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Leucocyte dysfunction associated with severe inflammatory stages and/or aging.
Instituto Politecnico Nacional
Immunochemistry Medical Research Unit, Specialties Hospital, National Medical Center “Siglo XXI”, Mexican Institute of Social Security, #330 Cuauhtémoc, Avenue “Doctores”, Mexico City Postcode: 06720
M.D, PhD Lourdes Andrea Arriaga Pizano
M.D, PhD Lourdes Andrea Arriaga Pizano
Type of Research Project
- Clinical Project with Laboratory work
What is the background of the project?
Severe inflammation processes such as Systemic inflammatory response syndrome are related with high mortality rates not just during the acute event (30-60% of mortality rate), but also after leaving the hospital (50% of mortality rate during the next 3 to 5 years). It has been proposed that this is related to a premature aging process induced by severe inflammation. We have previously published that during sepsis, leucocytes increased their expression of the “so called” activation markers, cells have diminished responses to inflammatory mediators and do not eliminate bacteria efficiently. If this leucocyte dysfunction initiates during the acute phase and prevails in sepsis survivors and if such premature aging involves a more compromised immune function is not yet known. Since leucocyte dysfunctional could lead to severe and life threatening infections and that premature immunology ageing predispose to chronic diseases, compromising both life length and quality, it is mandatory to increased our understanding of the effects that severe inflammation has in immunology dysfunction and/or ageing.
What is the aim of the project?
To evaluate, in the major subsets of circulating leukocytes from patients with severe inflammatory conditions, responses elicited by Toll-Like Receptor ligands, such as cytokine production, respiratory burst, and phagocytosis.
What techniques and methods are used?
Samples collection for circulating leukocytes obtention): after informed consent is signed, venous blood samples will be collected in heparinized tubes (16 Units/mililiters). Leucocyte Immunophenotyping: To establish the frequency of the different leukocytes in circulation and its expression of activation markers for leukocyte identification and characterization, 50 microliters of total blood (or 10x105 cells) will be incubated for 15 min at room temperature protected from light with fluorochrome conjugated antibodies (anti-CD45,CD3,CD14, CD16, Human Leukocyte Antigen-DR [HLA-DR], CD69, Triggering Receptors Expressed on Myeloid cells-1). After washing cells with phosphate buffered saline (PBS), at least 5,000 monocytes (CD45+CD14+CD16+/-) will be captured in a FACS Canto flow cytometer. The obtained FCS files will be analyzed with Infinicyt software version 8.0 Ex vivo leukocyte stimulation: For evaluating the response capability that leucocytes from patients with or without severe systemic inflammation have. Total blood will be stimulated or not with lipopolysaccharides “LPS” (100 nanograms/miliLiters) or InterLeukin-6 or Phorbol Myristate Acatate /Ionomicyn in presence or absence of brefeldin A. After 4, 8, 12 or 24 hours, supernatants and cells will be recovered. Cytokine quantification by multiple immunoassay based on beads analyzed by cytometry. To establish systemic cytokine profile from patients and secretion of cytokines after ex vivo stimulation. Serum or supernatant will be incubated with fluorochromed-beads covered with anti-cytokine antibodies (Capture antibodies: anti InterLeukina-1b, InterLeukina-4, InterLeukina-6, InterLeukina-8, InterLeukina-10 or anti-Tumor Necrosis Factor). After 3 hours, a detection antibody will be used for a sandwich like reaction and quantification will be calculated with the fluorescence intensity of the second antibody, according to a reference curve. Phagocytosis and oxidative burst evaluation: For establishing the functional state of phagocytes. Using inactivated bacteria stained with a fluorochrome that changes its emission according to internalization and milieu acidification, at single cell level for each leukocyte (identified by immunophenotyping) phagocytosis and oxidative burst will be evaluated. At least 5,000 monocytes will be capture using a flow cytometer and data analyze using Infinicyt software ELISA: To establish the serological concentration of inflammatory antagonists as Interleukin-Ra, and Enzime linked immunoassay will be used. Statistical analysis: To establish if the values are different among groups studied and that they are not random, T- test, Bonferroni, Man-Whitney U are going to be used for the analysis of parametric and non-parametric data. For association analysis, Spearman rho will be run using Statistical Analysis System/ Statistical software (SAS/STAT software).
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
It is expected that she/he will acquire the skills to work in a basic research laboratory and with clinical data. So she/he will be trained to understand the basics in blood sample manage, flow cytometry, Enzyme-Linked ImmunoSorbent Assay, and Cell culture techniques, all according to Good Laboratory Practices and with an strong translational view of medical sciences. The student will be involved in all the different techniques that we use in the project and that were mentioned before. The specific tasks that are expected to be accomplished by the student depends on each kind of technique, but in a brief way, she/he will participate and be highly involved in each of them as is mentioned in the techniques and methods section.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Yes, students will be trained as users of our Flow Cytometry core facility. Students will also participate in our weekly lab-seminars of article discussion and project dissertation from graduate students.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a poster - The student will prepare a scientific report - The student will prepare an abstract - The student’s name will be mentioned in a future publication
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
It is important that the student has basic knowledge of immunology, molecular biology, and biochemistry.
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system
- Function is Dissociated From Activation-Related Immunophenotype on Phagocytes From Patients With Sirs/Sepsis Syndrome. Flores-Mejía LA; et al. Shock. 2018 Dec 26. doi: 10.1097/SHK.0000000000001314
- Multiparameter flow cytometry analysis of leukocyte markers for diagnosis in preterm neonatal sepsis. Vázquez Rodríguez S;et al. J Matern Fetal Neonatal Med. 2019 Sep 19:1-11. doi: 10.1080/14767058.2019.1666100
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