A novel C5a receptor-tissue factor cross-talk in neutrophils links innate immunity to coagulation pathways

Neutrophils and complement are key sentinels of innate immunity and mediators of acute inflammation. Recent studies have suggested that inflammatory processes modulate thrombogenic pathways. To date, the potential cross-talk between innate immunity and thrombosis and the precise molecular pathway by which complement and neutrophils trigger the coagulation process have remained elusive. In this study, we demonstrate that antiphospholipid Ab-induced complement activation and downstream signaling via C5a receptors in neutrophils leads to the induction of tissue factor (TF), a key initiating component of the blood coagulation cascade. TF expression by neutrophils was associated with an enhanced procoagulant activity, as verified by a modified prothrombin time assay inhibited by anti-TF mAb. Inhibition studies using the complement inhibitor compstatin revealed that complement activation is triggered by antiphospholipid syndrome (APS) IgG and leads to the induction of a TF-dependent coagulant activity. Blockade studies using a selective C5a receptor antagonist and stimulation of neutrophils with recombinant human C5a demonstrated that C5a, and its receptor C5aR, mediate the expression of TF in neutrophils and thereby significantly enhance the procoagulant activity of neutrophils exposed to APS serum. These results identify a novel cross-talk between the complement and coagulation cascades that can potentially be exploited therapeutically in the treatment of APS and other complement-associated thrombotic diseases

A Tumor Mitochondria Vaccine Protects against Experimental Renal Cell Carcinoma

Mitochondria provide energy for cells via oxidative phosphorylation. Reactive oxygen species, a byproduct of this mitochondrial respiration, can damage mitochondrial DNA (mtDNA), and somatic mtDNA mutations have been found in all colorectal, ovarian, breast, urinary bladder, kidney, lung, and pancreatic tumors studied. The resulting altered mitochondrial proteins or tumor-associated mitochondrial Ags (TAMAs) are potentially immunogenic, suggesting that they may be targetable Ags for cancer immunotherapy. In this article, we show that the RENCA tumor cell line harbors TAMAs that can drive an antitumor immune response. We generated a cellular tumor vaccine by pulsing dendritic cells with enriched mitochondrial proteins from RENCA cells. Our dendritic cell-based RENCA mitochondrial lysate vaccine elicited a cytotoxic T cell response in vivo and conferred durable protection against challenge with RENCA cells when used in a prophylactic or therapeutic setting. By sequencing mtDNA from RENCA cells, we identified two mutated molecules: COX1 and ND5. Peptide vaccines generated from mitochondrial-encoded COX1 but not from ND5 had therapeutic properties similar to RENCA mitochondrial protein preparation. Thus, TAMAs can elicit effective antitumor immune responses, potentially providing a new immunotherapeutic strategy to treat cancer

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

The cytokine network in the initiation and/or proliferation of immune response: A study regarding the role of leptin in coagulation

Background: Leptin is a pleiotropic hormone involved in the regulation of energy balance, glucose homeostasis and inflammation pathways. The leptin receptor is expressed on leucocytes, and leptin has been shown to induce their functional activation and cytokine production. Human obesity is frequently associated with elevated circulating leptin levels and correlated with cardiovascular coagulopathy. Tissue factor (TF) is the primary in vivo initiator of the extrinsic coagulation cascade. Objective: Prompted by findings that different inflammatory mediators can induce expression of TF, we examined the role of leptin as an inducer of tissue factor in polymorphonuclear (PMN) and peripheral blood mononuclear cells (PBMC). Patients and Methods PMN and PBMC from healthy donors and Myeloproliferative disease (MPD) patients, carrying the V617F mutation resulting to constant JAK2 phosphorylation, were incubated with leptin and chemical inhibitors and TF expression was determined at functional, mRNA and protein levels. Results: TF was found to be over-expressed when both cell types were stimulated with leptin while chemical inhibition studies revealed that the effects of leptin on TF expression are mediated, at least in part, by signal transduction pathways involving JAK2 and PI-3 kinase, but not MAP kinases. Similarly, leucocytes from patients with MPD were unable to respond to leptin, probably due to a constant over-expression of SOCS3 in MPD myeloid cells. Conclusion: Our findings indicate a novel link between inflammation and thrombosis by showing that leptin can trigger the extrinsic coagulation cascade. Furthermore, they offer the knowledge that MPD cells could be used as a human model for further investigation of leptin resistance.Η λεπτίνη είναι μια πλειοτροπική ορμόνη που εμπλέκεται στον έλεγχο της ενεργειακής ισορροπίας, της ομοιόστασης της γλυκόζης και των οδών της φλεγμονής. Ο υποδοχέας της λεπτίνης εκφράζεται στα λευκά αιμοσφαίρια, και η λεπτίνη έχει αποδειχθεί ότι προκαλεί τη λειτουργική τους ενεργοποίηση οδηγώντας στην παραγωγή κυτταροκινών. Η ανθρώπινη παχυσαρκία συνδέεται με υψηλά επίπεδα λεπτίνης στην περιφέρεια και συσχετίζεται με καρδιαγγειακή θρομβοφιλία. Ο ιστικός παράγοντας (TF) είναι ο εκκινητής του εξωγενούς καταρράκτη της πήξης, ενώ παρουσιάζεται σε υψηλές συγκεντρώσεις σε παχύσαρκα άτομα. Παρακινούμενοι από τις παρατηρήσεις ότι πλήθος φλεγμονωδών παραγόντων μπορούν να προκαλέσουν την έκφραση του TF, εξετάσθηκε ο ρόλος της λεπτίνης ως επαγωγέας του ιστικού παράγοντα σε πολυμορφοπύρηνα (PMN) και μονοπύρηνα κύτταρα του περιφερικού αίματος (PBMC). PMN και PBMC από υγιείς δότες και ασθενείς με μυελοΰπερπλαστικά σύνδρομα (MPD), οι οποίοι εμφανίζουν τη μετάλλαξη V617F που οδηγεί σε συνεχή φωσφορυλίωση του JAK2, επωάστηκαν με λεπτίνη και χημικούς αναστολείς της οδού του JAK2 και η έκφραση του TF εξετάστηκε σε λειτουργικό, mRNA και πρωτεϊνικό επίπεδο. Ο TF βρέθηκε να υπέρ-εκφράζεται και στους δύο τύπους κυττάρων όταν διεγέρθηκαν με λεπτίνη, ενώ οι μελέτες χημικής αναστολής αποκάλυψαν ότι τα αποτελέσματα της λεπτίνης στην έκφραση του TF ελέγχονται, τουλάχιστον εν μέρει, από οδούς μετάδοσης σήματος που περιλαμβάνουν τον JAK2 και την κινάση PI-3, αλλά όχι τις κινάσεις MAP. Ομοίως, τα λευκοκύτταρα από ασθενείς με MPD ήταν ανίκανα να αποκριθούν στη λεπτίνη, πιθανώς λόγω της υπερέκφρασης του αναστολέα SOCS3 στα μυελικά κύτταρα των ασθενών με MPD. Τα αποτελέσματα αυτά δείχνουν μια νέα σύνδεση μεταξύ της φλεγμονής και της θρόμβωσης, αποδεικνύοντας ότι η λεπτίνη μπορεί να προκαλέσει την ενεργοποίηση του εξωγενή καταρράκτη της πήξης. Επιπλέον, προσφέρουν την πληροφορία ότι τα κύτταρα ασθενών με MPD θα μπορούσαν να χρησιμοποιηθούν ως ένα ανθρώπινο μοντέλο για την περαιτέρω έρευνα σχετικά με την αντίσταση στη λεπτίνη

Προς στρατηγική ελέγχου εμβολιασμού ενάντια στη νόσο COVID-19 με σκοπό τη βέλτιστη κατανομή πόρων μεταξύ διαφορετικών ηλικιακών ομάδων

Summarization: The rapid dissemination of the COVID-19 pandemic in recent times has turned the focus upon developing strategies for eradicating the COVID-19 disease. As development of vaccinations against SARS-CoV-2 virus has proven to be useful for the immunization and prevention of severe cases, it is important to employ optimal control policies regarding inoculation of a population while taking into consideration factors such as the available vaccine resources, the death counts and attaining herd immunity. In this work, an age-structured compartmental epidemiological model is proposed in order to model the dynamics of COVID-19. The model is then used to describe the dissemination of the disease in Greece based on available data using system identification techniques and a-priori knowledge of the disease behavior. The available data are processed to estimate the true number of infections and to best suit the model. The results are then presented for the data processing, the specification of the system parameters and the fitting of the system outputs to the data. Finally, preliminary work on the optimal vaccination control strategy based on Pontryagin’s Minimum Principle is presented for a controller which drives the system to herd immunity while minimizing the vaccine resource utilization and the number of deaths in the population.Περίληψη: Η ραγδαία εξάπλωση της πανδημίας COVID-19 πρόσφατα έχει στρέψει την προσοχή στην ανάπτυξη στρατηγικών εξάλειψης της ασθένειας. Καθώς η ανάπτυξη των εμβολίων ενάντια στον ιό SARS-CoV-2 αποδεικνύεται εξαιρετικά χρήσιμη για την ανοσοποίηση και την αποτροπή σοβαρής νόσου, κρίνεται σημαντική η υιοθέτηση βέλτιστης πολιτικής ελέγχου εμβολιασμού στον πληθυσμό λαμβάνοντας υπόψη παράγοντες όπως οι διαθέσιμοι πόροι εμβολίων, ο αριθμός των θανάτων ασθενών, καθώς και η επίτευξη της ανοσίας της αγέλης. Σε αυτή την εργασία, ένα ηλικιακά-δομημένο compartmental επιδημιολογικό μοντέλο προτείνεται με σκοπό την μοντελοποίηση της δυναμικής της ασθένειας COVID-19. Στη συνέχεια το μοντέλο αυτό χρησιμοποιείται για την περιγραφή της εξάπλωσης της ασθένειας στην Ελλάδα με βάση τα διαθέσιμα δεδομένα χρησιμοποιώντας τεχνικές system identification και την εκ-των-προτέρων γνώση για τη συμπεριφορά της ασθένειας. Τα διαθέσιμα δεδομένα υφίστανται επεξεργασία ώστε να μπορούν να εφαρμοστούν στο μοντέλο και ώστε να γίνει εκτίμηση του πραγματικού αριθμού των κρουσμάτων. Κατόπιν παρουσιάζονται τα αποτελέσματα από την επεξεργασία των δεδομένων, τον προσδιορισμό των παραμέτρων του συστήματος και την προσαρμογή των εξόδων του συστήματος στα δεδομένα. Τέλος, παρουσιάζεται μία αρχική μελέτη για την βέλτιστη πολιτική ελέγχου εμβολιασμού βασισμένη στην Ελάχιστη Αρχή του Pontryagin για έναν ελεγκτή που οδηγεί το σύστημα στην επίτευξη της ανοσίας της αγέλης με ταυτόχρονη ελαχιστοποίηση της χρήσης πόρων των εμβολίων και του αριθμού των θανάτων στον πληθυσμό

Numerical investigation of the role of heat transfer in bubble dynamics

[EN] Bubble dynamics is generally described by the well-known Rayleigh-Plesset (R-P) equation in which the bubble pressure (or equivalently the bubble density) is predefined by assuming a polytropic gas equation of state with common assumptions to include either isothermal or adiabatic bubble behaviour. The present study examines the applicability of this assumption by assuming that the bubble density obeys the ideal gas equation of state, while the heat exchange with the surrounding liquid is estimated as part of the numerical solution. The numerical model employed includes the solution of the Navier-Stokes equations along with the energy equation, while the liquidgas interface is tracked using the Volume of Fluid (VOF) methodology; phase-change mechanism is assumed to be insignificant compared to bubble heat transfer mechanism. To assess the effect of heat transfer and gas equation of state on bubble behaviour, simulations are also performed for the same initial conditions by using a polytropic equation of state for the bubble phase without solving the energy equation. The accuracy of computations is enhanced by using a dynamic local grid refinement technique which reduces the computational cost and allows for the accurate representation of the interface for the whole duration of the phenomenon in which the bubble size changes significantly. A parametric study performed for various initial bubble sizes and ambient conditions reveals the cases for which the bubble behaviour resembles that of an isothermal or the adiabatic one. Additional to the CFD simulations, a 0-D model is proposed to predict the bubble dynamics. This combines the solution of a modified R-P equation assuming ideal gas bubble content along with an equation for the bubble temperature based on the 1st law of thermodynamics; a correction factor is used to represent accurately the heat transfer between the two phases.Financial support from the MSCA-ITN-ETN of the European Union’s H2020 programme, under REA grant agreement n. 675676 is acknowledged.Fostiropoulos, SR.; Malgarinos, I.; Strotos, G.; Nikolopoulos, N.; Kakaras, E.; Koukouvinis, P.; Gavaises, M. (2017). Role of heat transfer in bubble dynamics neglecting phase change. A numerical study. En Ilass Europe. 28th european conference on Liquid Atomization and Spray Systems. Editorial Universitat Politècnica de València. 904-911. https://doi.org/10.4995/ILASS2017.2017.4691OCS90491

Inhibition of biomaterial-induced complement activation attenuates the inflammatory host response to implantation

Although complement is a known contributor to biomaterial-induced complications, pathological implications and therapeutic options remain to be explored. Here we investigated the involvement of complement in the inflammatory response to polypropylene meshes commonly used for hernia repair. In vitro assays revealed deposition of complement activation fragments on the mesh after incubation in plasma. Moreover, significant mesh-induced complement and granulocyte activation was observed in plasma and leukocyte preparations, respectively. Pretreatment of plasma with the complement inhibitor compstatin reduced opsonization &gt;2-fold, and compstatin and a C5a receptor antagonist (C5aRa) impaired granulocyte activation by 50 and 67%, respectively. We established a clinically relevant mouse model of implantation and could confirm deposition of C3 activation fragments on mesh implants in vivo using immunofluorescence. In meshes extracted after subcutaneous or peritoneal implantation, the amount of immune cell infiltrate in mice deficient in key complement components (C3, C5aR), or treated with C5aRa, was approximately half of that observed in wild-type littermates or mice treated with inactive C5aRa, respectively. Our data suggest that implantation of a widely used surgical mesh triggers the formation of an inflammatory cell microenvironment at the implant site through complement activation, and indicates a path for the therapeutic modulation of implant-related complications.Kourtzelis, I., Rafail, S., DeAngelis, R. A., Foukas, P. G., Ricklin, D., Lambris, J. D. Inhibition of biomaterial-induced complement activation attenuates the inflammatory host response to implantation