The effect of the anti-tumor lipid Inositol-C2-PAF on the mammary carcinoma cell line MCF-7 under migration and proliferation stimulating conditions

Das Mammakarzinom ist der häufigste Grund für alle krebsbedingten Todesfälle bei Frauen in Deutschland und auf der Welt. Der Großteil der mit dem Mammakarzinom assoziierten Todesfälle wird dabei durch Metastasen verursacht, deren Bildungen durch unkontrollierte Proliferation, Angiogenese und die epithelial-mesenchymale Transition (EMT) begünstigt werden. Zwei Signalwege, deren Aktivierung bei der Metastasierung bei Mammakarzinomzellen eine bedeutsame Rolle spielen, sind der IL-6/STAT3- und der IGF-1/PI3K/AKT-Signalweg. Die Antitumorlipide, deren bekanntester Vertreter Edelfosin ist, entfalten ihre Wirkung entgegen etablierter Chemotherapeutika nicht durch einen Angriff direkt auf die DNA oder den Spindelapparat, sondern nach Inkorporation in die Zellmembran. Auch die Gruppe der glykosidierten Phospholipide, zu der das hier betrachtete Inositol-C2-PAF zählt, gehören zu den Antitumorlipiden. Inositol-C2-PAF konnte seine anti-proliferatorischen und anti-migratorischen Effekte schon bei verschiedenen Zelltypen (zum Beispiel hämato-/onkologischen Tumorzelllinien und immortalisierten Keratinozyten) zeigen. Um das bisherige Wissen der Wirkung von Inositol-C2-PAF um eine solide Tumorform, das Mammakarzinom, zu erweitern, wurde ein 2D-Tumormodell für die schwach invasive Mammakarzinom-Zelllinie MCF 7 mit dem Ziel der Stimulation von Proliferation und Migration mit IL-6 (und CCL2) sowie IGF-1 etabliert; mit der Absicht, die Wirkung von Inositol-C2-PAF durch verschiedene Assays sowohl auf zellulärer Ebene, wie Wundheilungsassays, haptotaktischen Migrationsassays und Proliferationsassays, als auch auf molekularer Ebene (IL-6/STAT3- und IGF-1/PI3K/AKT-Signalweg) mittels Western Blot-Analyse, indirekter Immunfluoreszenz-Analyse und kapillarer isoelektrischer Fokussierung (cIEF, capillary isoelectric focusing) nachzuweisen. Es wurde gezeigt, dass die inhibitorische Wirkung von Inositol-C2-PAF auf die Proliferation stärker war als auf die Migration. Ebenso war der hemmende Einfluss von Inositol-C2-PAF auf die Aktivierung des PI3K/AKT-Signalwegs stärker als auf die des STAT3-Signalwegs. Bei einer genaueren Untersuchung der AKT-Isoformen wurde nachgewiesen, dass die inhibitorische Wirkung von Inositol-C2-PAF die Phosphorylierung von mehreren AKT-Isoformen (zumindest die von AKT1 und AKT2) beeinflusst. Die eingehendere Betrachtung des PI3K/AKT-Signalwegs konnte die Wirkung von Inositol-C2-PAF über die Hemmung anderer relevanter Signalmoleküle wie mTORC1 und RB weiter untermauern. Die effektive Wirkung von Inositol-C2-PAF auf den PI3K/AKT-Signalweg spricht für die weitere Erforschung von Inositol-C2-PAF im Hinblick auf eine wirkungsstarke und möglicherweise nebenwirkungsärmere Therapie beim Mammakarzinom. Aufgrund einer in der Literatur vorgefundenen uneinheitlichen Klassifikation der Antitumorlipide wurde im Rahmen dieser Arbeit eine Neueinteilung der in dieser Gruppe enthaltenden Wirkstoffe vorgeschlagen, die sich am Glycerol-Grundgerüst dieser Moleküle orientiert.The mammary carcinoma is the most frequent reason for cancer-related deaths in women in Germany and in the world. The majority of breast cancer associated deaths are attributed to metastases, which result from uncontrolled proliferation, angiogenesis and epithelial-mesenchymal transition (EMT). The activation of two signal transduction pathways, the IL-6/STAT3 and the IGF-1/PI3K/AKT pathway, plays a significant role in forming these metastases of mammary carcinoma cells. Inositol-C2-PAF, a member of the glycosidated phospholipids which belong to the group of anti-tumor lipids, has been studied for its anti-proliferative and anti-migratory effects in different cell types including haemato-oncological tumor cells and immortalized keratinocytes. The anti-tumor lipids, whose well-known chemical lead is Edelfosine, are a group of compounds which are incorporated into the membrane. From here, they exert their effects, quite contrary to established chemotherapeutics which interfere with the DNA or the mitotic spindle apparatus. This thesis focuses on the effect of Inositol-C2-PAF in a solid tumor, the mammary carcinoma. For this purpose, a 2D tumor model for the low invasive mammary carcinoma cell line MCF-7 was established. MCF-7 cells were incubated with IL-6 (and CCL2) and IGF-1 to stimulate both proliferation and migration. This allowed studying the effect of Inositol-C2-PAF on the cellular level using various assays like wound healing assays, haptotactic migration assays and proliferation assays, and on the molecular level with a focus on the IL-6/STAT3 and IGF-1/PI3K/AKT pathways, using Western Blot analysis, indirect immunofluorescence analysis and capillary isoelectric focusing (cIEF). The results demonstrated, that the inhibitory effect of Inositol-C2-PAF on proliferation was more effective than on migration. Additionally, the inhibitory effect of Inositol-C2-PAF on the activated PI3K/AKT-pathway was greater than on the STAT3-pathway. Using the cIEF, it could be demonstrated that the inhibitory effect of Inositol-C2-PAF affected the phosphorylation of different AKT-isoforms (at least AKT1 and AKT2). The more detailed look on other parts of the PI3K/AKT-pathway upstream and downstream from AKT could also prove the effect of Inositol-C2-PAF through inhibition of the activation of other relevant signal transducers like mTORC1 and RB. The potent effect of Inositol-C2-PAF on the tumor relevant PI3K/AKT-pathway requires further investigation of Inositol-C2-PAF as a potential therapeutic candidate for the mammary carcinoma. Lastly, due to an inconsistent categorization of the antitumour lipids in literature, a reclassification was suggested, that is leant on the glycerol-backbone of these molecules

Comparison of existing aneurysm models and their path forward

The two most important aneurysm types are cerebral aneurysms (CA) and abdominal aortic aneurysms (AAA), accounting together for over 80\% of all fatal aneurysm incidences. To minimise aneurysm related deaths, clinicians require various tools to accurately estimate its rupture risk. For both aneurysm types, the current state-of-the-art tools to evaluate rupture risk are identified and evaluated in terms of clinical applicability. We perform a comprehensive literature review, using the Web of Science database. Identified records (3127) are clustered by modelling approach and aneurysm location in a meta-analysis to quantify scientific relevance and to extract modelling patterns and further assessed according to PRISMA guidelines (179 full text screens). Beside general differences and similarities of CA and AAA, we identify and systematically evaluate four major modelling approaches on aneurysm rupture risk: finite element analysis and computational fluid dynamics as deterministic approaches and machine learning and assessment-tools and dimensionless parameters as stochastic approaches. The latter score highest in the evaluation for their potential as clinical applications for rupture prediction, due to readiness level and user friendliness. Deterministic approaches are less likely to be applied in a clinical environment because of their high model complexity. Because deterministic approaches consider underlying mechanism for aneurysm rupture, they have improved capability to account for unusual patient-specific characteristics, compared to stochastic approaches. We show that an increased interdisciplinary exchange between specialists can boost comprehension of this disease to design tools for a clinical environment. By combining deterministic and stochastic models, advantages of both approaches can improve accessibility for clinicians and prediction quality for rupture risk.Comment: 46 pages, 5 figure

Dichloroacetate and PX-478 exhibit strong synergistic effects in a various number of cancer cell lines

Background: One key approach for anticancer therapy is drug combination. Drug combinations can help reduce doses and thereby decrease side effects. Furthermore, the likelihood of drug resistance is reduced. Distinct alterations in tumor metabolism have been described in past decades, but metabolism has yet to be targeted in clinical cancer therapy. Recently, we found evidence for synergism between dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, and the HIF-1 alpha inhibitor PX-478. In this study, we aimed to analyse this synergism in cell lines of different cancer types and to identify the underlying biochemical mechanisms. Methods: The dose-dependent antiproliferative effects of the single drugs and their combination were assessed using SRB assays. FACS, Western blot and HPLC analyses were performed to investigate changes in reactive oxygen species levels, apoptosis and the cell cycle. Additionally, real-time metabolic analyses (Seahorse) were performed with DCA-treated MCF-7 cells. Results: The combination of DCA and PX-478 produced synergistic effects in all eight cancer cell lines tested, including colorectal, lung, breast, cervical, liver and brain cancer. Reactive oxygen species generation and apoptosis played important roles in this synergism. Furthermore, cell proliferation was inhibited by the combination treatment. Conclusions: Here, we found that these tumor metabolism-targeting compounds exhibited a potent synergism across all tested cancer cell lines. Thus, we highly recommend the combination of these two compounds for progression to in vivo translational and clinical trials

Diagnostic accuracy and confounders of vagus nerve ultrasound in amyotrophic lateral sclerosis — a single-center case series and pooled individual patient data meta-analysis

Background Several single-center studies proposed utility of vagus nerve (VN) ultrasound for detecting disease severity, autonomic dysfunction, and bulbar phenotype in amyotrophic lateral sclerosis (ALS). However, the resulting body of literature shows opposing results, leaving considerable uncertainty on the clinical benefits of VN ultrasound in ALS. Methods Relevant studies were identified up to 04/2024 and individual patient data (IPD) obtained from the respective authors were pooled with a so far unpublished cohort (from Munich). An IPD meta-analysis of 109 patients with probable or definite ALS (El Escorial criteria) and available VN cross-sectional area (CSA) was performed, with age, sex, ALS Functional Rating Scale-revised (ALSFRS-R), disease duration, and bulbar phenotype as independent variables. Results Mean age was 65 years (± 12) and 47% of patients (± 12) had bulbar ALS. Mean ALSFRS-R was 38 (± 7), and mean duration was 18 months (± 18). VN atrophy was highly prevalent [left: 67% (± 5), mean CSA 1.6mm2 (± 0.6); right: 78% (± 21), mean CSA 1.8 mm2 (± 0.7)]. VN CSA correlated with disease duration (mean slope: left − 0.01; right − 0.01), but not with ALSFRS-R (mean slope: left 0.004; mean slope: right − 0.002). Test accuracy for phenotyping bulbar vs. non-bulbar ALS was poor (summary receiver operating characteristic area under the curve: left 0.496; right 0.572). Conclusion VN atrophy in ALS is highly prevalent and correlates with disease duration, but not with ALSFRS-R. VN CSA is insufficient to differentiate bulbar from non-bulbar ALS phenotypes. Further studies are warranted to analyze the link between VN atrophy, autonomic impairment, and survival in ALS

Decline in the number of patients with meningitis in German hospitals during the COVID-19 pandemic

BACKGROUND AND OBJECTIVES: In 2020, a wide range of hygiene measures was implemented to mitigate infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In consequence, pulmonary infections due to other respiratory pathogens also decreased. Here, we evaluated the number of bacterial and viral meningitis and encephalitis cases during the coronavirus disease 2019 (COVID-19) pandemic. METHODS: In a multicentre retrospective analysis of data from January 2016 until December 2020, numbers of patients diagnosed with bacterial meningitis and other types of CNS infections (such as viral meningitis and encephalitis) at 26 German hospitals were studied. Furthermore, the number of common meningitis-preceding ear-nose-throat infections (sinusitis, mastoiditis and otitis media) was evaluated. RESULTS: Compared to the previous years, the total number of patients diagnosed with pneumococcal meningitis was reduced (n = 64 patients/year in 2020 vs. n = 87 to 120 patients/year between 2016 and 2019, all p < 0.05). Additionally, the total number of patients diagnosed with otolaryngological infections was significantly lower (n = 1181 patients/year in 2020 vs. n = 1525 to 1754 patients/year between 2016 and 2019, all p < 0.001). We also observed a decline in viral meningitis and especially enterovirus meningitis (n = 25 patients/year in 2020 vs. n = 97 to 181 patients/year between 2016 and 2019, all p < 0.001). DISCUSSION: This multicentre retrospective analysis demonstrates a decline in the number of patients treated for viral and pneumococcal meningitis as well as otolaryngological infections in 2020 compared to previous years. Since the latter often precedes pneumococcal meningitis, this may point to the significance of the direct spread of pneumococci from an otolaryngological focus such as mastoiditis to the brain as one important pathophysiological route in the development of pneumococcal meningitis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00415-022-11034-w

Patterns of Alcohol Consumption Among Individuals With Alcohol Use Disorder During the COVID-19 Pandemic and Lockdowns in Germany

Importance Alcohol consumption (AC) leads to death and disability worldwide. Ongoing discussions on potential negative effects of the COVID-19 pandemic on AC need to be informed by real-world evidence. Objective To examine whether lockdown measures are associated with AC and consumption-related temporal and psychological within-person mechanisms. Design, Setting, and Participants This quantitative, intensive, longitudinal cohort study recruited 1743 participants from 3 sites from February 20, 2020, to February 28, 2021. Data were provided before and within the second lockdown of the COVID-19 pandemic in Germany: before lockdown (October 2 to November 1, 2020); light lockdown (November 2 to December 15, 2020); and hard lockdown (December 16, 2020, to February 28, 2021). Main Outcomes and Measures Daily ratings of AC (main outcome) captured during 3 lockdown phases (main variable) and temporal (weekends and holidays) and psychological (social isolation and drinking intention) correlates. Results Of the 1743 screened participants, 189 (119 [63.0%] male; median [IQR] age, 37 [27.5-52.0] years) with at least 2 alcohol use disorder (AUD) criteria according to the Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition) yet without the need for medically supervised alcohol withdrawal were included. These individuals provided 14 694 smartphone ratings from October 2020 through February 2021. Multilevel modeling revealed significantly higher AC (grams of alcohol per day) on weekend days vs weekdays (β = 11.39; 95% CI, 10.00-12.77; P < .001). Alcohol consumption was above the overall average on Christmas (β = 26.82; 95% CI, 21.87-31.77; P < .001) and New Year’s Eve (β = 66.88; 95% CI, 59.22-74.54; P < .001). During the hard lockdown, perceived social isolation was significantly higher (β = 0.12; 95% CI, 0.06-0.15; P < .001), but AC was significantly lower (β = −5.45; 95% CI, −8.00 to −2.90; P = .001). Independent of lockdown, intention to drink less alcohol was associated with lower AC (β = −11.10; 95% CI, −13.63 to −8.58; P < .001). Notably, differences in AC between weekend and weekdays decreased both during the hard lockdown (β = −6.14; 95% CI, −9.96 to −2.31; P = .002) and in participants with severe AUD (β = −6.26; 95% CI, −10.18 to −2.34; P = .002). Conclusions and Relevance This 5-month cohort study found no immediate negative associations of lockdown measures with overall AC. Rather, weekend-weekday and holiday AC patterns exceeded lockdown effects. Differences in AC between weekend days and weekdays evinced that weekend drinking cycles decreased as a function of AUD severity and lockdown measures, indicating a potential mechanism of losing and regaining control. This finding suggests that temporal patterns and drinking intention constitute promising targets for prevention and intervention, even in high-risk individuals

Measuring self-regulation in everyday life: Reliability and validity of smartphone-based experiments in alcohol use disorder

Self-regulation, the ability to guide behavior according to one's goals, plays an integral role in understanding loss of control over unwanted behaviors, for example in alcohol use disorder (AUD). Yet, experimental tasks that measure processes underlying self-regulation are not easy to deploy in contexts where such behaviors usually occur, namely outside the laboratory, and in clinical populations such as people with AUD. Moreover, lab-based tasks have been criticized for poor test-retest reliability and lack of construct validity. Smartphones can be used to deploy tasks in the field, but often require shorter versions of tasks, which may further decrease reliability. Here, we show that combining smartphone-based tasks with joint hierarchical modeling of longitudinal data can overcome at least some of these shortcomings. We test four short smartphone-based tasks outside the laboratory in a large sample (N = 488) of participants with AUD. Although task measures indeed have low reliability when data are analyzed traditionally by modeling each session separately, joint modeling of longitudinal data increases reliability to good and oftentimes excellent levels. We next test the measures' construct validity and show that extracted latent factors are indeed in line with theoretical accounts of cognitive control and decision-making. Finally, we demonstrate that a resulting cognitive control factor relates to a real-life measure of drinking behavior and yields stronger correlations than single measures based on traditional analyses. Our findings demonstrate how short, smartphone-based task measures, when analyzed with joint hierarchical modeling and latent factor analysis, can overcome frequently reported shortcomings of experimental tasks

A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%–58.7±14.4% when two indicators were combined and 40–48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost

Management of Cerebral Venous Thrombosis Due to Adenoviral COVID-19 Vaccination

Objective Cerebral venous thrombosis (CVT) caused by vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare adverse effect of adenovirus-based severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) vaccines. In March 2021, after autoimmune pathogenesis of VITT was discovered, treatment recommendations were developed. These comprised immunomodulation, non-heparin anticoagulants, and avoidance of platelet transfusion. The aim of this study was to evaluate adherence to these recommendations and its association with mortality. Methods We used data from an international prospective registry of patients with CVT after the adenovirus-based SARS-CoV-2 vaccination. We analyzed possible, probable, or definite VITT-CVT cases included until January 18, 2022. Immunomodulation entailed administration of intravenous immunoglobulins and/or plasmapheresis. Results Ninety-nine patients with VITT-CVT from 71 hospitals in 17 countries were analyzed. Five of 38 (13%), 11 of 24 (46%), and 28 of 37 (76%) of the patients diagnosed in March, April, and from May onward, respectively, were treated in-line with VITT recommendations (p < 0.001). Overall, treatment according to recommendations had no statistically significant influence on mortality (14/44 [32%] vs 29/55 [52%], adjusted odds ratio [OR] = 0.43, 95% confidence interval [CI] = 0.16-1.19). However, patients who received immunomodulation had lower mortality (19/65 [29%] vs 24/34 [70%], adjusted OR = 0.19, 95% CI = 0.06-0.58). Treatment with non-heparin anticoagulants instead of heparins was not associated with lower mortality (17/51 [33%] vs 13/35 [37%], adjusted OR = 0.70, 95% CI = 0.24-2.04). Mortality was also not significantly influenced by platelet transfusion (17/27 [63%] vs 26/72 [36%], adjusted OR = 2.19, 95% CI = 0.74-6.54). Conclusions In patients with VITT-CVT, adherence to VITT treatment recommendations improved over time. Immunomodulation seems crucial for reducing mortality of VITT-CVT. ANN NEUROL 2022Peer reviewe

Cerebral venous sinus thrombosis due to vaccine-induced immune thrombotic thrombocytopenia in middle-income countries

Background: Adenovirus-based COVID-19 vaccines are extensively used in low- and middle-income countries (LMICs). Remarkably, cases of cerebral venous sinus thrombosis due to vaccine-induced immune thrombotic thrombocytopenia (CVST-VITT) have rarely been reported from LMICs. Aims: We studied the frequency, manifestations, treatment, and outcomes of CVST-VITT in LMICs. Methods: We report data from an international registry on CVST after COVID-19 vaccination. VITT was classified according to the Pavord criteria. We compared CVST-VITT cases from LMICs to cases from high-income countries (HICs). Results: Until August 2022, 228 CVST cases were reported, of which 63 were from LMICs (all middle-income countries [MICs]: Brazil, China, India, Iran, Mexico, Pakistan, Turkey). Of these 63, 32 (51%) met the VITT criteria, compared to 103 of 165 (62%) from HICs. Only 5 of the 32 (16%) CVST-VITT cases from MICs had definite VITT, mostly because anti-platelet factor 4 antibodies were often not tested. The median age was 26 (interquartile range [IQR] 20–37) versus 47 (IQR 32–58) years, and the proportion of women was 25 of 32 (78%) versus 77 of 103 (75%) in MICs versus HICs, respectively. Patients from MICs were diagnosed later than patients from HICs (1/32 [3%] vs. 65/103 [63%] diagnosed before May 2021). Clinical manifestations, including intracranial hemorrhage, were largely similar as was intravenous immunoglobulin use. In-hospital mortality was lower in MICs (7/31 [23%, 95% confidence interval (CI) 11–40]) than in HICs (44/102 [43%, 95% CI 34–53], p = 0.039). Conclusions: The number of CVST-VITT cases reported from LMICs was small despite the widespread use of adenoviral vaccines. Clinical manifestations and treatment of CVST-VITT cases were largely similar in MICs and HICs, while mortality was lower in patients from MICs.</p