriaceae in daily clinical practice.FUNDING: Netherlands Organisation for Health Research and Development.

Improvement of risk scoring is particularly important for patients with preserved left ventricular ejection fraction (LVEF) who generally lack efficient monitoring of progressing heart failure. Here, we evaluated whether the combination of serum biomarkers and echocardiographic parameters may be useful to predict the remodeling-related outcomes in patients with ST-segment elevation myocardial infarction (STEMI) and preserved LVEF (HFpEF) as compared to those with reduced LVEF (HFrEF). Echocardiographic assessment and measurement of the serum levels of NT-proBNP, sST2, galectin-3, matrix metalloproteinases, and their inhibitors (MMP-1, MMP-2, MMP-3, TIMP-1) was performed at the time of admission (1st day) and on the 10th&ndash, 12th day upon STEMI onset. We found a reduction in NT-proBNP, sST2, galectin-3, and TIMP-1 in both patient categories from hospital admission to the discharge, as well as numerous correlations between the indicated biomarkers and echocardiographic parameters, testifying to the ongoing ventricular remodeling. In patients with HFpEF, NT-proBNP, sST2, galectin-3, and MMP-3 correlated with the parameters reflecting the diastolic dysfunction, while in patients with HFrEF, these markers were mainly associated with LVEF and left ventricular end-systolic volume/diameter. Therefore, the combination of the mentioned serum biomarkers and echocardiographic parameters might be useful for the prediction of adverse cardiac remodeling in patients with HFpEF.

Background Questionnaires to detect emotional and behavioral problems (EBP) in Preventive Child Healthcare (PCH) should be short which potentially affects validity and reliability. Simulation studies have shown that Computerized Adaptive Testing (CAT) could overcome these weaknesses. We studied the applicability (using the measures participation rate, satisfaction, and efficiency) and the validity of CAT in routine PCH practice. Methods We analyzed data on 461 children aged 10–11 years (response 41%), who were assessed during routine well-child examinations by PCH professionals. Before the visit, parents completed the CAT and the Child Behavior Checklist (CBCL). Satisfaction was measured by parent- and PCH professional-report. Efficiency of the CAT procedure was measured as number of items needed to assess whether a child has serious problems or not. Its validity was assessed using the CBCL as the criterion. Results Parents and PCH professionals rated the CAT on average as good. The procedure required at average 16 items to assess whether a child has serious problems or not. Agreement of scores on the CAT scales with corresponding CBCL scales was high (range of Spearman correlations 0.59–0.72). Area Under Curves (AUC) were high (range: 0.95–0.97) for the Psycat total, externalizing, and hyperactivity scales using corresponding CBCL scale scores as criterion. For the Psycat internalizing scale the AUC was somewhat lower but still high (0.86). Conclusions CAT is a valid procedure for the identification of emotional and behavioral problems in children aged 10–11 years. It may support the efficient and accurate identification of children with overall, and potentially also specific, emotional and behavioral problems in routine PCH.