Entwicklung einer standardisierbaren Methodik zur Dokumentation und Quantifizierung der Heterogenität und Dynamik des thrombozytären Formwandels

1. Background and objectives Blood platelets respond to many stimuli by changing shape from resting, discoid forms into more rounded structures processing blebs and pseudopodia. Morphological change of platelet structure during activation is generally referred as the platelet shape change (PSC) reaction. PSC is a dynamic process and shows a remarkable heterogeneity. Until now the dynamics and the heterogeneity of PSC could not be analyzed quantitatively. There is evidence that the nature and extent of PSC might bear a physiological and probably also pathophysioloigal meaning. 2. Materials and methods We present a largely automated and thus for standardization suitable procedure for accurate and traceable documentation and quantification of PSC. Platelet rich plasma from anticoagulated whole blood was analyzed by dark field microspcopy. A computer controlled digital camera was adapted to a dark field light microscope. A continiuous series of recordings was captured. PSC was categorized and the recordings were evaluated using a self-developed, computer-based algorithm for analysis of platelet shape. 3. Results PSC continues for several hours after preparation. PSC can be monitored continuously on a single-cell basis. We observed and documentated a continuing PSC for a maximum period of 145 hours. The extension of the hyalomer, the formation of pseudopodia and microparticles can be quantitatively analyzed and graphically visualized. Different samples showed significantly different and even partly contrary expression patterns of hyalomer and pseudopodia formation. 4. Conclusions It is now possible to continuously monitor PSC on a single-cell basis. This could lead to a better understanding of platelet function. PSC can exactly be examined after platelet activation during a prolonged period regarding specific pattern of PSC depending on given conditions. Therefore we might contribute to new diagnostic and therapeutic approaches for detection of platelet dysfunction in diseases such as diabetes mellitus and systemic inflammatory diseases. Furthermore the presented method could improve the testing of drugs with effect on PSC.1. Hintergrund und Ziele Thrombozyten reagieren auf eine Vielzahl von Reizen mit einer Änderung ihrer scheibchenförmigen Ruheform in eine Kugelform und mit der Ausbildung von Bläschen und Fortsätzen. Dieser thrombozytäre Formwandel (TFW) zeigt eine auffällige Heterogenität und eine protrahierte Dynamik, die bislang nicht quantitativ erfasst werden können. Es gibt Hinweise, dass der Art und dem Ausmaß des TFW eine physiologische und evtl. auch pathophysiologische Bedeutung zukommen könnten. 2. Methoden Wir stellen ein bereits in großen Teilen automatisiertes und somit standardisierbares Verfahren vor, dass eine exakte und nachvollziehbare Dokumentation und Quantifizierung von TFW-Prozesse ermöglicht. Thrombozytenreiches Plasma wird in den Strahlengang eines Dunkelfeldmikroskops eingebracht. Mittels einer an das Mikroskop adaptierten, computergesteuerten Digitalkamera wird eine kontinuierliche Aufnahmeserie erfasst. Der TFW wird kategorisiert und mittels eines computergestützten Vermessungsalgorithmus ausgewertet. 3. Ergebnisse und Beobachtungen Der TFW ist ein über viele Stunden anhaltender kontinuierlicher Prozess. Es konnte ein kontinuierlich anhaltender TFW über einen Beobachtungszeitraum von maximal 145 Stunden dokumentiert werden. Hyalomerausdehnung, Pseudopodienausbildung und Mikropartikelbildung können kontinuierlich quantitativ erfasst, visualisiert und ausgewertet werden. In unterschiedlichen Proben zeigen sich signifikant unterschiedliche Ausprägungsmuster und zum Teil gegenläufige Entwicklungen des TFW. 4. Praktische Schlussfolgerungen Der TFW kann jetzt auf Einzelzellbasis kontinuierlich erfasst werden. Hierdurch kann zu einem besseren Verständnis thrombozytärer Funktionen in einer protrahierten Phase nach der Aktivierung beigetragen werden. Es kann untersucht werden, ob sich spezifische, vom umgebenden Milieu abhängige TFW-Muster zeigen. Hieraus könnten sich neue diagnostische und therapeutische Ansätze für Krankheiten wie Diabetes mellitus und entzündliche Systemerkrankungen, sowie für die Testung formwandelmodulierender Medikamente ergeben

Hautkrankheiten

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Geschlechts-Krankheiten

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The United States COVID-19 Forecast Hub dataset

Academic researchers, government agencies, industry groups, and individuals have produced forecasts at an unprecedented scale during the COVID-19 pandemic. To leverage these forecasts, the United States Centers for Disease Control and Prevention (CDC) partnered with an academic research lab at the University of Massachusetts Amherst to create the US COVID-19 Forecast Hub. Launched in April 2020, the Forecast Hub is a dataset with point and probabilistic forecasts of incident cases, incident hospitalizations, incident deaths, and cumulative deaths due to COVID-19 at county, state, and national, levels in the United States. Included forecasts represent a variety of modeling approaches, data sources, and assumptions regarding the spread of COVID-19. The goal of this dataset is to establish a standardized and comparable set of short-term forecasts from modeling teams. These data can be used to develop ensemble models, communicate forecasts to the public, create visualizations, compare models, and inform policies regarding COVID-19 mitigation. These open-source data are available via download from GitHub, through an online API, and through R packages

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival