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Background:Aedes aegypti is a potential vector for several arboviruses including dengue and Zika viruses. The species seems to be restricted to subtropical/tropical habitats and has difficulties in establishing permanent populations in southern Europe, probably due to constraints during the winter season. The aim of this study was to systematically analyze the cold tolerance (CT) of Ae. aegypti in its most cold-resistant life stage, the eggs.
Methods: The CT of Ae. aegypti eggs was compared with that of Ae. albopictus which is well established in large parts of Europe. By systematically studying the literature (meta-analysis), we recognized that CT has been rarely tested in Ae. aegypti eggs, but eggs can survive at zero and sub-zero temperatures for certain exposure periods. To overcome potential bias from experimental differences between studies, we then conducted species comparisons using a harmonized high-resolution CT measuring method. From subtropical populations of the same origin, the survival (hatching in %) and emergence of adults of both species were measured after zero and sub-zero temperature exposures for up to 9 days (3 °C, 0 °C and − 2 °C: ≤ 9 days; − 6 °C: ≤ 2 days).
Results: Our data show that Ae. aegypti eggs can survive low and sub-zero temperatures for a short time period similar to or even better than those of Ae. albopictus. Moreover, after short sub-zero exposures of eggs of both species, individuals still developed into viable adults (Ae. aegypti: 3 adults emerged after 6 days at − 2 °C, Ae. albopictus: 1 adult emerged after 1 day at − 6 °C).
Conclusions: Thus, both the literature and the present experimental data indicate that a cold winter may not be the preventing factor for the re-establishment of the dengue vector Ae. aegypti in southern Europe.
Psoriasis (PsO) is one of the common chronic inflammatory skin diseases. Approximately 3% of the European Caucasian population is affected. Psoriatic arthritis (PsA) is a chronic immune-mediated disease associated with PsO characterized by distinct musculoskeletal inflammation. Due to its heterogeneous clinical manifestations (e.g., oligo- or polyarthritis, enthesitis, dactylitis, and axial inflammation), early diagnosis of PsA is often difficult and delayed. Approximately 30% of PsO patients will develop PsA. The responsible triggers for the transition from PsO only to PsA are currently unclear, and the impacts of different factors (e.g., genetic, environmental) on disease development are currently discussed. There is a high medical need, recently unmet, to specifically detect those patients with an increased risk for the development of clinically evident PsA early to initiate sufficient treatment to inhibit disease progression and avoid structural damage and loss of function or even intercept disease development. Increased neoangiogenesis and enthesial inflammation are hypothesized to be early pathological findings in PsO patients with PsA development. Different disease states describe the transition from PsO to PsA. Two of those phases are of value for early detection of PsA at-risk patients to prevent later development of PsA as changes in biomarker profiles are detectable: the subclinical phase (soluble and imaging biomarkers detectable, no clinical symptoms) and the prodromal phase (imaging biomarkers detectable, unspecific musculoskeletal symptoms such as arthralgia and fatigue). To target the unmet need for early detection of this at-risk population and to identify the subgroup of patients who will transition from PsO to PsA, imaging plays an important role in characterizing patients precisely. Imaging techniques such as ultrasound (US), magnetic resonance imaging (MRI), and computerized tomography (CT) are advanced techniques to detect sensitively inflammatory changes or changes in bone structure. With the use of these techniques, anatomic structures involved in inflammatory processes can be identified. These techniques are complemented by fluorescence optical imaging as a sensitive method for detection of changes in vascularization, especially in longitudinal measures. Moreover, high-resolution peripheral quantitative CT (HR-pQCT) and dynamic contrast-enhanced MRI (DCE-MRI) may give the advantage to identify PsA-related early characteristics in PsO patients reflecting transition phases of the disease.
The coronavirus disease 2019 COVID-19 pandemic is rapidly spreading worldwide and is becoming a major public health crisis. Increasing evidence demonstrates a strong correlation between obesity and the COVID-19 disease. We have summarized recent studies and addressed the impact of obesity on COVID-19 in terms of hospitalization, severity, mortality, and patient outcome. We discuss the potential molecular mechanisms whereby obesity contributes to the pathogenesis of COVID-19. In addition to obesity-related deregulated immune response, chronic inflammation, endothelium imbalance, metabolic dysfunction, and its associated comorbidities, dysfunctional mesenchymal stem cells/adipose-derived mesenchymal stem cells may also play crucial roles in fueling systemic inflammation contributing to the cytokine storm and promoting pulmonary fibrosis causing lung functional failure, characteristic of severe COVID-19. Moreover, obesity may also compromise motile cilia on airway epithelial cells and impair functioning of the mucociliary escalators, reducing the clearance of severe acute respiratory syndrome coronavirus (SARS-CoV-2). Obese diseased adipose tissues overexpress the receptors and proteases for the SARS-CoV-2 entry, implicating its possible roles as virus reservoir and accelerator reinforcing violent systemic inflammation and immune response. Finally, anti-inflammatory cytokines like anti-interleukin 6 and administration of mesenchymal stromal/stem cells may serve as potential immune modulatory therapies for supportively combating COVID-19. Obesity is conversely related to the development of COVID-19 through numerous molecular mechanisms and individuals with obesity belong to the COVID-19-susceptible population requiring more protective measures.
Objective: Spinal epidural abscess (SEA) is a severe and life-threatening disease. Although commonly performed, the effect of timing in surgical treatment on patient outcome is still unclear. With this study, we aim to provide evidence for early surgical treatment in patients with SEA.
Methods: Patients treated for SEA in the authors' department between 2007 and 2016 were included for analysis and retrospectively analyzed for basic clinical parameters and outcome. Pre- and postoperative neurological status were assessed using the American Spinal Injury Association Impairment Scale (AIS). The self-reported quality of life (QOL) based on the Short-Form Health Survey 36 (SF-36) was assessed prospectively. Surgery was defined as "early", when performed within 12 hours after admission and "late" when performed thereafter. Conservative therapy was preferred and recommend in patients without neurological deficits and in patients denying surgical intervention.
Results: One hundred and twenty-three patients were included in this study. Forty-nine patients (39.8%) underwent early, 47 patients (38.2%) delayed surgery and 27 (21.9%) conservative therapy. No significant differences were observed regarding mean age, sex, diabetes, prior history of spinal infection, and bony destruction. Patients undergoing early surgery revealed a significant better clinical outcome before discharge than patients undergoing late surgery (p=0.001) and conservative therapy. QOL based on SF-36 were significantly better in the early surgery cohort in two of four physical items (physical functioning and bodily pain) and in one of four psychological items (role limitation) after a mean follow-up period of 58 months. Readmission to the hospital and failure of conservative therapy were observed more often in patients undergoing conservative therapy.
Conclusion: Our data on both clinical outcome and QOL provide evidence for early surgery within 12 hours after admission in patients with SEA.
Background: The treatment of severely injured patients, especially in older age, is complex, and based on strict guidelines. Methods: We conducted a retrospective study by analyzing our internal registry for mortality risk factors in deceased trauma patients. All patients that were admitted to the trauma bay of our level-1-trauma center from 2014 to 2018, and that died during the in-hospital treatment, were included. The aim of this study was to carry out a quality assurance concerning the initial care of severely injured patients. Results: In the 5-year period, 135 trauma patients died. The median (IQR) age was 69 (38–83) years, 71% were male, and the median (IQR) Injury Severity Score (ISS) was 25 (17–34) points. Overall, 41% of the patients suffered from severe traumatic brain injuries (TBI) (AIShead ≥ 4 points). For 12.7%, therapy was finally limited owing to an existing patient’s decree; in 64.9% with an uncertain prognosis, a ‘therapia minima’ was established in consensus with the relatives. Conclusion: Although the mortality rate was primarily related to the severity of the injury, a significant number of deaths were not exclusively due to medical reasons, but also to a self-determined limitation of therapy for severely injured geriatric patients. The conscientious documentation concerning the will of the patient is increasingly important in supporting medical decisions.
Background: The epidermal growth factor receptor (EGFR) signaling pathway is genetically activated in approximately 50% of glioblastomas (GBs). Its inhibition has been explored clinically but produced disappointing results, potentially due to metabolic effects that protect GB cells against nutrient deprivation and hypoxia. Here, we hypothesized that EGFR activation could disable metabolic adaptation and define a GB cell population sensitive to starvation.
Methods: Using genetically engineered GB cells to model different types of EGFR activation, we analyzed changes in metabolism and cell survival under conditions of the tumor microenvironment.
Results: We found that expression of mutant EGFRvIII as well as EGF stimulation of EGFR-overexpressing cells impaired physiological adaptation to starvation and rendered cells sensitive to hypoxia-induced cell death. This was preceded by adenosine triphosphate (ATP) depletion and an increase in glycolysis. Furthermore, EGFRvIII mutant cells had higher levels of mitochondrial superoxides potentially due to decreased metabolic flux into the serine synthesis pathway which was associated with a decrease in the NADPH/NADP+ ratio.
Conclusions: The finding that EGFR activation renders GB cells susceptible to starvation could help to identify a subgroup of patients more likely to benefit from starvation-inducing therapies.
Background: Unwanted anticholinergic effects are both underestimated and frequently overlooked. Failure to identify adverse drug reactions (ADRs) can lead to prescribing cascades and the unnecessary use of over-thecounter products. The objective of this systematic review and meta-analysis is to explore and quantify the frequency and severity of ADRs associated with amitriptyline vs. placebo in randomized controlled trials (RCTs) involving adults with any indication, as well as healthy individuals. Methods: A systematic search in six electronic databases, forward/backward searches, manual searches, and searches for Food and Drug Administration (FDA) and European Medicines Agency (EMA) approval studies, will be performed. Placebo-controlled RCTs evaluating amitriptyline in any dosage, regardless of indication and without restrictions on the time and language of publication, will be included, as will healthy individuals. Studies of topical amitriptyline, combination therapies, or including <100 participants, will be excluded. Two investigators will screen the studies independently, assess methodological quality, and extract data on design, population, intervention, and outcomes ((non-)anticholinergic ADRs, e.g., symptoms, test results, and adverse drug events (ADEs) such as falls). The primary outcome will be the frequency of anticholinergic ADRs as a binary outcome (absolute number of patients with/without anticholinergic ADRs) in amitriptyline vs. placebo groups. Anticholinergic ADRs will be defined by an experienced clinical pharmacologist, based on literature and data from Martindale: The Complete Drug Reference. Secondary outcomes will be frequency and severity of (non-)anticholinergic ADRs and ADEs. The information will be synthesized in meta-analyses and narratives. We intend to assess heterogeneity using metaregression (for indication, outcome, and time points) and I2 statistics. Binary outcomes will be expressed as odds ratios, and continuous outcomes as standardized mean differences. Effect measures will be provided using 95% confidence intervals. We plan sensitivity analyses to assess methodological quality, outcome reporting etc., and subgroup analyses on age, dosage, and duration of treatment. Discussion: We will quantify the frequency of anticholinergic and other ADRs/ADEs in adults taking amitriptyline for any indication by comparing rates for amitriptyline vs. placebo, hence, preventing bias from disease symptoms and nocebo effects. As no standardized instrument exists to measure it, our overall estimate of anticholinergic ADRs may have limitations.
Background: Essential Tremor (ET) is a progressive neurological disorder characterized by postural and kinetic tremor most commonly affecting the hands and arms. Medically intractable ET can be treated by deep brain stimulation (DBS) of the ventral intermediate nucleus of thalamus (VIM). We investigated whether the location of the effective contact (most tremor suppression with at least side effects) in VIM-DBS for ET changes over time, indicating a distinct mechanism of loss of efficacy that goes beyond progression of tremor severity, or a mere reduction of DBS efficacy.
Methods: We performed programming sessions in 10 patients who underwent bilateral vim-DBS surgery between 2009 and 2017 at our department. In addition to the intraoperative (T1) and first clinical programming session (T2) a third programming session (T3) was performed to assess the effect- and side effect threshold (minimum voltage at which a tremor suppression or side effects occurred). Additionally, we compared the choice of the effective contact between T1 and T2 which might be affected by a surgical induced “brain shift.”
Discussion: Over a time span of about 4 years VIM-DBS in ET showed continuous efficacy in tremor suppression during stim-ON compared to stim-OFF. Compared to immediate postoperative programming sessions in ET-patients with DBS, long-term evaluationshowednorelevantchangeinthechoiceofcontactwithrespecttosideeffects andefficacy.InthemajorityofthecasestheactivecontactatT2didnotcorrespondtothe most effective intraoperative stimulation site T1, which might be explained by a brain-shift due to cerebral spinal fluid loss after neurosurgical procedure.
This case series assessed a commercial airline flight from Tel Aviv, Israel, to Frankfurt, Germany, that occurred on March 9th, 2020. Among 102 passengers on a Boeing 737-900 aircraft were 24 members of a tourist group. Starting 7 days earlier, the group had contact with a hotel manager who later received a diagnosis of coronavirus disease 2019 (COVID-19). No member of the group had received a diagnosis of COVID-19 before the flight, and no measures to prevent transmission (eg, wearing of masks) had been applied. The flight duration was 4 hours 40 minutes.
MicroRNA miR-181 - a rheostat for TCR signaling in thymic selection and peripheral T-Cell function
(2020)
The selection of T cells during intra-thymic d evelopment is crucial to obtain a functional and simultaneously not self-reactive peripheral T cell repertoire. However, selection is a complex process dependent on T cell receptor (TCR) thresholds that remain incompletely understood. In peripheral T cells, activation, clonal expansion, and contraction of the active T cell pool, as well as other processes depend on TCR signal strength. Members of the microRNA (miRNA) miR-181 family have been shown to be dynamically regulated during T cell development as well as dependent on the activation stage of T cells. Indeed, it has been shown that expression of miR-181a leads to the downregulation of multiple phosphatases, implicating miR-181a as ‘‘rheostat’’ of TCR signaling. Consistently, genetic models have revealed an essential role of miR-181a/b-1 for the generation of unconventional T cells as well as a function in tuning TCR sensitivity in peripheral T cells during aging. Here, we review these broad roles of miR-181 family members in T cell function via modulating TCR signal strength.
With obesity having doubled in the last decade, hypertension is on the rise. In one-third of hypertensive patients the metabolic syndrome is present. This might be one factor for the increasing number of prescriptions for angiotensin receptor blockers and calcium-channel blockers besides a more favorable risk-to-benefit ratio. The aim of the present study was to evaluate a therapeutic drug monitoring (TDM) method for assessment of adherence based on cut-offs in inpatients and to compare it to an established urine drug screening in outpatients. A method for quantification of calcium-channel blockers and angiotensin receptor blockers using high-performance liquid chromatography-tandem mass spectrometric analysis (LC-MS/MS) was developed and validated. The method was applied to serum samples of 32 patients under supervised medication to establish cut-off values for adherence assessment based on dose-related concentrations (DRC, calculated from pharmacokinetic data). Furthermore, corresponding urine and blood samples of 42 outpatients without supervised medication were analysed and the results compared with regard to adherence assessment. All serum concentrations measured for amlodipine (n = 40), lercanidipine (n = 14), candesartan (n = 10), telmisartan (n = 4) and valsartan (n = 10) in inpatients were above the patient specific lower DRC confirming adherence. Of 42 outpatients the identification of analytes in urine as well as the quantification in serum exhibited differing results. According to urinalysis, adherence was demonstrated in only 87.0% of prescriptions, compared to 91.3% for serum analyses. Differences were observed for amlodipine, lercanidipine and candesartan which can be explained by a higher specificity of the serum analysis approach due to pharmacokinetics. The present study confirms that assessing adherence based on serum drug concentrations with individually calculated lower DRCs is more accurate than using qualitative urine analysis. In particular, drugs with low bioavailability, low renal excretion or high metabolism rate such as lercanidipine and candesartan may lead to underestimation of adherence via urine analysis.
Emerging evidence suggests a complex relationship between sphingosine 1-phosphate (S1P) signaling and stroke. Here, we show the kinetics of S1P in the acute phase of ischemic stroke and highlight accompanying changes in immune cells and S1P receptors (S1PR). Using a C57BL/6 mouse model of middle cerebral artery occlusion (MCAO), we assessed S1P concentrations in the brain, plasma, and spleen. We found a steep S1P gradient from the spleen towards the brain. Results obtained by qPCR suggested that cells expressing the S1PR type 1 (S1P1+) were the predominant population deserting the spleen. Here, we report the cerebral recruitment of T helper (TH) and regulatory T (TREG) cells to the ipsilateral hemisphere, which was associated with differential regulation of cerebral S1PR expression patterns in the brain after MCAO. This study provides insight that the S1P-S1PR axis facilitates splenic T cell egress and is linked to the cerebral recruitment of S1PR+ TH and TREG cells. Further insights by which means the S1P-S1PR-axis orchestrates neuronal positioning may offer new therapeutic perspectives after ischemic stroke.
Background: Many patients suffering from exercise-induced asthma (EIA) have normal lung function at rest and show symptoms and a decline in FEV1 when they do sports or during exercise-challenge. It has been described that long-chain polyunsaturated fatty acids (LCPUFA) could exert a protective effect on EIA.
Methods: In this study the protective effect of supplementation with a special combination of n-3 and n-6 LCPUFA (sc-LCPUFA) (total 1.19 g/ day) were investigated in an EIA cold air provocation model. Primary outcome measure: Decrease in FEV1 after exercise challenge and secondary outcome measure: anti-inflammatory effects monitored by exhaled NO (eNO) before and after sc-LCPUFA supplementation versus placebo.
Results: Ninety-nine patients with exercise-induced symptoms aged 10 to 45 were screened by a standardized exercise challenge in a cold air chamber at 4 °C. Seventy-three patients fulfilled the inclusion criteria of a FEV1 decrease > 15% and were treated double-blind placebo-controlled for 4 weeks either with sc-LCPUFA or placebo. Thirty-two patients in each group completed the study. Mean FEV1 decrease after cold air exercise challenge and eNO were unchanged after 4 weeks sc-LCPUFA supplementation.
Conclusion: Supplementation with sc-LCPUFA at a dose of 1.19 g/d did not have any broncho-protective and anti-inflammatory effects on EIA.
Trial registration: Clinical trial registration number: NCT02410096. Registered 7 February 2015 at Clinicaltrial.gov
SARS-CoV-2 is the causative agent of COVID-19. Severe COVID-19 disease has been associated with disseminated intravascular coagulation and thrombosis, but the mechanisms underlying COVID-19-related coagulopathy remain unknown. The risk of severe COVID-19 disease is higher in males than in females and increases with age. To identify gene products that may contribute to COVID-19-related coagulopathy, we analyzed the expression of genes associated with the Gene Ontology (GO) term “blood coagulation” in the Genotype-Tissue Expression (GTEx) database and identified four procoagulants, whose expression is higher in males and increases with age (ADAMTS13, F11, HGFAC, KLKB1), and two anticoagulants, whose expression is higher in females and decreases with age (C1QTNF1, SERPINA5). However, the expression of none of these genes was regulated in a proteomics dataset of SARS-CoV-2-infected cells and none of the proteins have been identified as a binding partner of SARS-CoV-2 proteins. Hence, they may rather generally predispose individuals to thrombosis without directly contributing to COVID-19-related coagulopathy. In contrast, the expression of the procoagulant transferrin (not associated to the GO term “blood coagulation”) was higher in males, increased with age, and was upregulated upon SARS-CoV-2 infection. Hence, transferrin warrants further examination in ongoing clinic-pathological investigations.
miR-142-3p expression is predictive for severe traumatic brain injury (TBI) in trauma patients
(2020)
Background: Predictive biomarkers in biofluids are the most commonly used diagnostic method, but established markers in trauma diagnostics lack accuracy. This study investigates promisingmicroRNAs(miRNA)releasedfromaffectedtissueafterseveretraumathathavepredictive values for the effects of the injury.
Methods: A retrospective analysis of prospectively collected data and blood samples of n = 33 trauma patients (ISS≥16) is provided. Levels of miR-9-5p, -124-3p, -142-3p, -219a-5p, -338-3pand-423-3p inseverelyinjuredpatients (PT)withouttraumatic braininjury (TBI) or with severe TBI (PT + TBI) and patients with isolated TBI (isTBI) were measured within 6 h after trauma.
Results: The highest miR-423-3p expression was detected in patients with severe isTBI, followed by patients with PT + TBI, and lowest levels were found in PT patients without TBI (2−∆∆Ct,p = 0.009). ApositivecorrelationbetweenmiR-423-3plevelandincreasingAIShead (p = 0.001) and risk of mortality (RISC II, p = 0.062) in trauma patients (n = 33) was found. ROC analysis of miR-423-3p levels revealed them as statistically significant to predict the severity of brain injury in trauma patients (p = 0.006). miR-124-3p was only found in patients with severe TBI, miR-338-3p was shown in all trauma groups. miR-9-5p, miR-142-3p and miR-219a-5p could not be detected in any of the four groups. Conclusion: miR-423-3p expression is significantly elevated after isolated traumatic braininjuryandpredictableforsevereTBIinthefirsthoursaftertrauma. miR-423-3pcouldrepresent a promising new biomarker to identify severe isolated TBI.
A message from the human placenta: structural and immunomodulatory defense against SARS-CoV-2
(2020)
The outbreak of the coronavirus disease 2019 (COVID-19) pandemic has caused a global public health crisis. Viral infections may predispose pregnant women to a higher rate of pregnancy complications, including preterm births, miscarriage and stillbirth. Despite reports of neonatal COVID-19, definitive proof of vertical transmission is still lacking. In this review, we summarize studies regarding the potential evidence for transplacental transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), characterize the expression of its receptors and proteases, describe the placental pathology and analyze virus-host interactions at the maternal-fetal interface. We focus on the syncytium, the barrier between mother and fetus, and describe in detail its physical andstructuraldefenseagainstviralinfections. Wefurtherdiscussthepotentialmolecularmechanisms, whereby the placenta serves as a defense front against pathogens by regulating the interferon type III signaling, microRNA-triggered autophagy and the nuclear factor-κB pathway. Based on these data, we conclude that vertical transmission may occur but rare, ascribed to the potent physical barrier, the fine-regulatedplacentalimmunedefenseandmodulationstrategies. Particularly,immunomodulatory mechanismsemployedbytheplacentamaymitigateviolentimmuneresponse,maybesoftencytokine storm tightly associated with severely ill COVID-19 patients, possibly minimizing cell and tissue damages, and potentially reducing SARS-CoV-2 transmission.
Chronic treatment with the mTOR inhibitor, everolimus, fails long-term in preventing tumor growth and dissemination in cancer patients. Thus, patients experiencing treatment resistance seek complementary measures, hoping to improve therapeutic efficacy. This study investigated metastatic characteristics of bladder carcinoma cells exposed to everolimus combined with the isothiocyanate sulforaphane (SFN), which has been shown to exert cancer inhibiting properties. RT112, UMUC3, or TCCSUP bladder carcinoma cells were exposed short- (24 h) or long-term (8 weeks) to everolimus (0.5 nM) or SFN (2.5 µM), alone or in combination. Adhesion and chemotaxis along with profiling details of CD44 receptor variants (v) and integrin α and β subtypes were evaluated. The functional impact of CD44 and integrins was explored by blocking studies and siRNA knock-down. Long-term exposure to everolimus enhanced chemotactic activity, whereas long-term exposure to SFN or the SFN-everolimus combination diminished chemotaxis. CD44v4 and v7 increased on RT112 cells following exposure to SFN or SFN-everolimus. Up-regulation of the integrins α6, αV, and β1 and down-regulation of β4 that was present with everolimus alone could be prevented by combining SFN and everolimus. Down-regulation of αV, β1, and β4 reduced chemotactic activity, whereas knock-down of CD44 correlated with enhanced chemotaxis. SFN could, therefore, inhibit resistance-related tumor dissemination during everolimus-based bladder cancer treatment.
Depletion of yeast/fly Ataxin-2 rescues TDP-43 overexpression toxicity. In mouse models of Amyotrophic Lateral Sclerosis via TDP-43 overexpression, depletion of its ortholog ATXN2 mitigated motor neuron degeneration and extended lifespan from 25 days to >300 days. There is another ortholog in mammals, named ATXN2L (Ataxin-2-like), which is almost uncharacterized but also functions in RNA surveillance at stress granules. We generated mice with Crispr/Cas9-mediated deletion of Atxn2l exons 5-8, studying homozygotes prenatally and heterozygotes during aging. Our novel findings indicate that ATXN2L absence triggers mid-gestational embryonic lethality, affecting female animals more strongly. Weight and development stages of homozygous mutants were reduced. Placenta phenotypes were not apparent, but brain histology showed lamination defects and apoptosis. Aged heterozygotes showed no locomotor deficits or weight loss over 12 months. Null mutants in vivo displayed compensatory efforts to maximize Atxn2l expression, which were prevented upon nutrient abundance in vitro. Mouse embryonal fibroblast cells revealed more multinucleated giant cells upon ATXN2L deficiency. In addition, in human neural cells, transcript levels of ATXN2L were induced upon starvation and glucose and amino acids exposure, but this induction was partially prevented by serum or low cholesterol administration. Neither ATXN2L depletion triggered dysregulation of ATXN2, nor a converse effect was observed. Overall, this essential role of ATXN2L for embryogenesis raises questions about its role in neurodegenerative diseases and neuroprotective therapies.
Manufacturing processes of custom implant abutments may contaminate their surfaces with micro wear deposits and generic pollutants. Such particulate debris, if not removed, might be detrimental and provoke inflammatory reactions in peri-implant tissues. Although regulatory guidelines for adequate cleaning, disinfection, or sterilization exist, there does not appear to be a consistent application and data on the amount and extent of such contaminants is lacking. The aim of the present in vitro study was to evaluate the quality and quantity of processing-related surface contamination of computer-aided design/computer-aided manufacturing (CAD/CAM) abutments in the state of delivery and after ultrasonic cleaning. A total of 28 CAD/CAM monotype and hybrid abutments were cleaned and disinfected applying a three-stage ultrasonic protocol (Finevo protocol). Before and after cleaning, the chemical composition and the contamination of the abutments were assessed using scanning electron microscopy (SEM), dispersive X-ray spectroscopy(EDX),andcomputer-aidedplanimetricmeasurement(CAPM).Inthedeliverycondition, monotype abutments showed a significantly higher amount of debris compared to hybrid abutments (4.86±6.10% vs. 0.03 ± 0.03%, p < 0.001). The polishing process applied in the laboratory after bonding the hybrid abutment components reduces the surface roughness and thus contributes substantially to their purity. The extent of contamination caused by computer-aided manufacturing of custom abutments can be substantially minimized using a three-stage ultrasonic protocol.
Alcoholism is one of the leading and increasingly prevalent reasons of liver associated morbidity and mortality worldwide. Alcoholic hepatitis (AH) constitutes a severe disease with currently no satisfying treatment options. Lipoxin A4 (LXA4), a 15-lipoxygenase (ALOX15)-dependent lipid mediator involved in resolution of inflammation, showed promising pre-clinical results in the therapy of several inflammatory diseases. Since inflammation is a main driver of disease progression in alcoholic hepatitis, we investigated the impact of endogenous ALOX15-dependent lipid mediators and exogenously applied LXA4 on AH development. A mouse model for alcoholic steatohepatitis (NIAAA model) was tested in Alox12/15+/+ and Alox12/15−/− mice, with or without supplementation of LXA4. Absence of Alox12/15 aggravated parameters of liver disease, increased hepatic immune cell infiltration in AH, and elevated systemic neutrophils as a marker for systemic inflammation. Interestingly, i.p. injections of LXA4 significantly lowered transaminase levels only in Alox12/15−/− mice and reduced hepatic immune cell infiltration as well as systemic inflammatory cytokine expression in both genotypes, even though steatosis progressed. Thus, while LXA4 injection attenuated selected parameters of disease progression in Alox12/15−/− mice, its beneficial impact on immunity was also apparent in Alox12/15+/+ mice. In conclusion, pro-resolving lipid mediators may be beneficial to reduce inflammation in alcoholic hepatitis.
Autism spectrum disorders (ASD) are highly heritable and are characterized by deficits in social communication and restricted and repetitive behaviors. Twin studies on phenotypic subdomains suggest a differing underlying genetic etiology. Studying genetic variation explaining phenotypic variance will help to identify specific underlying pathomechanisms. We investigated the effect of common variation on ASD subdomains in two cohorts including >2500 individuals. Based on the Autism Diagnostic Interview-Revised (ADI-R), we identified and confirmed six subdomains with a SNP-based genetic heritability h2SNP = 0.2–0.4. The subdomains nonverbal communication (NVC), social interaction (SI), and peer interaction (PI) shared genetic risk factors, while the subdomains of repetitive sensory-motor behavior (RB) and restricted interests (RI) were genetically independent of each other. The polygenic risk score (PRS) for ASD as categorical diagnosis explained 2.3–3.3% of the variance of SI, joint attention (JA), and PI, 4.5% for RI, 1.2% of RB, but only 0.7% of NVC. We report eight genome-wide significant hits—partially replicating previous findings—and 292 known and novel candidate genes. The underlying biological mechanisms were related to neuronal transmission and development. At the SNP and gene level, all subdomains showed overlap, with the exception of RB. However, no overlap was observed at the functional level. In summary, the ADI-R algorithm-derived subdomains related to social communication show a shared genetic etiology in contrast to restricted and repetitive behaviors. The ASD-specific PRS overlapped only partially, suggesting an additional role of specific common variation in shaping the phenotypic expression of ASD subdomains.
Recent studies suggested an important contribution of sphingosine-1-phospate (S1P) signaling via its specific receptors (S1PRs) in the production of pro-inflammatory mediators such as Interleukin (IL)-1β in cancer and inflammation. In an inflammation-driven cancer setting, we previously reported that myeloid S1PR1 signaling induces IL-1β production by enhancing NLRP3 (NOD-, LRR- and Pyrin Domain-Containing Protein 3) inflammasome activity. However, the autocrine role of S1P and enzymes acting on the S1P rheostat in myeloid cells are unknown. Using human and mouse macrophages with pharmacological or genetic intervention we explored the relative contribution of sphingosine kinases (SPHKs) in NLRP3 inflammasome activity regulation. We noticed redundancy in SPHK1 and SPHK2 activities towards macrophage NLRP3 inflammasome transcriptional induction and IL-1β secretion. However, pharmacological blockade of both kinases in unison completely abrogated NLRP3 inflammasome induction and IL-1β secretion. Interestingly, human and mouse macrophages demonstrate varied responses towards SPHKs inhibition and IL-1β secretion. Clinical datasets of renal cell carcinoma and psoriasis patients showed a positive correlation between enzymes affecting the S1P rheostat with NLRP3 inflammasome components expression, which corroborates our finding. Our data provide a better understanding on the role of SPHKs and de novo synthesized S1P in macrophage NLRP3 inflammasome activation
Pulmonary failure is the main cause of morbidity and mortality in the human chromosomal instability syndrome Ataxia-telangiectasia (A-T). Major phenotypes include recurrent respiratory tract infections and bronchiectasis, aspiration, respiratory muscle abnormalities, interstitial lung disease, and pulmonary fibrosis. At present, no effective pulmonary therapy for A-T exists. Cell therapy using adipose-derived mesenchymal stromal/stem cells (ASCs) might be a promising approach for tissue regeneration. The aim of the present project was to investigate whether ASCs migrate into the injured lung parenchyma of Atm-deficient mice as an indication of incipient tissue damage during A-T. Therefore, ASCs isolated from luciferase transgenic mice (mASCs) were intravenously transplanted into Atm-deficient and wild-type mice. Retention kinetics of the cells were monitored using in vivo bioluminescence imaging (BLI) and completed by subsequent verification using quantitative real-time polymerase chain reaction (qRT-PCR). The in vivo imaging and the qPCR results demonstrated migration accompanied by a significantly longer retention time of transplanted mASCs in the lung parenchyma of Atm-deficient mice compared to wild type mice. In conclusion, our study suggests incipient damage in the lung parenchyma of Atm-deficient mice. In addition, our data further demonstrate that a combination of luciferase-based PCR together with BLI is a pivotal tool for tracking mASCs after transplantation in models of inflammatory lung diseases such as A-T.
Progressive bladder cancer growth is associated with abnormal activation of the mammalian target of the rapamycin (mTOR) pathway, but treatment with an mTOR inhibitor has not been as effective as expected. Rather, resistance develops under chronic drug use, prompting many patients to lower their relapse risk by turning to natural, plant-derived products. The present study was designed to evaluate whether the natural compound, sulforaphane (SFN), combined with the mTOR inhibitor everolimus, could block the growth and proliferation of bladder cancer cells in the short- and long-term. The bladder cancer cell lines RT112, UMUC3, and TCCSUP were exposed short- (24 h) or long-term (8 weeks) to everolimus (0.5 nM) or SFN (2.5 µM) alone or in combination. Cell growth, proliferation, apoptosis, cell cycle progression, and cell cycle regulating proteins were evaluated. siRNA blockade was used to investigate the functional impact of the proteins. Short-term application of SFN and/or everolimus resulted in significant tumor growth suppression, with additive inhibition on clonogenic tumor growth. Long-term everolimus treatment resulted in resistance development characterized by continued growth, and was associated with elevated Akt-mTOR signaling and cyclin-dependent kinase (CDK)1 phosphorylation and down-regulation of p19 and p27. In contrast, SFN alone or SFN+everolimus reduced cell growth and proliferation. Akt and Rictor signaling remained low, and p19 and p27 expressions were high under combined drug treatment. Long-term exposure to SFN+everolimus also induced acetylation of the H3 and H4 histones. Phosphorylation of CDK1 was diminished, whereby down-regulation of CDK1 and its binding partner, Cyclin B, inhibited tumor growth. In conclusion, the addition of SFN to the long-term everolimus application inhibits resistance development in bladder cancer cells in vitro. Therefore, sulforaphane may hold potential for treating bladder carcinoma in patients with resistance to an mTOR inhibitor.
Treatment of large bone defects is one of the great challenges in contemporary orthopedic and traumatic surgery. Grafts are necessary to support bone healing. A well-established allograft is demineralized bone matrix (DBM) prepared from donated human bone tissue. In this study, a fibrous demineralized bone matrix (f-DBM) with a high surface-to-volume ratio has been analyzed for toxicity and immunogenicity. f-DBM was transplanted to a 5-mm, plate-stabilized, femoral critical-size-bone-defect in Sprague-Dawley (SD)-rats. Healthy animals were used as controls. After two months histology, hematological analyses, immunogenicity as well as serum biochemistry were performed. Evaluation of free radical release and hematological and biochemical analyses showed no significant differences between the control group and recipients of f-DBM. Histologically, there was no evidence of damage to liver and kidney and good bone healing was observed in the f-DBM group. Reactivity against human HLA class I and class II antigens was detected with mostly low fluorescence values both in the serum of untreated and treated animals, reflecting rather a background reaction. Taken together, these results provide evidence for no systemic toxicity and the first proof of no basic immunogenic reaction to bone allograft and no sensitization of the recipient.
Although the therapeutic armamentarium for bladder cancer has considerably widened in the last few years, severe side effects and the development of resistance hamper long-term treatment success. Thus, patients turn to natural plant products as alternative or complementary therapeutic options. One of these is curcumin, the principal component of Curcuma longa that has shown chemopreventive effects in experimental cancer models. Clinical and preclinical studies point to its role as a chemosensitizer, and it has been shown to protect organs from toxicity induced by chemotherapy. These properties indicate that curcumin could hold promise as a candidate for additive cancer treatment. This review evaluates the relevance of curcumin as an integral part of therapy for bladder cancer.
Genetic association studies have shown their usefulness in assessing the role of ion channels in human thermal pain perception. We used machine learning to construct a complex phenotype from pain thresholds to thermal stimuli and associate it with the genetic information derived from the next-generation sequencing (NGS) of 15 ion channel genes which are involved in thermal perception, including ASIC1, ASIC2, ASIC3, ASIC4, TRPA1, TRPC1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM8, TRPV1, TRPV2, TRPV3, and TRPV4. Phenotypic information was complete in 82 subjects and NGS genotypes were available in 67 subjects. A network of artificial neurons, implemented as emergent self-organizing maps, discovered two clusters characterized by high or low pain thresholds for heat and cold pain. A total of 1071 variants were discovered in the 15 ion channel genes. After feature selection, 80 genetic variants were retained for an association analysis based on machine learning. The measured performance of machine learning-mediated phenotype assignment based on this genetic information resulted in an area under the receiver operating characteristic curve of 77.2%, justifying a phenotype classification based on the genetic information. A further item categorization finally resulted in 38 genetic variants that contributed most to the phenotype assignment. Most of them (10) belonged to the TRPV3 gene, followed by TRPM3 (6). Therefore, the analysis successfully identified the particular importance of TRPV3 and TRPM3 for an average pain phenotype defined by the sensitivity to moderate thermal stimuli.
NIMA (never-in-mitosis gene A)-related kinase 1 (Nek1) is shown to impact on different cellular pathways such as DNA repair, checkpoint activation, and apoptosis. Its role as a molecular target for radiation sensitization of malignant cells, however, remains elusive. Stably transduced doxycycline (Dox)-inducible Nek1 shRNA HeLa cervix and siRNA-transfected HCT-15 colorectal carcinoma cells were irradiated in vitro and 3D clonogenic radiation survival, residual DNA damage, cell cycle distribution, and apoptosis were analyzed. Nek1 knockdown (KD) sensitized both cell lines to ionizing radiation following a single dose irradiation and more pronounced in combination with a 6 h fractionation (3 × 2 Gy) regime. For preclinical analyses we focused on cervical cancer. Nek1 shRNA HeLa cells were grafted into NOD/SCID/IL-2Rγc−/− (NSG) mice and Nek1 KD was induced by Dox-infused drinking water resulting in a significant cytostatic effect if combined with a 6 h fractionation (3 × 2 Gy) regime. In addition, we correlated Nek1 expression in biopsies of patients with cervical cancer with histopathological parameters and clinical follow-up. Our results indicate that elevated levels of Nek1 were associated with an increased rate of local or distant failure, as well as with impaired cancer-specific and overall survival in univariate analyses and for most endpoints in multivariable analyses. Finally, findings from The Cancer Genome Atlas (TCGA) validation cohort confirmed a significant association of high Nek1 expression with a reduced disease-free survival. In conclusion, we consider Nek1 to represent a novel biomarker and potential therapeutic target for drug development in the context of optimized fractionation intervals.
Non-alcoholic fatty liver disease (NAFLD) is gaining in importance and is linked to obesity. Especially,thedevelopmentoffibrosisandportalhypertensioninNAFLDpatientsrequirestreatment. Transgenic TGR(mREN2)27 rats overexpressing mouse renin spontaneously develop NAFLD with portal hypertension but without obesity. This study investigated the additional role of obesity in this model on the development of portal hypertension and fibrosis. Obesity was induced in twelve-week old TGR(mREN2)27 rats after receiving Western diet (WD) for two or four weeks. Liver fibrosis was assessed using standard techniques. Hepatic expression of transforming growth factor-β1 (TGF-β1), collagen type Iα1, α-smooth muscle actin, and the macrophage markers Emr1, as well as the chemoattractant Ccl2, interleukin-1β (IL1β) and tumor necrosis factor-α (TNFα) were analyzed. Assessment of portal and systemic hemodynamics was performed using the colored microsphere technique. Asexpected,WDinducedobesityandliverfibrosisasconfirmedbySiriusRedandOilRed O staining. The expression of the monocyte-macrophage markers, Emr1, Ccl2, IL1β and TNFα were increasedduringfeedingofWD,indicatinginfiltrationofmacrophagesintotheliver,eventhoughthis increase was statistically not significant for the EGF module-containing mucin-like receptor (Emr1) mRNA expression levels. Of note, portal pressure increased with the duration of WD compared to animals that received a normal chow. Besides obesity, WD feeding increased systemic vascular resistance reflecting systemic endothelial and splanchnic vascular dysfunction. We conclude that transgenic TGR(mREN2)27 rats are a suitable model to investigate NAFLD development with liver fibrosis and portal hypertension. Tendency towards elevated expression of Emr1 is associated with macrophage activity point to a significant role of macrophages in NAFLD pathogenesis, probably due to a shift of the renin–angiotensin system towards a higher activation of the classical pathway. The hepatic injury induced by WD in TGR(mREN2)27 rats is suitable to evaluate different stages of fibrosis and portal hypertension in NAFLD with obesity.
Background: The purpose of this pilot study was to create a valid and reliable set of assessment questions for examining Evidence-based Dentistry (EbD) knowledge. For this reason, we adapted and validated for dental students the Berlin Questionnaire (BQ), which assesses Evidence-based Medicine (EbM) abilities.
Methods: The Berlin Questionnaire was validated with medical residents. We adapted it for use in a dentistry setting. An expert panel reviewed the adapted BQ for content validity. A cross-sectional cohort representing four training levels (EbD-novice dental students, EbD-trained dental students, dentists, and EbM−/EbD-expert faculty) completed the questionnaire. A total of 140 participants comprised the validation set. Internal reliability, item difficulty and item discrimination were assessed. Construct validity was assessed by comparing the mean total scores of students to faculty and comparing proportions of students and faculty who passed each item.
Results: Among the 133 participants (52 EbD-novice dental students, 53 EbD-trained dental students, 12 dentists, and 16 EbM-/ EbD-expert faculty), a statistically significant (p < 0.001) difference was evident in the total score corresponding to the training level. The total score reliability and psychometric properties of items modified for discipline-specific content were acceptable. Cronbach’s alpha was 0.648.
Conclusion: The adapted Berlin Questionnaire is a reliable and valid instrument to assess competence in Evidence-based Dentistry in dental students. Future research will focus on refining the instrument further.
Background: Radiotherapy dose and target volume prescriptions for anal squamous cell carcinoma (ASCC) vary considerably in daily practice and guidelines, including those from NCCN, UK, Australasian, and ESMO. We conducted a pattern-of-care survey to assess the patient management in German speaking countries.
Methods: We developed an anonymous questionnaire comprising 18 questions on diagnosis and treatment of ASCC. The survey was sent to 361 DEGRO-associated institutions, including 41 university hospitals, 118 non-university institutions, and 202 private practices.
Results: We received a total of 101 (28%) surveys, including 20 (19.8%) from university, 36 (35.6%) from non-university clinics, and 45 (44.6%) from private practices. A total of 28 (27.8%) institutions reported to treat more than 5 patients with early-stage ASCC and 42 (41.6%) institutions treat more than 5 patients with locoregionally-advanced ASCC per year. Biopsy of suspicious inguinal nodes was advocated in only 12 (11.8%) centers. Screening for human immunodeficiency virus (HIV) is done in 28 (27.7%). Intensity modulated radiotherapy or similar techniques are used in 97%. The elective lymph node dose ranged from 30.6 Gy to 52.8 Gy, whereas 87% prescribed 50.4–55. 8 Gy (range: 30.6 to 59.4 Gy) to the involved lymph nodes. The dose to gross disease of cT1 or cT2 ASCC ranged from 50 to ≥60 Gy. For cT3 or cT4 tumors the target dose ranged from 54 Gy to more than 60 Gy, with 76 (75.2%) institutions prescribing 59.4 Gy. The preferred concurrent chemotherapy regimen was 5-FU/Mitomycin C, whereas 6 (6%) prescribed Capecitabine/Mitomycin C. HIV-positive patients are treated with full-dose CRT in 87 (86.1%) institutions. First assessment for clinical response is reported to be performed at 4–6 weeks after completion of CRT in 2 (2%) institutions, at 6–8 weeks in 20 (19.8%), and 79 (78%) institutions wait up to 5 months.
Conclusions: We observed marked differences in radiotherapy doses and treatment technique in patients with ASCC, and also variable approaches for patients with HIV. These data underline the need for an consensus treatment guideline for ASCC.
Objective: Phenotypic (Sensititre Myco, pDST) and genotypic drug susceptibility testing (GenoType NTM DR, gDST) in M. avium complex (MAC) have become available as standardized assays, but comparable data is needed. This study aimed to investigate the phenotypic and genotypic drug susceptibility patterns in MAC clinical isolates.
Methods: Overall, 98 isolates from 85 patients were included. pDST and gDST were performed on all isolates and results compared regarding specificity and sensitivity using pDST as a reference method. The impact of drug instability on pDST results was studied using a biological assay over 14 days. In addition, the evolution of antimicrobial resistance was investigated in sequential isolates of 13 patients.
Results: Macrolide resistance was rare, 1.2% (95% CI 0.7–7.3) of isolates in the base cohort. No aminoglycoside resistances were found, but 14.1% of the studied isolates (95% CI 7.8–23.8) showed intermediate susceptibility. The GenoType NTM DR identified two out of four macrolide-resistant isolates. Antibiotic stability was demonstrated to be poor in rifampicin, rifabutin, and doxycycylin.
Conclusions: pDST results in NTM for unstable antibiotics must be interpreted with care. A combination of pDST and gDST will be useful for the guidance of antimicrobial therapy in MAC-disease.
Molecular and cellular research modalities for the study of liver pathologies have been tremendously improved over the recent decades. Advanced technologies offer novel opportunities to establish cell isolation techniques with excellent purity, paving the path for 2D and 3D microscopy and high-throughput assays (e.g., bulk or single-cell RNA sequencing). The use of stem cell and organoid research will help to decipher the pathophysiology of liver diseases and the interaction between various parenchymal and non-parenchymal liver cells. Furthermore, sophisticated animal models of liver disease allow for the in vivo assessment of fibrogenesis, portal hypertension and hepatocellular carcinoma (HCC) and for the preclinical testing of therapeutic strategies. The purpose of this review is to portray in detail novel in vitro and in vivo methods for the study of liver cell biology that had been presented at the workshop of the 8th meeting of the European Club for Liver Cell Biology (ECLCB-8) in October of 2018 in Bonn, Germany.
Objective: To study the effect of total hip replacement (THR) on serum cartilage oligomeric matrix protein concentration (sCOMP) and its correlation with joint loading during gait in patients with unilateral hip osteoarthritis.
Design: In this prospective multimodal (clinical, biomechanical, biochemical) study blood samples from 15 patients were taken before and up to three times after THR (7 days, 3 months and 1 year), each after a resting period of at least 30 min, for analysis of sCOMP. Gait analysis was performed before and 1 year after THR to determine hip and knee joint moments.
Results: Seven days after THR, sCOMP decreased significantly compared to the preoperative measurement (p < 0.001). Three months and 1 year postoperatively, sCOMP reverted to concentrations in the range of the preoperative value. One year postoperatively, a linear correlation between sCOMP and the maximum hip flexion moment was indicated in the first half of the stance phase on the unaffected side (r = −0.736, p = 0.024). No further correlations could be determined.
Conclusions: Surprisingly, the removal of a joint affected by osteoarthritis did not have a sustained effect on sCOMP. Both before and after THR there was no scientifically substantiated correlation between sCOMP and joint moments from gait analysis. Consequently, the examination of sCOMP is not useful to detect altered joint loads that may influence degenerative changes of adjacent joints after THR.
The registration number in the German Registry of Clinical Trials is DRKS00015053.
Background: Experienced and anticipated regret influence physicians’ decision-making. In medicine, diagnostic decisions and diagnostic errors can have a severe impact on both patients and physicians. Little empirical research exists on regret experienced by physicians when they make diagnostic decisions in primary care that later prove inappropriate or incorrect. The aim of this study was to explore the experience of regret following diagnostic decisions in primary care.
Methods: In this qualitative study, we used an online questionnaire on a sample of German primary care physicians. We asked participants to report on cases in which the final diagnosis differed from their original opinion, and in which treatment was at the very least delayed, possibly resulting in harm to the patient. We asked about original and final diagnoses, illness trajectories, and the reactions of other physicians, patients and relatives. We used thematic analysis to assess the data, supported by MAXQDA 11 and Microsoft Excel 2016.
Results: 29 GPs described one case each (14 female/15 male patients, aged 1.5–80 years, response rate < 1%). In 26 of 29 cases, the final diagnosis was more serious than the original diagnosis. In two cases, the diagnoses were equally serious, and in one case less serious. Clinical trajectories and the reactions of patients and relatives differed widely. Although only one third of cases involved preventable harm to patients, the vast majority (27 of 29) of physicians expressed deep feelings of regret.
Conclusion: Even if harm to patients is unavoidable, regret following diagnostic decisions can be devastating for clinicians, making them ‘second victims’. Procedures and tools are needed to analyse cases involving undesirable diagnostic events, so that ‘true’ diagnostic errors, in which harm could have been prevented, can be distinguished from others. Further studies should also explore how physicians can be supported in dealing with such events in order to prevent them from practicing defensive medicine.
Background: Point of care devices for performing targeted coagulation substitution in patients who are bleeding have become increasingly important in recent years. New on the market is the Quantra. It is a device that uses sonorheometry, a sonic estimation of elasticity via resonance, which is a novel ultrasound-based technology that measures viscoelastic properties of whole blood. Several studies have already shown the comparability of the Quantra with devices already established on the market, such as the rotational thromboelastometry (ROTEM) device.
Objective: In contrast to existing studies, this study is the first prospective interventional study using this new system in a cardiac surgical patient cohort. We will investigate the noninferiority between an already existing coagulation algorithm based on the ROTEM/Multiplate system and a new algorithm based on the Quantra system for the treatment of coagulopathic cardiac surgical patients.
Methods: The study is divided into two phases. In an initial observation phase, whole blood samples of 20 patients obtained at three defined time points (prior to surgery, after completion of cardiopulmonary bypass, and on arrival in the intensive care unit) will be analyzed using both the ROTEM/Multiplate and Quantra systems. The obtained threshold values will be used to develop a novel algorithm for hemotherapy. In a second intervention phase, the new algorithm will be tested for noninferiority against an algorithm used routinely for years in our department.
Results: The main objective of the examination is the cumulative loss of blood within 24 hours after surgery. Statistical calculations based on the literature and in-house data suggest that the new algorithm is not inferior if the difference in cumulative blood loss is <150 mL/24 hours.
Conclusions: Because of the comparability of the Quantra sonorheometry system with the ROTEM measurement methods, the existing hemotherapy treatment algorithm can be adapted to the Quantra device with proof of noninferiority.
Trial Registration: ClinicalTrials.gov NCT03902275; https://clinicaltrials.gov/ct2/show/NCT03902275
International Registered Report Identifier (IRRID): DERR1-10.2196/17206
Background: Computerized virtual patients (VP) have spread into many areas of healthcare delivery and medical education. They provide various advantages like flexibility in pace and space of learning, a high degree of teaching reproducibility and a cost effectiveness. However, the educational benefit of VP as an additive or also as an alternative to traditional teaching formats remains unclear. Moreover, there are no randomized-controlled studies that investigated the use of VP in a dental curriculum. Therefore, this study investigates VP as an alternative to lecturer-led small-group teaching in a curricular, randomized and controlled setting.
Methods: Randomized and controlled cohort study. Four VP cases were created according to previously published design principles and compared with lecturer-led small group teaching (SGT) within the Oral and Maxillofacial Surgery clerkship for dental students at the Department for Cranio-, Oral and Maxillofacial Plastic Surgery, Goethe University, Frankfurt, Germany. Clinical competence was measured prior (T0), directly (T1) and 6 weeks (T2) after the intervention using theoretical tests and a self-assessment questionnaire. Furthermore, VP design was evaluated using a validated toolkit.
Results: Fifty-seven students (VP = 32; SGT = 25) agreed to participate in the study. No competence differences were found at T0 (p = 0.56). The VP group outperformed (p < .0001) the SGT group at T1. At T2 there was no difference between both groups (p = 0.55). Both interventions led to a significant growth in self-assessed competence. The VP group felt better prepared to diagnose and treat real patients and regarded VP cases as a rewarding learning experience.
Conclusions: VP cases are an effective alternative to lecture-led SGT in terms of learning efficacy in the short and long-term as well as self-assessed competence growth and student satisfaction. Furthermore, integrating VP cases within a curricular Oral and Maxillofacial Surgery Clerkship is feasible and leads to substantial growth of clinical competence in undergraduate dental students.
Aims: Long non-coding RNAs (lncRNAs) have been shown to regulate numerous processes in the human genome, but the function of these transcripts in vascular aging is largely unknown. We aim to characterize the expression of lncRNAs in endothelial aging and analyse the function of the highly conserved lncRNA H19.
Methods and results: H19 was downregulated in endothelium of aged mice. In human, atherosclerotic plaques H19 was mainly expressed by endothelial cells and H19 was significantly reduced in comparison to healthy carotid artery biopsies. Loss of H19 led to an upregulation of p16 and p21, reduced proliferation and increased senescence in vitro. Depletion of H19 in aortic rings of young mice inhibited sprouting capacity. We generated endothelial-specific inducible H19 deficient mice (H19iEC-KO), resulting in increased systolic blood pressure compared with control littermates (Ctrl). These H19iEC-KO and Ctrl mice were subjected to hindlimb ischaemia, which showed reduced capillary density in H19iEC-KO mice. Mechanistically, exon array analysis revealed an involvement of H19 in IL-6 signalling. Accordingly, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 were upregulated upon H19 depletion. A luciferase reporter screen for differential transcription factor activity revealed STAT3 as being induced upon H19 depletion and repressed after H19 overexpression. Furthermore, depletion of H19 increased the phosphorylation of STAT3 at TYR705 and pharmacological inhibition of STAT3 activation abolished the effects of H19 silencing on p21 and vascular cell adhesion molecule 1 expression as well as proliferation.
Conclusion: These data reveal a pivotal role for the lncRNA H19 in controlling endothelial cell aging.
Proton-pumping complex I of the mitochondrial respiratory chain is among the largest and most complex membrane protein complexes. The enzyme contributes substantially to oxidative energy-conversion in eukaryotic cells. Its malfunctions are implicated in many hereditary and degenerative disorders. Here, we report the X-ray structure of mitochondrial complex I at 3.6- 3.9 Å resolution describing in detail the central subunits that execute the bioenergetic function. A continuous axis of basic and acidic residues running centrally through the membrane arm connects the ubiquinone reduction site in the hydrophilic arm to four putative proton-pumping units. The binding position for a substrate analogous inhibitor and blockage of the predicted ubiquinone binding site provide a model for the ‘deactive’ form of the enzyme. The proposed transition into the active form is based on a concerted structural rearrangement at the ubiquinone reduction site rendering support for a two-state stabilization-change mechanism of protonpumping.
CD4+CD25+ regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti-CD4 monoclonal antibodies (mAbs), the humanized CD4-specific monoclonal antibody tregalizumab (BT-061) is able to selectively activate the suppressive properties of Tregs in vitro. BT-061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT-061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti-CD4 mAbs. We found that, due to this special epitope binding, BT-061 induces a unique phosphorylation of T-cell receptor complex-associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT-061 does not induce the release of pro-inflammatory cytokines. These results demonstrate that BT-061 stimulation via the CD4 receptor is able to induce T-cell receptor-independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing.
Background: Following acute coronary syndrome (ACS), the risk for future cardiovascular events is high and is related to levels of low-density lipoprotein cholesterol (LDL-C) even within the setting of intensive statin treatment. Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates LDL receptor expression and circulating levels of LDL-C. Antibodies to PCSK9 can produce substantial and sustained reductions of LDL-C. The ODYSSEY Outcomes trial tests the hypothesis that treatment with alirocumab, a fully human monoclonal antibody to PCSK9, improves cardiovascular outcomes after ACS.
Design: This Phase 3 study will randomize approximately 18,000 patients to receive biweekly injections of alirocumab (75-150 mg) or matching placebo beginning 1 to 12 months after an index hospitalization for acute myocardial infarction or unstable angina. Qualifying patients are treated with atorvastatin 40 or 80 mg daily, rosuvastatin 20 or 40 mg daily, or the maximum tolerated and approved dose of one of these agents and fulfill one of the following criteria: LDL-C ≥ 70 mg/dL, non-high-density lipoprotein cholesterol ≥ 100 mg/dL, or apolipoprotein B ≥ 80 mg/dL. The primary efficacy measure is time to first occurrence of coronary heart disease death, acute myocardial infarction, hospitalization for unstable angina, or ischemic stroke. The trial is expected to continue until 1613 primary end point events have occurred with minimum follow-up of at least 2 years, providing 90% power to detect a 15% hazard reduction. Adverse events of special interest include allergic events and injection site reactions. Interim analyses are planned when approximately 50% and 75% of the targeted number of primary end points have occurred.
Summary: ODYSSEY Outcomes will determine whether the addition of the PCSK9 antibody alirocumab to intensive statin therapy reduces cardiovascular morbidity and mortality after ACS.
Background: Candida spp. are a frequent cause of nosocomial bloodstream infections worldwide.
Objective: To evaluate the use patterns and outcomes associated with intravenous (IV) fluconazole therapy in intensive care units in Spain and Germany.
Patients and methods: The research reported here was a prospective multicenter longitudinal observational study in adult intensive care unit patients receiving IV fluconazole. Demographic, microbiologic, therapy success, length of hospital stay, adverse event, and all-cause mortality data were collected at 14 sites in Spain and five in Germany, from February 2004 to November 2005.
Results: Patients (n = 303) received prophylaxis (n = 29), empiric therapy (n = 140), preemptive therapy (n = 85), or definitive therapy (n = 49). A total of 298 patients (98.4%) were treated with IV fluconazole as first-line therapy. The treating physicians judged therapy successful in 66% of prophylactic, 55% of empiric, 45% of preemptive, and 43% of definitive group patients. In the subgroup of 152 patients with proven and specified Candida infection only, 32% suffered from Candida specified as potentially resistant to IV fluconazole. The overall mortality rate was 42%.
Conclusion: Our study informs treatment decision makers that approximately 32% of the patients with microbiological results available suffered from Candida specified as potentially resistant to IV fluconazole, highlighting the importance of appropriate therapy.
Background
Cytochrome-P450 (CYP450) epoxygenases metabolise arachidonic acid (AA) into four different biologically active epoxyeicosatrienoic acid (EET) regioisomers. Three of the EETs (i.e., 8,9-, 11,12- and 14,15-EET) are rapidly hydrolysed by the enzyme soluble epoxide hydrolase (sEH). Here, we investigated the role of sEH in nociceptive processing during peripheral inflammation.
Results
In dorsal root ganglia (DRG), we found that sEH is expressed in medium and large diameter neurofilament 200-positive neurons. Isolated DRG-neurons from sEH-/- mice showed higher EET and lower DHET levels. Upon AA stimulation, the largest changes in EET levels occurred in culture media, indicating both that cell associated EET concentrations quickly reach saturation and EET-hydrolyzing activity mostly effects extracellular EET signaling. In vivo, DRGs from sEH-deficient mice exhibited elevated 8,9-, 11,12- and 14,15-EET-levels. Interestingly, EET levels did not increase at the site of zymosan-induced inflammation. Cellular imaging experiments revealed direct calcium flux responses to 8,9-EET in a subpopulation of nociceptors. In addition, 8,9-EET sensitized AITC-induced calcium increases in DRG neurons and AITC-induced calcitonin gene related peptide (CGRP) release from sciatic nerve axons, indicating that 8,9-EET sensitizes TRPA1-expressing neurons, which are known to contribute to mechanical hyperalgesia. Supporting this, sEH-/- mice showed increased nociceptive responses to mechanical stimulation during zymosan-induced inflammation and 8,9-EET injection reduced mechanical thresholds in naive mice.
Conclusion
Our results show that the sEH can regulate mechanical hyperalgesia during inflammation by inactivating 8,9-EET, which sensitizes TRPA1-expressing nociceptors. Therefore we suggest that influencing the CYP450 pathway, which is actually highly considered to treat cardiovascular diseases, may cause pain side effects.
Background: The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings: We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal alpha-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions: Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.