|News and Events||Working Groups||Short-Term Scientific Missions||Management Committee||Members|
COST Action CA15203 (2016-2021): MitoEAGLE
Evolution-Age-Gender-Lifestyle-Environment: mitochondrial fitness mapping
MitoPedia topics: EAGLE
COST WG4: WG4
|Name||Garcia-Souza Luiz Felipe, MSc, PhD|
Luiz joined Oroboros Instruments in August 2016.
|Address||Schoepfstrasse 18, A-6020|
|O2k-Network Lab||AT Innsbruck Oroboros, BR Rio de Janeiro Oliveira MF|
Topics: platelet, thrombin, activation
|BEC 2020.1 doi10.26124bec2020-0001.v1||BEC 2020.1||Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. https://doi.org/10.26124/bec:2020-0001.v1|
|Fischer 2022 Metabolites||2022||Fischer C, Valente de Souza L, Komlódi T, Garcia-Souza LF, Volani C, Tymoszuk P, Demetz E, Seifert M, Auer K, Hilbe R, Brigo N, Petzer V, Asshoff M, Gnaiger E, Weiss G (2022) Mitochondrial respiration in response to iron deficiency anemia. Comparison of peripheral blood mononuclear cells and liver. https://doi.org/10.3390/metabo12030270|
|Vernerova 2021 Biomedicines||2021||Vernerova A, Garcia-Souza LF, Soucek O, Kostal M, Rehacek V, Krcmova LK, Gnaiger E, Sobotka O (2021) Mitochondrial respiration of platelets: comparison of isolation methods. https://doi.org/10.3390/biomedicines9121859|
|Hoppel 2021 Cells||2021||Hoppel F, Garcia-Souza LF, Kantner-Rumplmair W, Burtscher M, Gnaiger E, Pesta D, Calabria E (2021) Human platelet mitochondrial function reflects systemic mitochondrial alterations: a protocol for application in field studies. https://doi.org/10.3390/cells10082088|
|MitoEAGLE blood cells 1||2020||Åsander Frostner Eleonor*, Aburel Oana M*, Avram Vlad F*, Calabria Elisa, Castelo Rueda Maria Paulina*, Chamkha Imen*, Čižmárová Beata, Danila Maria-Daniela*, Doerrier Carolina*, Eckert Gunter P*, Ehinger Johannes K, Elmer Eskil*, Garcia-Souza Luiz F*, Gnaiger Erich*, Hoppel Florian*, Karabatsiakis Alexander*, Keppner Gloria, Kidere Dita*, Krako Jakovljević Nina, Labieniec-Watala Magdalena*, Lelcu Theia*, Micankova Petra, Michalak Slawomir*, Molina Anthony JA*, Pavlovic Kasja, Pichler Irene*, Piel Sarah, Rousar Tomas, Rybacka-Mossakowska Joanna, Schartner Melanie, Siewiera Karolina*, Silaidos Carmina*, Sjövall Fredrik*, Sobotka Ondrej*, Sumbalova Zuzana*, Swiniuch Daria, Vernerova Andrea*, Volani Chiara*, Vujacic-Mirski Ksenija*, Watala Cezary* (2020) Interlaboratory guide to mitochondrial respiratory studies with peripheral blood mononuclear cells and platelets. - Updated: 2020-03-06 - *Confirmed|
|Doerrier 2018 Methods Mol Biol||2018||Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. https://doi.org/10.1007/978-1-4939-7831-1_3|
|Gatterer 2018 J Sports Sci Med||2018||Gatterer H, Menz V, Salazar-Martinez E, Sumbalova Z, Garcia-Souza LF, Cizmarova B, Gnaiger E, Burtscher M (2018) Exercise performance, muscle oxygen extraction and blood cell mitochondrial respiration after repeated-sprint and sprint interval training in hypoxia: a pilot study. J Sports Sci Med 17:339-347.|
|Sumbalova 2018 Nova Sciences||2018||Sumbalová Z, Garcia-Souza LF, Cizmarova B, Volani C, Gnaiger E (2018) Analysis of mitochondrial function in human blood cells. In: Recent advances in mitochondrial medicine and coenzyme Q10. Gvozdjáková A, Cornelissen G, Singh RB eds, Nova Sciences:255-68.|
|Rochael 2015 Sci Rep||2015||Rochael NC, Guimarães-Costa AB, Nascimento MT, DeSouza-Vieira TS, Oliveira MP, Garcia-Souza LF, Oliveira MF, Saraiva EM (2015) Classical ROS-dependent and early/rapid ROS-independent release of neutrophil extracellular traps triggered by Leishmania parasites. Sci Rep 5:18302.|
|Garcia-Souza 2014 Int J Biochem Cell Biol||2014||Garcia-Souza LF, Oliveira MF (2014) Mitochondria: Biological roles in platelet physiology and pathology. Int J Biochem Cell Biol 50:156-60.|
|Japiassu 2011 Crit Care Med||2011||Japiassu AM, Santiago AP, d'Avila Jda C, Garcia-Souza LF, Galina A, Castro Faria-Neto HC, Bozza FA, Oliveira MF (2011) Bioenergetic failure of human peripheral blood monocytes in patients with septic shock is mediated by reduced F1Fo adenosine-5'-triphosphate synthase activity. Crit Care Med 39:1056-63.|
MitoEAGLE Short-Term Scientific Mission
- Work Plan summary
- Examination of platelet (PLT) function is a topic neglected in hematology mainly due to the unavailability of suitable methods. The gold standard today is the examination of optical aggregometry, which is an easily available method, but has low w and is usually difficult to interpret. Electron microscopy and other examinations do not clearly target PLT functionality. A simple PLT count (especially in the case of deep thrombocytopenia) creates a false impression of bleeding diathesis. Even a small number of PLT, usually about 10 to 30 mio/mL, is enough for effective hemostasis, as shown in the case of immune thrombocytopenia. Knowledge of functional activity in these cases could eliminate the need to discontinue anticoagulant treatment or prophylaxis and thus improve patient quality of life.
- Thanks to modern high-resolution respirometers, we can analyze and study the metabolic profile and function of PLT and peripheral blood mononuclear cells (PBMC) isolated from a small amount of venous blood. Respirometry with PLT isolated from peripheral blood by differential centrifugation provides an adequate alternative for the study of human and non-hematologic diseases with minimal risk to patients. As a result, this research has attracted the attention of the scientific community in recent years as a possible experimental model in various medical applications and cell research. As an example, outside the hematology field, it was shown that increased respiration of PLT from septic patients correlates with an increase in pro-inflammatory cytokines in the bloodstream. Similarly, the change in mitochondrial PBMC function was correlated with systemic dysfunctions during hemorrhagic shock and resuscitation. A recent study demonstrated a change in the metabolic profile of PLT in diabetic rats.
- With an increasing database collected by various groups worldwide, we encounter methodological problems discrediting data reproducibility and disallowing further progress of this method to clinical practice. The main concerns and discrepancies were observed in the preparation of the sample, specifically the isolation procedure used to obtain quality control of PLT and PBMC. Different types of anticoagulants, different isolation procedures and the respiratory measurement protocol itself have not been standardized until recently and are therefore the subject of intensive research and discussion in expert forums in the frame of international project MitoEAGLE (COST Action CA15203).
- Blood transfusions are necessary for the treatment of various pathological conditions and have been used for decades worldwide. The therapeutic benefits of PLT transfusions are generally acknowledged by medical professionals and the process of separating and storing PLT concentrates has been documented in detail. However, improvement of the function and life of PLT transfusions is still under investigation.
- The aim of this project is to introduce differential centrifugation methodology in the University Hospital in Hradec Kralove and to compare mitochondrial respiration of PLT obtained by this method and by single donor apheresis from healthy donors.
- After developing a reproducible quality control, we plan to use this methodology mainly in patients after chemotherapy, bone marrow infiltration or septic conditions. For these patients, thrombocytopenia is usually transient but especially limiting because of the apparent risk of intensive bleeding. Another area of use is the diagnosis of immune thrombocytopenia, which is currently still a diagnosis “per exclusionem” due to lack of diagnostic methods. Immune thrombocytopenia is characterized by an increased production and destruction of otherwise functional platelets. Unfortunately, there is no test that would otherwise demonstrate good platelet function and thus facilitate differential diagnosis of platelet pathologies. Including a functional test for platelet activity and verifying its relevance to individual diagnoses would greatly facilitate orientation in the subject and would have clear clinical applications in hematological practice. Thanks to the metabolic role of mitochondria, this methodology can be also applied to study metabolic diseases in both clinical and basic research: 1) in septic patients receiving full parenteral nutrition; 2) in the development of acute light and severe pancreatitis; 3) obese diabetic patients during two-week weight reduction hospitalization.
- Work Plan summary
- Work Plan summary
- A rapidly increasing number of human pathologies is linked to mitochondrial dysfunction. During the past few years,blood cells such as peripheral blood mononuclear cells (PBMCs) and platelets (PLTs) have become the targets in investigations on various pathologies, particularly as potential alternatives to the invasive sampling of tissue biopsies. However, standard operating procedures for mitochondrial respiratory studies of blood cells are still missing. A key towards achieving comparability among data sets and the applicability of human blood cells for the evaluation of mitochondrial fitness in health and disease is the standardization of the procedures to separate or isolate blood cells, the experimental procedure for the evaluation of mitochondrial respiratory characteristics, and the format for reporting. Publications regarding PBMC and PLT respiration, lack harmonization. Application of different anticoagulants, different isolation methods and storage, and different respiratory protocols make it impossible to compare results on a quantitative basis. In a previous WG4 meeting (Lund 2018-05-28), it was reported that during measurements of H2O2 production of Permeabilized PLT, it was observed a rapid increase in H2O2 production after titration of digitonin. However, other groups reported that this phenomenon was not observed in their experiments. To achieve comparability of results, we propose in this STSM to compare PLT isolation methods from Prof. Dr. Eskil Elmer’s lab (MitoEAGLE Lund’s protocol) and Prof. Dr. Erich Gnaiger’s lab (MitoEAGLE Innsbruck protocol). Comparing these protocols in the same subjects, we can define a standard protocol for future projects in Lund, Innsbruck, and perhaps among other labs participating in MitoEAGLE. For this project, PLTs will be isolated from whole blood using both protocol from the same blood sample. After isolation, PLTs will be submitted to flow cytometry, respirometry and fluorometry. Remnants cell will be cryopreserved. Flow cytometry: Activation markers, such as CD-62P and Annexin-V. To be certain that our new viability method can be applied to platelets, it is necessary to use different (activation) markers to correlate with our respirometric data. Suggested markers: (A) Pselectin (CD62P) - activation marker, the more stressed/activated, more expression is detected on the surface of platelets. Respirometry: A respiration protocol was developed a substrate-uncoupler-inhibitor titration protocol to measure respiratory control in intact cells and cell viability in an integrated assay: the coupling control and cell viability protocol (SUIT 7, CCVP). The respirometric CCVP viability test can be applied to platelets. Due to their small size there is no comparable method available, since standard tests are linked to activation markers rather than cell viability. The combination of these experiments provide data to compare activation induced by PLT isolation and validate the respirometric viability test to PLT simultaneously. Fluorometry: H2O2 assessment will be performed in parallel to respiration. SUIT 9 is a short, simple protocol that can address the issue of “oxidative burst” reported in WG4 meeting. With this experiment, we can investigate and adjust our isolation protocols to avoid unpredictable measurement artifacts. Experimental details (respirometry): • Number of donors: 4 to 5 • Respiration media: MiR05 •Cell type: freshly isolated PLT Protocols (respirometry/Fluorometry): • AmR_SUIT 9_pce: 1S;2D;3P;4Ama • SUIT 7: ce1;ce2P;ce3Omy;ce4U*;ce5Glc;ce6M2;ce7Rot;ce8S;1Dig*;1U*;1c; 2Ama;3AsTm;4Azd Experimental details (flow cytometry): • Number of donors: 4 to 5 Incubation: 20 min, RT, Tyrode modified (137 mM NaCl, 2,68 mM KCI, 5 mM HEPES, 1 mM MgCl2, 11,9 mM NaHCO3, 0,42 mM NaH2PO4 and 5,6 mM glucose; pH 7,4) Marker: • CD62P Cryopreservation: • Cryopreservation media: Autologous plasma+10 mM EGTA +10%DMSO • Storage temperature: -80 ºC, for 1 week
- Work Plan summary
- IOC154 Innsbruck AT
- Bioblast 2022: BEC Inaugural Conference
- IOC145 Innsbruck AT
- IOC144 Innsbruck AT
- IOC141 Schroecken AT
- Life Science PhD Meeting 2019 Innsbruck AT
- MitoEAGLE 2019 Matrei am Brenner AT
- DSL Retreat 2019 Innsbruck AT
- MitoEAGLE 2018 Innsbruck AT
- MitoEAGLE 2018 Lund SE
- MitoEAGLE 2018 Poznan PL
- MitoEAGLE 2018 Innsbruck AT
- MitoFit Workshop ATP 2017 Innsbruck AT
- MitoEAGLE 2017 Hradec Kralove CZ
- MiPschool 2017 Obergurgl AT
- MitoFit Open Seminar 2017 Innsbruck AT
- IOC122 Schroecken AT
- MitoEAGLE 2016 Verona IT
- IOC114 Innsbruck AT
- MitoFit Science Camp 2016 Kuehtai AT
- IOC80 Schroecken AT
Visiting scientist in the Oroboros O2k-Laboratory
- 2016, February 15 to March 15